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Current state of stem cell-based therapies: an overview

Riham mohamed aly.

1 Department of Basic Dental Science, National Research Centre, Cairo, Egypt;

2 Stem Cell Laboratory, Center of Excellence for Advanced Sciences, National Research Centre, Cairo, Egypt

Recent research reporting successful translation of stem cell therapies to patients have enriched the hope that such regenerative strategies may one day become a treatment for a wide range of vexing diseases. In fact, the past few years witnessed, a rather exponential advancement in clinical trials revolving around stem cell-based therapies. Some of these trials resulted in remarkable impact on various diseases. In this review, the advances and challenges for the development of stem-cell-based therapies are described, with focus on the use of stem cells in dentistry in addition to the advances reached in regenerative treatment modalities in several diseases. The limitations of these treatments and ongoing challenges in the field are also discussed while shedding light on the ethical and regulatory challenges in translating autologous stem cell-based interventions, into safe and effective therapies.

Introduction

Cell-based therapy as a modality of regenerative medicine is considered one of the most promising disciplines in the fields of modern science & medicine. Such an advanced technology offers endless possibilities for transformative and potentially curative treatments for some of humanities most life threatening diseases. Regenerative medicine is rapidly becoming the next big thing in health care with the particular aim of repairing and possibly replacing diseased cells, tissues or organs and eventually retrieving normal function. Fortunately, the prospect of regenerative medicine as an alternative to conventional drug-based therapies is becoming a tangible reality by the day owing to the vigorous commitment of the research communities in studying the potential applications across a wide range of diseases like neurodegenerative diseases and diabetes, among many others ( 1 ).

Recent research reporting successful translation of stem cell therapies to patients have enriched the hope that such regenerative strategies may one day become a treatment for a wide range of vexing diseases ( 2 ). In fact, the past few years witnessed, a rather exponential advancement in clinical trials revolving around stem cell-based therapies. Some of these trials resulted in remarkable impact on various diseases ( 3 ). For example, a case of Epidermolysis Bullosa manifested signs of skin recovery after treatment with keratinocyte cultures of epidermal stem cells ( 4 ). Also, a major improvement in eyesight of patients suffering from macular degeneration was reported after transplantation of patient-derived induced pluripotent stem cells (iPSCs) that were induced to differentiate into pigment epithelial cells of the retina ( 5 ).

However, in spite of the increased amount of publications reporting successful cases of stem cell-based therapies, a major number of clinical trials have not yet acquired full regulatory approvals for validation as stem cell therapies. To date, the most established stem cell treatment is bone marrow transplants to treat blood and immune system disorders ( 1 , 6 , 7 ).

In this review, the advances and challenges for the development of stem-cell-based therapies are described, with focus on the use of stem cells in dentistry in addition to the advances reached in regenerative treatment modalities in several diseases. The limitations of these treatments and ongoing challenges in the field are also discussed while shedding light on the ethical and regulatory challenges in translating autologous stem cell-based interventions, into safe and effective therapies.

Stem cell-based therapies

Stem cell-based therapies are defined as any treatment for a disease or a medical condition that fundamentally involves the use of any type of viable human stem cells including embryonic stem cells (ESCs), iPSCs and adult stem cells for autologous and allogeneic therapies ( 8 ). Stem cells offer the perfect solution when there is a need for tissue and organ transplantation through their ability to differentiate into the specific cell types that are required for repair of diseased tissues.

However, the complexity of stem cell-based therapies often leads researchers to search for stable, safe and easily accessible stem cells source that has the potential to differentiate into several lineages. Thus, it is of utmost importance to carefully select the type of stem cells that is suitable for clinical application ( 7 , 9 ).

Stem cells hierarchy

There are mainly three types of stem cells. All three of them share the significant property of self-renewal in addition to a unique ability to differentiate. However, it should be noted that stem cells are not homogeneous, but rather exist in a developmental hierarchy ( 10 ). The most basic and undeveloped of stem cells are the totipotent stem cells. These cells are capable of developing into a complete embryo while forming the extra-embryonic tissue at the same time. This unique property is brief and starts with the fertilization of the ovum and ends when the embryo reaches the four to eight cells stage. Following that cells undergo subsequent divisions until reaching the blastocyst stage where they lose their totipotency property and assume a pluripotent identity where cells are only capable of differentiating into every embryonic germ layer (ectoderm, mesoderm and endoderm). Cells of this stage are termed “embryonic stem cells” and are obtained by isolation from the inner cell mass of the blastocyst in a process that involves the destruction of the forming embryo. After consecutive divisions, the property of pluripotency is lost and the differentiation capability becomes more lineage restricted where the cells become multipotent meaning that they can only differentiate into limited types of cells related to the tissue of origin. This is the property of “adult stem cells”, which helps create a state of homeostasis throughout the lifetime of the organism. Adult stem cells are present in a metabolically quiescent state in almost all specialized tissues of the body, which includes bone marrow and oral and dental tissues among many others ( 11 ).

Many authors consider adult stem cells the gold standard in stem cell-based therapies ( 12 , 13 ). Adult stem cells demonstrated signs of clinical success especially in hematopoietic transplants ( 14 , 15 ). In contrast to ESCs, adult stem cells are not subjected to controversial views regarding their origin. The fact that ESCs derivation involves destruction of human embryos renders them unacceptable for a significant proportion of the population for ethical and religious convictions ( 16 - 18 ).

Turning point in stem cell research

It was in 2006 when Shinya Yamanka achieved a scientific breakthrough in stem cell research by succeeding in generating cells that have the same properties and genetic profile of ESCs. This was achieved via the transient over-expression of a cocktail of four transcription factors; OCT4, SOX2, KLF4 and MYC in, fully differentiated somatic cells, namely fibroblasts ( 19 , 20 ). These cells were called iPSCs and has transformed the field of stem cell research ever since ( 21 ). The most important feature of these cells is their ability to differentiate into any of the germ layers just like ESCs precluding the ethical debate surrounding their use. The development of iPSCs technology has created an innovative way to both identify and treat diseases. Since they can be generated from the patient’s own cells, iPSCs thus present a promising potential for the production of pluripotent derived patient-matched cells that could be used for autologous transplantation. True these cells symbolize a paradigm shift since they enable researchers to directly observe and treat relevant patient cells; nevertheless, a number of challenges still need to be addressed before iPSCs-derived cells can be applied in cell therapies. Such challenges include; the detection and removal of incompletely differentiated cells, addressing the genomic and epigenetic alterations in the generated cells and overcoming the tumorigenicity of these cells that could arise on transplantation ( 22 ).

Therapeutic translation of stem cell research

With the rapid increase witnessed in stem cell basic research over the past years, the relatively new research discipline “Translational Research” has evolved significantly building up on the outcomes of basic research in order to develop new therapies. The clinical translation pathway starts after acquiring the suitable regulatory approvals. The importance of translational research lies in it’s a role as a filter to ensure that only safe and effective therapies reach the clinic ( 23 ). It bridges the gap from bench to bed. Currently, some stem cell-based therapies utilizing adult stem cells are clinically available and mainly include bone marrow transplants of hematopoietic stem cells and skin grafts for severe burns ( 23 ). To date, there are more than 3,000 trials involving the use of adult stem cells registered in WHO International Clinical Trials Registry. Additionally, initial trials involving the new and appealing iPSCs based therapies are also registered. In fact, the first clinical attempt employing iPSCs reported successful results in treating macular degeneration ( 24 ). Given the relative immaturity in the field of cellular therapy, the outcomes of such trials shall facilitate the understanding of the timeframes needed to achieve successful therapies and help in better understanding of the diseases. However, it is noteworthy that evaluation of stem cell-based therapies is not an easy task since transplantation of cells is ectopic and may result in tumor formation and other complications. This accounts for the variations in the results reported from previous reports. The following section discusses the published data of some of the most important clinical trials involving the use of different types of stem cells both in medicine and in dentistry.

Stem cell-based therapy for neurodegenerative diseases

The successful generation of neural cells from stem cells in vitro paved the way for the current stem cell-based clinical trials targeting neurodegenerative diseases ( 25 , 26 ). These therapies do not just target detaining the progression of irrecoverable neuro-degenerative diseases like Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), but are also focused on completely treating such disorders.

Parkinson’s disease (PD)

PD is characterized by a rapid loss of midbrain dopaminergic neurons. The first attempt for using human ESC cells to treat PD was via the generation of dopaminergic-like neurons, later human iPSCs was proposed as an alternative to overcome ESCs controversies ( 27 ). Both cells presented hope for obtaining an endless source of dopaminergic neurons instead of the previously used fetal brain tissues. Subsequently, protocols that mimicked the development of dopaminergic neurons succeeded in generating dopaminergic neurons similar to that of the midbrain which were able to survive, integrate and functionally mature in animal models of PD preclinically ( 28 ). Based on the research presented by different groups; the “Parkinson’s Global Force” was formed which aimed at guiding researchers to optimize their cell characterization and help promote the clinical progress toward successful therapy. Recently, In August 2018, Shinya Yamanka initiated the first approved clinical trial to treat PD using iPSCs. Seven patients suffering from moderate PD were recruited ( 29 ). Donor matched allogeneic cells were used to avoid any genetic influence of the disease. The strategy behind the trial involved the generation of dopaminergic progenitors followed by surgical transplantation into the brains of patients by a special device. In addition, immunosuppressant medications were given to avoid any adverse reaction. Preliminary results so far revealed the safety of the treatment.

MS is an inflammatory and neurodegenerative autoimmune disease of the central nervous system. Stem cell-based therapies are now exploring the possibility of halting the disease progression and reverse the neural damage. A registered phase 1 clinical trial was conducted by the company Celgene TM in 2014 using placental-derived mesenchymal stem cells (MSCs) infusion to treat patients suffering from MS ( 30 ). This trial was performed at 6 centers in the United States and 2 centers in Canada and included 16 patients. Results demonstrated that cellular infusions were safe with no signs of paradoxical aggravation. However, clinical responses from patients indicated that the cellular treatment did not improve the MS condition ( 31 ). For the last decade immunoablative therapy demonstrated accumulative evidence of inducing long-term remission and improvement of disability caused by MS. This approach involves the replacement of the diseased immune system through administration of high-dose immunosuppressive therapy followed by hematopoietic stem cells infusion ( 32 ). However, immunoablation strategies demonstrated several complications such as infertility and neurological disabilities. A number of randomized controlled trials are planned to address these concerns ( 32 ). Currently, new and innovative stem cell-based therapies for MS are only in the initial stages, and are based on different mechanisms exploring the possibility of replacing damaged neuronal tissue with neural cells derived from iPSCs however, the therapeutic potential of iPSCs is still under research ( 33 ).

ALS is a neurodegenerative disease that causes degeneration of the motor neurons which results in disturbance in muscle performance. The first attempt to treat ALS was through the transplantation of MSCs in a mouse model. The outcomes of this experiment were promising and resulted in a decrease of the disease manifestations and thus providing proof of principal ( 34 ). Based on these results, several planned/ongoing clinical trials are on the way. These trials mainly assess the safety of the proposed concept and have not proved clinical success to date. Notably, while pre-clinical studies have reported that cells derived from un-diseased individuals are superior to cells from ALS patients; most of the clinical trials attempted have employed autologous transplantation. This information may account for the absence of therapeutic improvement reported ( 35 ).

Spinal cord injury

Other neurologic indications for the use of stem cells are spinal cord injuries. Though the transplantation of different forms of neural stem cells and oligo-dendrocyte progenitors has led to growth in the axons in addition to neural connectivity which presents a possibility for repair ( 36 ), proof of recovered function has yet to be established in stringent clinical trials. Nevertheless, Japan has recently given approval to stem-cell treatment for spinal-cord injuries. This approval was based on clinical trials that are yet to be published and involves 13 patients, who are suffering from recent spinal-cord injury. The Japanese team discovered that injection of stem cells isolated from the patients’ bone marrow aided in regaining some lost sensation and mobility. This is the first stem cell-based therapy targeting spinal-cord injuries to gain governmental approval to offer to patients ( 37 ).

Stem cell-based therapies for ocular diseases

A huge number of the currently registered clinical trials for stem cell-based therapies target ocular diseases. This is mainly due to the fact that the eye is an immune privileged site. Most of these trials span various countries including Japan, China, Israel, Korea, UK, and USA and implement allogeneic ESC lines ( 35 , 36 ). Notably, the first clinical trial to implement the use autologous iPSCs-derived retinal cells was in Japan which followed the new regulatory laws issued in 2014 by Japan’s government to regulate regenerative medicine applications. Two patients were recruited in this trial, the first one received treatment for macular degeneration using iPSCs-generated retinal cell sheet ( 37 ). After 1 year of follow-up, there were no signs of serious complications including abnormal proliferation and systemic malignancy. Moreover, there were no signs of rejection of the transplanted retinal epithelial sheet in the second year follow-up. Most importantly, the signs of corrected visual acuity of the treated eye were reported. These results were enough to conclude that iPSCs-based autologous transplantation was safe and feasible ( 38 ). It is worthy to mention that the second patient was withdrawn from the study due to detectable genetic variations the patient’s iPSCs lines which was not originally present in the patient’s original fibroblasts. Such alterations may jeopardize the overall safety of the treatment. The fact that this decision was taken, even though the performed safety assays did not demonstrate tumorgenicity in the iPSCs-derived retinal pigment epithelium (RPE) cells, indicates that researchers in the field of iPSCs have full awareness of the importance of safety issues ( 39 ).

Stem cell-based therapies for treatment of diabetes

Pancreatic beta cells are destructed in type 1 diabetes mellitus, because of disorders in the immune system while in type 2 insulin insufficiency is caused by failure of the beta-cell to normally produce insulin. In both cases the affected cell is the beta cell, and since the pancreas does not efficiently regenerate islets from endogenous adult stem cells, other cell sources were tested ( 38 ). Pluripotent stem cells (PSCs) are considered the cells of choice for beta cell replacement strategies ( 39 ). Currently, there are a few industry-sponsored clinical trials that are registered targeting beta cell replacement using ESCs. These trials revolve around the engraftment of insulin-producing beta cells in an encapsulating device subcutaneously to protect the cells from autoimmunity in patients with type 1 diabetes ( 40 ). The company ViaCyte TM in California recently initiated a phase I/II trial ( {"type":"clinical-trial","attrs":{"text":"NCT02239354","term_id":"NCT02239354"}} NCT02239354 ) in 2014 in collaboration with Harvard University. This trial involves 40 patients and employs two subcutaneous capsules of insulin producing beta cells generated from ESCs. The results shall be interesting due to the ease of monitoring and recovery of the transplanted cells. The preclinical studies preceding this trial demonstrated successful glycemic correction and the devices were successfully retrieved after 174 days and contained viable insulin-producing cells ( 41 ).

Stem cells in dentistry

Stem cells have been successfully isolated from human teeth and were studied to test their ability to regenerate dental structures and periodontal tissues. MSCs were reported to be successfully isolated from dental tissues like dental pulp of permanent and deciduous teeth, periodontal ligament, apical papilla and dental follicle ( 42 - 44 ). These cells were described as an excellent cell source owing to their ease of accessibility, their ability to differentiate into osteoblasts and odontoblasts and lack of ethical controversies ( 45 ). Moreover, dental stem cells demonstrated superior abilities in immunomodulation properties either through cell to cell interaction or via a paracrine effect ( 46 ). Stem cells of non-dental origin were also suggested for dental tissue and bone regeneration. Different approaches were investigated for achieving dental and periodontal regeneration ( 47 ); however, assessments of stem cells after transplantation still require extensive studying. Clinical trials have only recently begun and their results are yet to be fully evaluated. However, by carefully applying the knowledge acquired from the extensive basic research in dental and periodontal regeneration, stem cell-based dental and periodontal regeneration may soon be a readily available treatment. To date, there are more than 6,000 clinical trials involving the use of with stem cells, however only a total of 44 registered clinical trials address oral diseases worldwide ( 48 ). Stem cell-based clinical trials with reported results targeting the treatment of oral disease are discussed below.

Dental pulp regeneration

The first human clinical study using autologous dental pulp stem cells (DPSCs) for complete pulp regeneration was reported by Nakashima et al. in 2017 ( 49 ). This pilot study was based on extensive preclinical studies conducted by the same group ( 50 ). Patients with irreversible pulpitis were recruited and followed up for 6 months following DPSCs’ transplantation. Granulocyte colony-stimulating factor was administered to induce stem cell mobilization to enrich the stem cell populations. The research team reported that the use of DPSCs seeded on collagen scaffold in molars and premolars undergoing pulpectomy was safe. No adverse events or toxicity were demonstrated in the clinical and laboratory evaluations. Positive electric pulp testing was obtained after cell transplantation in all patients. Moreover, magnetic resonance imaging of the de - novo tissues formed in the root canal demonstrated similar results to normal pulp, which indicated successful pulp regeneration. A different group conducted a clinical trial that recruited patients diagnosed with necrotic pulp. Autologous stem cells from deciduous teeth were employed to induce pulp regeneration ( 51 ). Follow-up of the cases after a year from the intervention reported evidence of pulp regeneration with vascular supply and innervation. In addition, no signs of adverse effects were observed in patients receiving DPSCs transplantation. Both trials are proceeding with the next phases, however the results obtained are promising.

Periodontal tissue regeneration

Aimetti et al. performed a study which included eleven patients suffering from chronic periodontitis and have one deep intra bony defect in addition to the presence of one vital tooth that needs extraction ( 52 ). Pulp tissue was passed through 50-µm filters in presence of collagen sponge scaffold and was followed by transplantation in the bony defects caused by periodontal disease. Both clinical and radiographic evaluations confirmed the efficacy of this therapeutic intervention. Periodontal examination, attachment level, and probe depth showed improved results in addition to significant stability of the gingival margin. Moreover, radiographic analysis demonstrated bone regeneration.

Regeneration of mandibular bony defects

The first clinical study using DPSCs for oro-maxillo-facial bone regeneration was conducted in 2009 ( 53 ). Patients in this study suffered from extreme bone loss following extraction of third molars. A bio-complex composed of DPSCs cultured on collagen sponge scaffolds was applied to the affected sites. Vertical repair of the damaged area with complete restoration of the periodontal tissue was demonstrated six months after the treatment. Three years later, the same group published a report evaluating the stability and quality of the regenerated bone after DPSCs transplantation ( 54 ). Histological and advanced holotomography demonstrated that newly formed bone was uniformly vascularized. However, it was of compact type, rather than a cancellous type which is usually the type of bone in this region.

Stem cells for treatment of Sjögren’s syndrome

Sjögren’s syndrome (SS) is a systemic autoimmune disease marked by dry mouth and eyes. A novel therapeutic approach for SS. utilizing the infusion of MSCs in 24 patients was reported by Xu et al. in 2012 ( 55 ). The strategy behind this treatment was based on the immunologic regulatory functions of MSCs. Infused MSCs migrated toward the inflammatory sites in a stromal cell-derived factor-1-dependent manner. Results reported from this clinical trial demonstrated suppressed autoimmunity with subsequent restoration of salivary gland secretion in SS patients.

Stem cells and tissue banks

The ability to bank autologous stem cells at their most potent state for later use is an essential adjuvant to stem cell-based therapies. In order to be considered valid, any novel stem cell-based therapy should be as effective as the routine treatment. Thus, when appraising a type of stem cells for application in cellular therapies, issues like immune rejection must be avoided and at the same time large numbers of stem cells must be readily available before clinical implementation. iPSCs theoretically possess the ability to proliferate unlimitedly which pose them as an attractive source for use in cell-based therapies. Unlike, adult stem cells iPSCs ability to propagate does not decrease with time ( 22 ). Recently, California Institute for Regenerative Medicine (CIRM) has inaugurated an iPSCs repository to provide researchers with versatile iPSCs cell lines in order to accelerate stem cell treatments through studying genetic variation and disease modeling. Another important source for stem cells banking is the umbilical cord. Umbilical cord is immediately cryopreserved after birth; which permits stem cells to be successfully stored and ready for use in cell-based therapies for incurable diseases of a given individuals. However, stem cells of human exfoliated deciduous teeth (SHEDs) are more attractive as a source for stem cell banking. These cells have the capacity to differentiate into further cell types than the rest of the adult stem cells ( 56 ). Moreover, procedures involving the isolation and cryopreservation of these cells are un-complicated and not aggressive. The most important advantage of banking SHEDs is the insured autologous transplant which avoids the possibility of immune rejection ( 57 ). Contrary to cord blood stem cells, SHEDs have the ability to differentiate into connective tissues, neural and dental tissues ( 58 ) Finally, the ultimate goal of stem cell banking, is to establish a repository of high-quality stem cell lines derived from many individuals for future use in therapy.

Current regulatory guidelines for stem cell-based therapies

With the increased number of clinical trials employing stem cells as therapeutic approaches, the need for developing regulatory guidelines and standards to ensure patients safety is becoming more and more essential. However, the fact that stem cell therapy is rather a new domain makes it subject to scientific, ethical and legal controversies that are yet to be regulated. Leading countries in the field have devised guidelines serving that purpose. Recently, the Food and Drug Administration (FDA) has released regulatory guidelines to ensure that these treatments are safe and effective ( 59 ). These guidelines state that; treatments involving stem cells that have been minimally manipulated and are intended for homogeneous use do not require premarket approval to come into action and shall only be subjected to regulatory guidelines against disease transmission. In 2014, a radical regulatory reform in Japan occurred with the passing of two new laws that permitted conditional approval of cell-based treatments following early phase clinical trials on the condition that clinical safety data are provided from at least ten patients. These laws allow skipping most of the traditional criteria of clinical trials in what was described as “fast track approvals” and treatments were classified according to risk ( 60 ). To date, the treatments that acquired conditional approval include those targeting; spinal-cord injury, cardiac disease and limb ischemia ( 61 ). Finally, regulatory authorities are now demanding application of standardization and safety regulations protocols for cellular products, which include the use of Xeno-free culture media, recombinant growth factors in addition to “Good Manufacturing Practice” (GMP) culture supplies.

Challenges & ethical issues facing stem cell-based therapies

Stem cell-based therapies face many obstacles that need to be urgently addressed. The most persistent concern is the ethical conflict regarding the use of ESCs. As previously mentioned, ESCs are far superior regarding their potency; however, their derivation requires destruction human embryos. True, the discovery of iPSCs overcame this concern; nevertheless, iPSCs themselves currently face another ethical controversy of their own which addresses their unlimited capacity of differentiation with concerns that these cells could one day be applied in human cloning. The use of iPSCs in therapy is still considered a high-risk treatment modality, since transplantation of these cells could induce tumor formation. Such challenge is currently addressed through developing optimized protocols to ensure their safety in addition to developing global clinical-grade iPSCs cell lines before these cells are available for clinical use ( 61 ). As for MSCs, these cells have been universally considered safe, however continuous monitoring and prolonged follow-up should be the focus of future research to avoid the possibility of tumor formation after treatments ( 62 ). Finally, it could be postulated that one of the most challenging ethical issues faced in the field of stem cell-based therapies at the moment, is the increasing number of clinics offering unproven stem cell-based treatments. Researchers are thus morally obligated to ensure that ethical considerations are not undermined in pursuit of progress in clinical translation.

Conclusions

Stem cell therapy is becoming a tangible reality by the day, thanks to the mounting research conducted over the past decade. With every research conducted the possibilities of stem cells applications increased in spite of the many challenges faced. Currently, progress in the field of stem cells is very promising with reports of clinical success in treating various diseases like; neurodegenerative diseases and macular degeneration progressing rapidly. iPSCs are conquering the field of stem cells research with endless possibilities of treating diseases using patients own cells. Regeneration of dental and periodontal tissues using MSCs has made its way to the clinic and soon enough will become a valid treatment. Although, challenges might seem daunting, stem cell research is advancing rapidly and cellular therapeutics is soon to be applicable. Fortunately, there are currently tremendous efforts exerted globally towards setting up regulatory guidelines and standards to ensure patients safety. In the near future, stem cell-based therapies shall significantly impact human health.

Acknowledgments

Funding: None.

Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/ .

Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/sci-2020-001 ). The author has no conflicts of interest to declare.

• Open access
• Published: 26 February 2019

Stem cells: past, present, and future

• Wojciech Zakrzewski 1 ,
• Maciej Dobrzyński 2 ,
• Maria Szymonowicz 1 &
• Zbigniew Rybak 1  

Stem Cell Research & Therapy volume  10 , Article number:  68 ( 2019 ) Cite this article

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In recent years, stem cell therapy has become a very promising and advanced scientific research topic. The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and derivation. Quality control and teratoma formation assays are important procedures in assessing the properties of the stem cells tested. Derivation methods and the utilization of culturing media are crucial to set proper environmental conditions for controlled differentiation. Among many types of stem tissue applications, the use of graphene scaffolds and the potential of extracellular vesicle-based therapies require attention due to their versatility. The review is summarized by challenges that stem cell therapy must overcome to be accepted worldwide. A wide variety of possibilities makes this cutting edge therapy a turning point in modern medicine, providing hope for untreatable diseases.

Stem cell classification

Stem cells are unspecialized cells of the human body. They are able to differentiate into any cell of an organism and have the ability of self-renewal. Stem cells exist both in embryos and adult cells. There are several steps of specialization. Developmental potency is reduced with each step, which means that a unipotent stem cell is not able to differentiate into as many types of cells as a pluripotent one. This chapter will focus on stem cell classification to make it easier for the reader to comprehend the following chapters.

Totipotent stem cells are able to divide and differentiate into cells of the whole organism. Totipotency has the highest differentiation potential and allows cells to form both embryo and extra-embryonic structures. One example of a totipotent cell is a zygote, which is formed after a sperm fertilizes an egg. These cells can later develop either into any of the three germ layers or form a placenta. After approximately 4 days, the blastocyst’s inner cell mass becomes pluripotent. This structure is the source of pluripotent cells.

Pluripotent stem cells (PSCs) form cells of all germ layers but not extraembryonic structures, such as the placenta. Embryonic stem cells (ESCs) are an example. ESCs are derived from the inner cell mass of preimplantation embryos. Another example is induced pluripotent stem cells (iPSCs) derived from the epiblast layer of implanted embryos. Their pluripotency is a continuum, starting from completely pluripotent cells such as ESCs and iPSCs and ending on representatives with less potency—multi-, oligo- or unipotent cells. One of the methods to assess their activity and spectrum is the teratoma formation assay. iPSCs are artificially generated from somatic cells, and they function similarly to PSCs. Their culturing and utilization are very promising for present and future regenerative medicine.

Multipotent stem cells have a narrower spectrum of differentiation than PSCs, but they can specialize in discrete cells of specific cell lineages. One example is a haematopoietic stem cell, which can develop into several types of blood cells. After differentiation, a haematopoietic stem cell becomes an oligopotent cell. Its differentiation abilities are then restricted to cells of its lineage. However, some multipotent cells are capable of conversion into unrelated cell types, which suggests naming them pluripotent cells.

Oligopotent stem cells can differentiate into several cell types. A myeloid stem cell is an example that can divide into white blood cells but not red blood cells.

Unipotent stem cells are characterized by the narrowest differentiation capabilities and a special property of dividing repeatedly. Their latter feature makes them a promising candidate for therapeutic use in regenerative medicine. These cells are only able to form one cell type, e.g. dermatocytes.

Stem cell biology

A blastocyst is formed after the fusion of sperm and ovum fertilization. Its inner wall is lined with short-lived stem cells, namely, embryonic stem cells. Blastocysts are composed of two distinct cell types: the inner cell mass (ICM), which develops into epiblasts and induces the development of a foetus, and the trophectoderm (TE). Blastocysts are responsible for the regulation of the ICM microenvironment. The TE continues to develop and forms the extraembryonic support structures needed for the successful origin of the embryo, such as the placenta. As the TE begins to form a specialized support structure, the ICM cells remain undifferentiated, fully pluripotent and proliferative [ 1 ]. The pluripotency of stem cells allows them to form any cell of the organism. Human embryonic stem cells (hESCs) are derived from the ICM. During the process of embryogenesis, cells form aggregations called germ layers: endoderm, mesoderm and ectoderm (Fig.  1 ), each eventually giving rise to differentiated cells and tissues of the foetus and, later on, the adult organism [ 2 ]. After hESCs differentiate into one of the germ layers, they become multipotent stem cells, whose potency is limited to only the cells of the germ layer. This process is short in human development. After that, pluripotent stem cells occur all over the organism as undifferentiated cells, and their key abilities are proliferation by the formation of the next generation of stem cells and differentiation into specialized cells under certain physiological conditions.

Oocyte development and formation of stem cells: the blastocoel, which is formed from oocytes, consists of embryonic stem cells that later differentiate into mesodermal, ectodermal, or endodermal cells. Blastocoel develops into the gastrula

Signals that influence the stem cell specialization process can be divided into external, such as physical contact between cells or chemical secretion by surrounding tissue, and internal, which are signals controlled by genes in DNA.

Stem cells also act as internal repair systems of the body. The replenishment and formation of new cells are unlimited as long as an organism is alive. Stem cell activity depends on the organ in which they are in; for example, in bone marrow, their division is constant, although in organs such as the pancreas, division only occurs under special physiological conditions.

Stem cell functional division

Whole-body development.

During division, the presence of different stem cells depends on organism development. Somatic stem cell ESCs can be distinguished. Although the derivation of ESCs without separation from the TE is possible, such a combination has growth limits. Because proliferating actions are limited, co-culture of these is usually avoided.

ESCs are derived from the inner cell mass of the blastocyst, which is a stage of pre-implantation embryo ca. 4 days after fertilization. After that, these cells are placed in a culture dish filled with culture medium. Passage is an inefficient but popular process of sub-culturing cells to other dishes. These cells can be described as pluripotent because they are able to eventually differentiate into every cell type in the organism. Since the beginning of their studies, there have been ethical restrictions connected to the medical use of ESCs in therapies. Most embryonic stem cells are developed from eggs that have been fertilized in an in vitro clinic, not from eggs fertilized in vivo.

Somatic or adult stem cells are undifferentiated and found among differentiated cells in the whole body after development. The function of these cells is to enable the healing, growth, and replacement of cells that are lost each day. These cells have a restricted range of differentiation options. Among many types, there are the following:

Mesenchymal stem cells are present in many tissues. In bone marrow, these cells differentiate mainly into the bone, cartilage, and fat cells. As stem cells, they are an exception because they act pluripotently and can specialize in the cells of any germ layer.

Neural cells give rise to nerve cells and their supporting cells—oligodendrocytes and astrocytes.

Haematopoietic stem cells form all kinds of blood cells: red, white, and platelets.

Skin stem cells form, for example, keratinocytes, which form a protective layer of skin.

The proliferation time of somatic stem cells is longer than that of ESCs. It is possible to reprogram adult stem cells back to their pluripotent state. This can be performed by transferring the adult nucleus into the cytoplasm of an oocyte or by fusion with the pluripotent cell. The same technique was used during cloning of the famous Dolly sheep.

hESCs are involved in whole-body development. They can differentiate into pluripotent, totipotent, multipotent, and unipotent cells (Fig.  2 ) [ 2 ].

Changes in the potency of stem cells in human body development. Potency ranges from pluripotent cells of the blastocyst to unipotent cells of a specific tissue in a human body such as the skin, CNS, or bone marrow. Reversed pluripotency can be achieved by the formation of induced pluripotent stem cells using either octamer-binding transcription factor (Oct4), sex-determining region Y (Sox2), Kruppel-like factor 4 (Klf4), or the Myc gene

Pluripotent cells can be named totipotent if they can additionally form extraembryonic tissues of the embryo. Multipotent cells are restricted in differentiating to each cell type of given tissue. When tissue contains only one lineage of cells, stem cells that form them are called either called oligo- or unipotent.

iPSC quality control and recognition by morphological differences

The comparability of stem cell lines from different individuals is needed for iPSC lines to be used in therapeutics [ 3 ]. Among critical quality procedures, the following can be distinguished:

Short tandem repeat analysis—This is the comparison of specific loci on the DNA of the samples. It is used in measuring an exact number of repeating units. One unit consists of 2 to 13 nucleotides repeating many times on the DNA strand. A polymerase chain reaction is used to check the lengths of short tandem repeats. The genotyping procedure of source tissue, cells, and iPSC seed and master cell banks is recommended.

Identity analysis—The unintentional switching of lines, resulting in other stem cell line contamination, requires rigorous assay for cell line identification.

Residual vector testing—An appearance of reprogramming vectors integrated into the host genome is hazardous, and testing their presence is a mandatory procedure. It is a commonly used procedure for generating high-quality iPSC lines. An acceptable threshold in high-quality research-grade iPSC line collections is ≤ 1 plasmid copies per 100 cells. During the procedure, 2 different regions, common to all plasmids, should be used as specific targets, such as EBNA and CAG sequences [ 3 ]. To accurately represent the test reactions, a standard curve needs to be prepared in a carrier of gDNA from a well-characterized hPSC line. For calculations of plasmid copies per cell, it is crucial to incorporate internal reference gDNA sequences to allow the quantification of, for example, ribonuclease P (RNaseP) or human telomerase reverse transcriptase (hTERT).

Karyotype—A long-term culture of hESCs can accumulate culture-driven mutations [ 4 ]. Because of that, it is crucial to pay additional attention to genomic integrity. Karyotype tests can be performed by resuscitating representative aliquots and culturing them for 48–72 h before harvesting cells for karyotypic analysis. If abnormalities are found within the first 20 karyotypes, the analysis must be repeated on a fresh sample. When this situation is repeated, the line is evaluated as abnormal. Repeated abnormalities must be recorded. Although karyology is a crucial procedure in stem cell quality control, the single nucleotide polymorphism (SNP) array, discussed later, has approximately 50 times higher resolution.

Viral testing—When assessing the quality of stem cells, all tests for harmful human adventitious agents must be performed (e.g. hepatitis C or human immunodeficiency virus). This procedure must be performed in the case of non-xeno-free culture agents.

Bacteriology—Bacterial or fungal sterility tests can be divided into culture- or broth-based tests. All the procedures must be recommended by pharmacopoeia for the jurisdiction in which the work is performed.

Single nucleotide polymorphism arrays—This procedure is a type of DNA microarray that detects population polymorphisms by enabling the detection of subchromosomal changes and the copy-neutral loss of heterozygosity, as well as an indication of cellular transformation. The SNP assay consists of three components. The first is labelling fragmented nucleic acid sequences with fluorescent dyes. The second is an array that contains immobilized allele-specific oligonucleotide (ASO) probes. The last component detects, records, and eventually interprets the signal.

Flow cytometry—This is a technique that utilizes light to count and profile cells in a heterogeneous fluid mixture. It allows researchers to accurately and rapidly collect data from heterogeneous fluid mixtures with live cells. Cells are passed through a narrow channel one by one. During light illumination, sensors detect light emitted or refracted from the cells. The last step is data analysis, compilation and integration into a comprehensive picture of the sample.

Phenotypic pluripotency assays—Recognizing undifferentiated cells is crucial in successful stem cell therapy. Among other characteristics, stem cells appear to have a distinct morphology with a high nucleus to cytoplasm ratio and a prominent nucleolus. Cells appear to be flat with defined borders, in contrast to differentiating colonies, which appear as loosely located cells with rough borders [ 5 ]. It is important that images of ideal and poor quality colonies for each cell line are kept in laboratories, so whenever there is doubt about the quality of culture, it can always be checked according to the representative image. Embryoid body formation or directed differentiation of monolayer cultures to produce cell types representative of all three embryonic germ layers must be performed. It is important to note that colonies cultured under different conditions may have different morphologies [ 6 ].

Histone modification and DNA methylation—Quality control can be achieved by using epigenetic analysis tools such as histone modification or DNA methylation. When stem cells differentiate, the methylation process silences pluripotency genes, which reduces differentiation potential, although other genes may undergo demethylation to become expressed [ 7 ]. It is important to emphasize that stem cell identity, together with its morphological characteristics, is also related to its epigenetic profile [ 8 , 9 ]. According to Brindley [ 10 ], there is a relationship between epigenetic changes, pluripotency, and cell expansion conditions, which emphasizes that unmethylated regions appear to be serum-dependent.

hESC derivation and media

hESCs can be derived using a variety of methods, from classic culturing to laser-assisted methodologies or microsurgery [ 11 ]. hESC differentiation must be specified to avoid teratoma formation (see Fig.  3 ).

Spontaneous differentiation of hESCs causes the formation of a heterogeneous cell population. There is a different result, however, when commitment signals (in forms of soluble factors and culture conditions) are applied and enable the selection of progenitor cells

hESCs spontaneously differentiate into embryonic bodies (EBs) [ 12 ]. EBs can be studied instead of embryos or animals to predict their effects on early human development. There are many different methods for acquiring EBs, such as bioreactor culture [ 13 ], hanging drop culture [ 12 ], or microwell technology [ 14 , 15 ]. These methods allow specific precursors to form in vitro [ 16 ].

The essential part of these culturing procedures is a separation of inner cell mass to culture future hESCs (Fig.  4 ) [ 17 ]. Rosowski et al. [ 18 ] emphasizes that particular attention must be taken in controlling spontaneous differentiation. When the colony reaches the appropriate size, cells must be separated. The occurrence of pluripotent cells lasts for 1–2 days. Because the classical utilization of hESCs caused ethical concerns about gastrulas used during procedures, Chung et al. [ 19 ] found out that it is also possible to obtain hESCs from four cell embryos, leaving a higher probability of embryo survival. Additionally, Zhang et al. [ 20 ] used only in vitro fertilization growth-arrested cells.

Culturing of pluripotent stem cells in vitro. Three days after fertilization, totipotent cells are formed. Blastocysts with ICM are formed on the sixth day after fertilization. Pluripotent stem cells from ICM can then be successfully transmitted on a dish

Cell passaging is used to form smaller clusters of cells on a new culture surface [ 21 ]. There are four important passaging procedures.

Enzymatic dissociation is a cutting action of enzymes on proteins and adhesion domains that bind the colony. It is a gentler method than the manual passage. It is crucial to not leave hESCs alone after passaging. Solitary cells are more sensitive and can easily undergo cell death; collagenase type IV is an example [ 22 , 23 ].

Manual passage , on the other hand, focuses on using cell scratchers. The selection of certain cells is not necessary. This should be done in the early stages of cell line derivation [ 24 ].

Trypsin utilization allows a healthy, automated hESC passage. Good Manufacturing Practice (GMP)-grade recombinant trypsin is widely available in this procedure [ 24 ]. However, there is a risk of decreasing the pluripotency and viability of stem cells [ 25 ]. Trypsin utilization can be halted with an inhibitor of the protein rho-associated protein kinase (ROCK) [ 26 ].

Ethylenediaminetetraacetic acid ( EDTA ) indirectly suppresses cell-to-cell connections by chelating divalent cations. Their suppression promotes cell dissociation [ 27 ].

Stem cells require a mixture of growth factors and nutrients to differentiate and develop. The medium should be changed each day.

Traditional culture methods used for hESCs are mouse embryonic fibroblasts (MEFs) as a feeder layer and bovine serum [ 28 ] as a medium. Martin et al. [ 29 ] demonstrated that hESCs cultured in the presence of animal products express the non-human sialic acid, N -glycolylneuraminic acid (NeuGc). Feeder layers prevent uncontrolled proliferation with factors such as leukaemia inhibitory factor (LIF) [ 30 ].

First feeder layer-free culture can be supplemented with serum replacement, combined with laminin [ 31 ]. This causes stable karyotypes of stem cells and pluripotency lasting for over a year.

Initial culturing media can be serum (e.g. foetal calf serum FCS), artificial replacement such as synthetic serum substitute (SSS), knockout serum replacement (KOSR), or StemPro [ 32 ]. The simplest culture medium contains only eight essential elements: DMEM/F12 medium, selenium, NaHCO 3, l -ascorbic acid, transferrin, insulin, TGFβ1, and FGF2 [ 33 ]. It is not yet fully known whether culture systems developed for hESCs can be allowed without adaptation in iPSC cultures.

Turning point in stem cell therapy

The turning point in stem cell therapy appeared in 2006, when scientists Shinya Yamanaka, together with Kazutoshi Takahashi, discovered that it is possible to reprogram multipotent adult stem cells to the pluripotent state. This process avoided endangering the foetus’ life in the process. Retrovirus-mediated transduction of mouse fibroblasts with four transcription factors (Oct-3/4, Sox2, KLF4, and c-Myc) [ 34 ] that are mainly expressed in embryonic stem cells could induce the fibroblasts to become pluripotent (Fig.  5 ) [ 35 ]. This new form of stem cells was named iPSCs. One year later, the experiment also succeeded with human cells [ 36 ]. After this success, the method opened a new field in stem cell research with a generation of iPSC lines that can be customized and biocompatible with the patient. Recently, studies have focused on reducing carcinogenesis and improving the conduction system.

Retroviral-mediated transduction induces pluripotency in isolated patient somatic cells. Target cells lose their role as somatic cells and, once again, become pluripotent and can differentiate into any cell type of human body

The turning point was influenced by former discoveries that happened in 1962 and 1987.

The former discovery was about scientist John Gurdon successfully cloning frogs by transferring a nucleus from a frog’s somatic cells into an oocyte. This caused a complete reversion of somatic cell development [ 37 ]. The results of his experiment became an immense discovery since it was previously believed that cell differentiation is a one-way street only, but his experiment suggested the opposite and demonstrated that it is even possible for a somatic cell to again acquire pluripotency [ 38 ].

The latter was a discovery made by Davis R.L. that focused on fibroblast DNA subtraction. Three genes were found that originally appeared in myoblasts. The enforced expression of only one of the genes, named myogenic differentiation 1 (Myod1), caused the conversion of fibroblasts into myoblasts, showing that reprogramming cells is possible, and it can even be used to transform cells from one lineage to another [ 39 ].

Although pluripotency can occur naturally only in embryonic stem cells, it is possible to induce terminally differentiated cells to become pluripotent again. The process of direct reprogramming converts differentiated somatic cells into iPSC lines that can form all cell types of an organism. Reprogramming focuses on the expression of oncogenes such as Myc and Klf4 (Kruppel-like factor 4). This process is enhanced by a downregulation of genes promoting genome stability, such as p53. Additionally, cell reprogramming involves histone alteration. All these processes can cause potential mutagenic risk and later lead to an increased number of mutations. Quinlan et al. [ 40 ] checked fully pluripotent mouse iPSCs using whole genome DNA sequencing and structural variation (SV) detection algorithms. Based on those studies, it was confirmed that although there were single mutations in the non-genetic region, there were non-retrotransposon insertions. This led to the conclusion that current reprogramming methods can produce fully pluripotent iPSCs without severe genomic alterations.

During the course of development from pluripotent hESCs to differentiated somatic cells, crucial changes appear in the epigenetic structure of these cells. There is a restriction or permission of the transcription of genes relevant to each cell type. When somatic cells are being reprogrammed using transcription factors, all the epigenetic architecture has to be reconditioned to achieve iPSCs with pluripotency [ 41 ]. However, cells of each tissue undergo specific somatic genomic methylation. This influences transcription, which can further cause alterations in induced pluripotency [ 42 ].

Source of iPSCs

Because pluripotent cells can propagate indefinitely and differentiate into any kind of cell, they can be an unlimited source, either for replacing lost or diseased tissues. iPSCs bypass the need for embryos in stem cell therapy. Because they are made from the patient’s own cells, they are autologous and no longer generate any risk of immune rejection.

At first, fibroblasts were used as a source of iPSCs. Because a biopsy was needed to achieve these types of cells, the technique underwent further research. Researchers investigated whether more accessible cells could be used in the method. Further, other cells were used in the process: peripheral blood cells, keratinocytes, and renal epithelial cells found in urine. An alternative strategy to stem cell transplantation can be stimulating a patient’s endogenous stem cells to divide or differentiate, occurring naturally when skin wounds are healing. In 2008, pancreatic exocrine cells were shown to be reprogrammed to functional, insulin-producing beta cells [ 43 ].

The best stem cell source appears to be the fibroblasts, which is more tempting in the case of logistics since its stimulation can be fast and better controlled [ 44 ].

• Teratoma formation assay

The self-renewal and differentiation capabilities of iPSCs have gained significant interest and attention in regenerative medicine sciences. To study their abilities, a quality-control assay is needed, of which one of the most important is the teratoma formation assay. Teratomas are benign tumours. Teratomas are capable of rapid growth in vivo and are characteristic because of their ability to develop into tissues of all three germ layers simultaneously. Because of the high pluripotency of teratomas, this formation assay is considered an assessment of iPSC’s abilities [ 45 ].

Teratoma formation rate, for instance, was observed to be elevated in human iPSCs compared to that in hESCs [ 46 ]. This difference may be connected to different differentiation methods and cell origins. Most commonly, the teratoma assay involves an injection of examined iPSCs subcutaneously or under the testis or kidney capsule in mice, which are immune-deficient [ 47 ]. After injection, an immature but recognizable tissue can be observed, such as the kidney tubules, bone, cartilage, or neuroepithelium [ 30 ]. The injection site may have an impact on the efficiency of teratoma formation [ 48 ].

There are three groups of markers used in this assay to differentiate the cells of germ layers. For endodermal tissue, there is insulin/C-peptide and alpha-1 antitrypsin [ 49 ]. For the mesoderm, derivatives can be used, e.g. cartilage matrix protein for the bone and alcian blue for the cartilage. As ectodermal markers, class III B botulin or keratin can be used for keratinocytes.

Teratoma formation assays are considered the gold standard for demonstrating the pluripotency of human iPSCs, demonstrating their possibilities under physiological conditions. Due to their actual tissue formation, they could be used for the characterization of many cell lineages [ 50 ].

Directed differentiation

To be useful in therapy, stem cells must be converted into desired cell types as necessary or else the whole regenerative medicine process will be pointless. Differentiation of ESCs is crucial because undifferentiated ESCs can cause teratoma formation in vivo. Understanding and using signalling pathways for differentiation is an important method in successful regenerative medicine. In directed differentiation, it is likely to mimic signals that are received by cells when they undergo successive stages of development [ 51 ]. The extracellular microenvironment plays a significant role in controlling cell behaviour. By manipulating the culture conditions, it is possible to restrict specific differentiation pathways and generate cultures that are enriched in certain precursors in vitro. However, achieving a similar effect in vivo is challenging. It is crucial to develop culture conditions that will allow the promotion of homogenous and enhanced differentiation of ESCs into functional and desired tissues.

Regarding the self-renewal of embryonic stem cells, Hwang et al. [ 52 ] noted that the ideal culture method for hESC-based cell and tissue therapy would be a defined culture free of either the feeder layer or animal components. This is because cell and tissue therapy requires the maintenance of large quantities of undifferentiated hESCs, which does not make feeder cells suitable for such tasks.

Most directed differentiation protocols are formed to mimic the development of an inner cell mass during gastrulation. During this process, pluripotent stem cells differentiate into ectodermal, mesodermal, or endodermal progenitors. Mall molecules or growth factors induce the conversion of stem cells into appropriate progenitor cells, which will later give rise to the desired cell type. There is a variety of signal intensities and molecular families that may affect the establishment of germ layers in vivo, such as fibroblast growth factors (FGFs) [ 53 ]; the Wnt family [ 54 ] or superfamily of transforming growth factors—β(TGFβ); and bone morphogenic proteins (BMP) [ 55 ]. Each candidate factor must be tested on various concentrations and additionally applied to various durations because the precise concentrations and times during which developing cells in embryos are influenced during differentiation are unknown. For instance, molecular antagonists of endogenous BMP and Wnt signalling can be used for ESC formation of ectoderm [ 56 ]. However, transient Wnt and lower concentrations of the TGFβ family trigger mesodermal differentiation [ 57 ]. Regarding endoderm formation, a higher activin A concentration may be required [ 58 , 59 ].

There are numerous protocols about the methods of forming progenitors of cells of each of germ layers, such as cardiomyocytes [ 60 ], hepatocytes [ 61 ], renal cells [ 62 ], lung cells [ 63 , 64 ], motor neurons [ 65 ], intestinal cells [ 66 ], or chondrocytes [ 67 ].

Directed differentiation of either iPSCs or ESCs into, e.g. hepatocytes, could influence and develop the study of the molecular mechanisms in human liver development. In addition, it could also provide the possibility to form exogenous hepatocytes for drug toxicity testing [ 68 ].

Levels of concentration and duration of action with a specific signalling molecule can cause a variety of factors. Unfortunately, for now, a high cost of recombinant factors is likely to limit their use on a larger scale in medicine. The more promising technique focuses on the use of small molecules. These can be used for either activating or deactivating specific signalling pathways. They enhance reprogramming efficiency by creating cells that are compatible with the desired type of tissue. It is a cheaper and non-immunogenic method.

One of the successful examples of small-molecule cell therapies is antagonists and agonists of the Hedgehog pathway. They show to be very useful in motor neuron regeneration [ 69 ]. Endogenous small molecules with their function in embryonic development can also be used in in vitro methods to induce the differentiation of cells; for example, retinoic acid, which is responsible for patterning the nervous system in vivo [ 70 ], surprisingly induced retinal cell formation when the laboratory procedure involved hESCs [ 71 ].

The efficacy of differentiation factors depends on functional maturity, efficiency, and, finally, introducing produced cells to their in vivo equivalent. Topography, shear stress, and substrate rigidity are factors influencing the phenotype of future cells [ 72 ].

The control of biophysical and biochemical signals, the biophysical environment, and a proper guide of hESC differentiation are important factors in appropriately cultured stem cells.

Stem cell utilization and their manufacturing standards and culture systems

The European Medicines Agency and the Food and Drug Administration have set Good Manufacturing Practice (GMP) guidelines for safe and appropriate stem cell transplantation. In the past, protocols used for stem cell transplantation required animal-derived products [ 73 ].

The risk of introducing animal antigens or pathogens caused a restriction in their use. Due to such limitations, the technique required an obvious update [ 74 ]. Now, it is essential to use xeno-free equivalents when establishing cell lines that are derived from fresh embryos and cultured from human feeder cell lines [ 75 ]. In this method, it is crucial to replace any non-human materials with xeno-free equivalents [ 76 ].

NutriStem with LN-511, TeSR2 with human recombinant laminin (LN-511), and RegES with human foreskin fibroblasts (HFFs) are commonly used xeno-free culture systems [ 33 ]. There are many organizations and international initiatives, such as the National Stem Cell Bank, that provide stem cell lines for treatment or medical research [ 77 ].

Stem cell use in medicine

Stem cells have great potential to become one of the most important aspects of medicine. In addition to the fact that they play a large role in developing restorative medicine, their study reveals much information about the complex events that happen during human development.

The difference between a stem cell and a differentiated cell is reflected in the cells’ DNA. In the former cell, DNA is arranged loosely with working genes. When signals enter the cell and the differentiation process begins, genes that are no longer needed are shut down, but genes required for the specialized function will remain active. This process can be reversed, and it is known that such pluripotency can be achieved by interaction in gene sequences. Takahashi and Yamanaka [ 78 ] and Loh et al. [ 79 ] discovered that octamer-binding transcription factor 3 and 4 (Oct3/4), sex determining region Y (SRY)-box 2 and Nanog genes function as core transcription factors in maintaining pluripotency. Among them, Oct3/4 and Sox2 are essential for the generation of iPSCs.

Many serious medical conditions, such as birth defects or cancer, are caused by improper differentiation or cell division. Currently, several stem cell therapies are possible, among which are treatments for spinal cord injury, heart failure [ 80 ], retinal and macular degeneration [ 81 ], tendon ruptures, and diabetes type 1 [ 82 ]. Stem cell research can further help in better understanding stem cell physiology. This may result in finding new ways of treating currently incurable diseases.

Haematopoietic stem cell transplantation

Haematopoietic stem cells are important because they are by far the most thoroughly characterized tissue-specific stem cell; after all, they have been experimentally studied for more than 50 years. These stem cells appear to provide an accurate paradigm model system to study tissue-specific stem cells, and they have potential in regenerative medicine.

Multipotent haematopoietic stem cell (HSC) transplantation is currently the most popular stem cell therapy. Target cells are usually derived from the bone marrow, peripheral blood, or umbilical cord blood [ 83 ]. The procedure can be autologous (when the patient’s own cells are used), allogenic (when the stem cell comes from a donor), or syngeneic (from an identical twin). HSCs are responsible for the generation of all functional haematopoietic lineages in blood, including erythrocytes, leukocytes, and platelets. HSC transplantation solves problems that are caused by inappropriate functioning of the haematopoietic system, which includes diseases such as leukaemia and anaemia. However, when conventional sources of HSC are taken into consideration, there are some important limitations. First, there is a limited number of transplantable cells, and an efficient way of gathering them has not yet been found. There is also a problem with finding a fitting antigen-matched donor for transplantation, and viral contamination or any immunoreactions also cause a reduction in efficiency in conventional HSC transplantations. Haematopoietic transplantation should be reserved for patients with life-threatening diseases because it has a multifactorial character and can be a dangerous procedure. iPSC use is crucial in this procedure. The use of a patient’s own unspecialized somatic cells as stem cells provides the greatest immunological compatibility and significantly increases the success of the procedure.

Stem cells as a target for pharmacological testing

Stem cells can be used in new drug tests. Each experiment on living tissue can be performed safely on specific differentiated cells from pluripotent cells. If any undesirable effect appears, drug formulas can be changed until they reach a sufficient level of effectiveness. The drug can enter the pharmacological market without harming any live testers. However, to test the drugs properly, the conditions must be equal when comparing the effects of two drugs. To achieve this goal, researchers need to gain full control of the differentiation process to generate pure populations of differentiated cells.

Stem cells as an alternative for arthroplasty

One of the biggest fears of professional sportsmen is getting an injury, which most often signifies the end of their professional career. This applies especially to tendon injuries, which, due to current treatment options focusing either on conservative or surgical treatment, often do not provide acceptable outcomes. Problems with the tendons start with their regeneration capabilities. Instead of functionally regenerating after an injury, tendons merely heal by forming scar tissues that lack the functionality of healthy tissues. Factors that may cause this failed healing response include hypervascularization, deposition of calcific materials, pain, or swelling [ 84 ].

Additionally, in addition to problems with tendons, there is a high probability of acquiring a pathological condition of joints called osteoarthritis (OA) [ 85 ]. OA is common due to the avascular nature of articular cartilage and its low regenerative capabilities [ 86 ]. Although arthroplasty is currently a common procedure in treating OA, it is not ideal for younger patients because they can outlive the implant and will require several surgical procedures in the future. These are situations where stem cell therapy can help by stopping the onset of OA [ 87 ]. However, these procedures are not well developed, and the long-term maintenance of hyaline cartilage requires further research.

Osteonecrosis of the femoral hip (ONFH) is a refractory disease associated with the collapse of the femoral head and risk of hip arthroplasty in younger populations [ 88 ]. Although total hip arthroplasty (THA) is clinically successful, it is not ideal for young patients, mostly due to the limited lifetime of the prosthesis. An increasing number of clinical studies have evaluated the therapeutic effect of stem cells on ONFH. Most of the authors demonstrated positive outcomes, with reduced pain, improved function, or avoidance of THA [ 89 , 90 , 91 ].

Rejuvenation by cell programming

Ageing is a reversible epigenetic process. The first cell rejuvenation study was published in 2011 [ 92 ]. Cells from aged individuals have different transcriptional signatures, high levels of oxidative stress, dysfunctional mitochondria, and shorter telomeres than in young cells [ 93 ]. There is a hypothesis that when human or mouse adult somatic cells are reprogrammed to iPSCs, their epigenetic age is virtually reset to zero [ 94 ]. This was based on an epigenetic model, which explains that at the time of fertilization, all marks of parenteral ageing are erased from the zygote’s genome and its ageing clock is reset to zero [ 95 ].

In their study, Ocampo et al. [ 96 ] used Oct4, Sox2, Klf4, and C-myc genes (OSKM genes) and affected pancreas and skeletal muscle cells, which have poor regenerative capacity. Their procedure revealed that these genes can also be used for effective regenerative treatment [ 97 ]. The main challenge of their method was the need to employ an approach that does not use transgenic animals and does not require an indefinitely long application. The first clinical approach would be preventive, focused on stopping or slowing the ageing rate. Later, progressive rejuvenation of old individuals can be attempted. In the future, this method may raise some ethical issues, such as overpopulation, leading to lower availability of food and energy.

For now, it is important to learn how to implement cell reprogramming technology in non-transgenic elder animals and humans to erase marks of ageing without removing the epigenetic marks of cell identity.

Cell-based therapies

Stem cells can be induced to become a specific cell type that is required to repair damaged or destroyed tissues (Fig.  6 ). Currently, when the need for transplantable tissues and organs outweighs the possible supply, stem cells appear to be a perfect solution for the problem. The most common conditions that benefit from such therapy are macular degenerations [ 98 ], strokes [ 99 ], osteoarthritis [ 89 , 90 ], neurodegenerative diseases, and diabetes [ 100 ]. Due to this technique, it can become possible to generate healthy heart muscle cells and later transplant them to patients with heart disease.

Stem cell experiments on animals. These experiments are one of the many procedures that proved stem cells to be a crucial factor in future regenerative medicine

In the case of type 1 diabetes, insulin-producing cells in the pancreas are destroyed due to an autoimmunological reaction. As an alternative to transplantation therapy, it can be possible to induce stem cells to differentiate into insulin-producing cells [ 101 ].

Stem cells and tissue banks

iPS cells with their theoretically unlimited propagation and differentiation abilities are attractive for the present and future sciences. They can be stored in a tissue bank to be an essential source of human tissue used for medical examination. The problem with conventional differentiated tissue cells held in the laboratory is that their propagation features diminish after time. This does not occur in iPSCs.

The umbilical cord is known to be rich in mesenchymal stem cells. Due to its cryopreservation immediately after birth, its stem cells can be successfully stored and used in therapies to prevent the future life-threatening diseases of a given patient.

Stem cells of human exfoliated deciduous teeth (SHED) found in exfoliated deciduous teeth has the ability to develop into more types of body tissues than other stem cells [ 102 ] (Table  1 ). Techniques of their collection, isolation, and storage are simple and non-invasive. Among the advantages of banking, SHED cells are:

Guaranteed donor-match autologous transplant that causes no immune reaction and rejection of cells [ 103 ]

Simple and painless for both child and parent

Less than one third of the cost of cord blood storage

Not subject to the same ethical concerns as embryonic stem cells [ 104 ]

In contrast to cord blood stem cells, SHED cells are able to regenerate into solid tissues such as connective, neural, dental, or bone tissue [ 105 , 106 ]

SHED can be useful for close relatives of the donor

Fertility diseases

In 2011, two researchers, Katsuhiko Hayashi et al. [ 107 ], showed in an experiment on mice that it is possible to form sperm from iPSCs. They succeeded in delivering healthy and fertile pups in infertile mice. The experiment was also successful for female mice, where iPSCs formed fully functional eggs .

Young adults at risk of losing their spermatogonial stem cells (SSC), mostly cancer patients, are the main target group that can benefit from testicular tissue cryopreservation and autotransplantation. Effective freezing methods for adult and pre-pubertal testicular tissue are available [ 108 ].

Qiuwan et al. [ 109 ] provided important evidence that human amniotic epithelial cell (hAEC) transplantation could effectively improve ovarian function by inhibiting cell apoptosis and reducing inflammation in injured ovarian tissue of mice, and it could be a promising strategy for the management of premature ovarian failure or insufficiency in female cancer survivors.

For now, reaching successful infertility treatments in humans appears to be only a matter of time, but there are several challenges to overcome. First, the process needs to have high efficiency; second, the chances of forming tumours instead of eggs or sperm must be maximally reduced. The last barrier is how to mature human sperm and eggs in the lab without transplanting them to in vivo conditions, which could cause either a tumour risk or an invasive procedure.

Therapy for incurable neurodegenerative diseases

Thanks to stem cell therapy, it is possible not only to delay the progression of incurable neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease (AD), and Huntington disease, but also, most importantly, to remove the source of the problem. In neuroscience, the discovery of neural stem cells (NSCs) has nullified the previous idea that adult CNS were not capable of neurogenesis [ 110 , 111 ]. Neural stem cells are capable of improving cognitive function in preclinical rodent models of AD [ 112 , 113 , 114 ]. Awe et al. [ 115 ] clinically derived relevant human iPSCs from skin punch biopsies to develop a neural stem cell-based approach for treating AD. Neuronal degeneration in Parkinson’s disease (PD) is focal, and dopaminergic neurons can be efficiently generated from hESCs. PD is an ideal disease for iPSC-based cell therapy [ 116 ]. However, this therapy is still in an experimental phase ( https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4539501 /). Brain tissue from aborted foetuses was used on patients with Parkinson’s disease [ 117 ]. Although the results were not uniform, they showed that therapies with pure stem cells are an important and achievable therapy.

Stem cell use in dentistry

Teeth represent a very challenging material for regenerative medicine. They are difficult to recreate because of their function in aspects such as articulation, mastication, or aesthetics due to their complicated structure. Currently, there is a chance for stem cells to become more widely used than synthetic materials. Teeth have a large advantage of being the most natural and non-invasive source of stem cells.

For now, without the use of stem cells, the most common periodontological treatments are either growth factors, grafts, or surgery. For example, there are stem cells in periodontal ligament [ 118 , 119 ], which are capable of differentiating into osteoblasts or cementoblasts, and their functions were also assessed in neural cells [ 120 ]. Tissue engineering is a successful method for treating periodontal diseases. Stem cells of the root apical areas are able to recreate periodontal ligament. One of the possible methods of tissue engineering in periodontology is gene therapy performed using adenoviruses-containing growth factors [ 121 ].

As a result of animal studies, dentin regeneration is an effective process that results in the formation of dentin bridges [ 122 ].

Enamel is more difficult to regenerate than dentin. After the differentiation of ameloblastoma cells into the enamel, the former is destroyed, and reparation is impossible. Medical studies have succeeded in differentiating bone marrow stem cells into ameloblastoma [ 123 ].

Healthy dental tissue has a high amount of regular stem cells, although this number is reduced when tissue is either traumatized or inflamed [ 124 ]. There are several dental stem cell groups that can be isolated (Fig.  7 ).

Localization of stem cells in dental tissues. Dental pulp stem cells (DPSCs) and human deciduous teeth stem cells (SHED) are located in the dental pulp. Periodontal ligaments stem cells are located in the periodontal ligament. Apical papilla consists of stem cells from the apical papilla (SCAP)

Dental pulp stem cell (DPSC)

These were the first dental stem cells isolated from the human dental pulp, which were [ 125 ] located inside dental pulp (Table  2 ). They have osteogenic and chondrogenic potential. Mesenchymal stem cells (MSCs) of the dental pulp, when isolated, appear highly clonogenic; they can be isolated from adult tissue (e.g. bone marrow, adipose tissue) and foetal (e.g. umbilical cord) [ 126 ] tissue, and they are able to differentiate densely [ 127 ]. MSCs differentiate into odontoblast-like cells and osteoblasts to form dentin and bone. Their best source locations are the third molars [ 125 ]. DPSCs are the most useful dental source of tissue engineering due to their easy surgical accessibility, cryopreservation possibility, increased production of dentin tissues compared to non-dental stem cells, and their anti-inflammatory abilities. These cells have the potential to be a source for maxillofacial and orthopaedic reconstructions or reconstructions even beyond the oral cavity. DPSCs are able to generate all structures of the developed tooth [ 128 ]. In particular, beneficial results in the use of DPSCs may be achieved when combined with other new therapies, such as periodontal tissue photobiomodulation (laser stimulation), which is an efficient technique in the stimulation of proliferation and differentiation into distinct cell types [ 129 ]. DPSCs can be induced to form neural cells to help treat neurological deficits.

Stem cells of human exfoliated deciduous teeth (SHED) have a faster rate of proliferation than DPSCs and differentiate into an even greater number of cells, e.g. other mesenchymal and non-mesenchymal stem cell derivatives, such as neural cells [ 130 ]. These cells possess one major disadvantage: they form a non-complete dentin/pulp-like complex in vivo. SHED do not undergo the same ethical concerns as embryonic stem cells. Both DPSCs and SHED are able to form bone-like tissues in vivo [ 131 ] and can be used for periodontal, dentin, or pulp regeneration. DPSCs and SHED can be used in treating, for example, neural deficits [ 132 ]. DPSCs alone were tested and successfully applied for alveolar bone and mandible reconstruction [ 133 ].

Periodontal ligament stem cells (PDLSCs)

These cells are used in periodontal ligament or cementum tissue regeneration. They can differentiate into mesenchymal cell lineages to produce collagen-forming cells, adipocytes, cementum tissue, Sharpey’s fibres, and osteoblast-like cells in vitro. PDLSCs exist both on the root and alveolar bone surfaces; however, on the latter, these cells have better differentiation abilities than on the former [ 134 ]. PDLSCs have become the first treatment for periodontal regeneration therapy because of their safety and efficiency [ 135 , 136 ].

Stem cells from apical papilla (SCAP)

These cells are mesenchymal structures located within immature roots. They are isolated from human immature permanent apical papilla. SCAP are the source of odontoblasts and cause apexogenesis. These stem cells can be induced in vitro to form odontoblast-like cells, neuron-like cells, or adipocytes. SCAP have a higher capacity of proliferation than DPSCs, which makes them a better choice for tissue regeneration [ 137 , 138 ].

Dental follicle stem cells (DFCs)

These cells are loose connective tissues surrounding the developing tooth germ. DFCs contain cells that can differentiate into cementoblasts, osteoblasts, and periodontal ligament cells [ 139 , 140 ]. Additionally, these cells proliferate after even more than 30 passages [ 141 ]. DFCs are most commonly extracted from the sac of a third molar. When DFCs are combined with a treated dentin matrix, they can form a root-like tissue with a pulp-dentin complex and eventually form tooth roots [ 141 ]. When DFC sheets are induced by Hertwig’s epithelial root sheath cells, they can produce periodontal tissue; thus, DFCs represent a very promising material for tooth regeneration [ 142 ].

Pulp regeneration in endodontics

Dental pulp stem cells can differentiate into odontoblasts. There are few methods that enable the regeneration of the pulp.

The first is an ex vivo method. Proper stem cells are grown on a scaffold before they are implanted into the root channel [ 143 ].

The second is an in vivo method. This method focuses on injecting stem cells into disinfected root channels after the opening of the in vivo apex. Additionally, the use of a scaffold is necessary to prevent the movement of cells towards other tissues. For now, only pulp-like structures have been created successfully.

Methods of placing stem cells into the root channel constitute are either soft scaffolding [ 144 ] or the application of stem cells in apexogenesis or apexification. Immature teeth are the best source [ 145 ]. Nerve and blood vessel network regeneration are extremely vital to keep pulp tissue healthy.

The potential of dental stem cells is mainly regarding the regeneration of damaged dentin and pulp or the repair of any perforations; in the future, it appears to be even possible to generate the whole tooth. Such an immense success would lead to the gradual replacement of implant treatments. Mandibulary and maxillary defects can be one of the most complicated dental problems for stem cells to address.

Acquiring non-dental tissue cells by dental stem cell differentiation

In 2013, it was reported that it is possible to grow teeth from stem cells obtained extra-orally, e.g. from urine [ 146 ]. Pluripotent stem cells derived from human urine were induced and generated tooth-like structures. The physical properties of the structures were similar to natural ones except for hardness [ 127 ]. Nonetheless, it appears to be a very promising technique because it is non-invasive and relatively low-cost, and somatic cells can be used instead of embryonic cells. More importantly, stem cells derived from urine did not form any tumours, and the use of autologous cells reduces the chances of rejection [ 147 ].

Use of graphene in stem cell therapy

Over recent years, graphene and its derivatives have been increasingly used as scaffold materials to mediate stem cell growth and differentiation [ 148 ]. Both graphene and graphene oxide (GO) represent high in-plane stiffness [ 149 ]. Because graphene has carbon and aromatic network, it works either covalently or non-covalently with biomolecules; in addition to its superior mechanical properties, graphene offers versatile chemistry. Graphene exhibits biocompatibility with cells and their proper adhesion. It also tested positively for enhancing the proliferation or differentiation of stem cells [ 148 ]. After positive experiments, graphene revealed great potential as a scaffold and guide for specific lineages of stem cell differentiation [ 150 ]. Graphene has been successfully used in the transplantation of hMSCs and their guided differentiation to specific cells. The acceleration skills of graphene differentiation and division were also investigated. It was discovered that graphene can serve as a platform with increased adhesion for both growth factors and differentiation chemicals. It was also discovered that π-π binding was responsible for increased adhesion and played a crucial role in inducing hMSC differentiation [ 150 ].

Therapeutic potential of extracellular vesicle-based therapies

Extracellular vesicles (EVs) can be released by virtually every cell of an organism, including stem cells [ 151 ], and are involved in intercellular communication through the delivery of their mRNAs, lipids, and proteins. As Oh et al. [ 152 ] prove, stem cells, together with their paracrine factors—exosomes—can become potential therapeutics in the treatment of, e.g. skin ageing. Exosomes are small membrane vesicles secreted by most cells (30–120 nm in diameter) [ 153 ]. When endosomes fuse with the plasma membrane, they become exosomes that have messenger RNAs (mRNAs) and microRNAs (miRNAs), some classes of non-coding RNAs (IncRNAs) and several proteins that originate from the host cell [ 154 ]. IncRNAs can bind to specific loci and create epigenetic regulators, which leads to the formation of epigenetic modifications in recipient cells. Because of this feature, exosomes are believed to be implicated in cell-to-cell communication and the progression of diseases such as cancer [ 155 ]. Recently, many studies have also shown the therapeutic use of exosomes derived from stem cells, e.g. skin damage and renal or lung injuries [ 156 ].

In skin ageing, the most important factor is exposure to UV light, called “photoageing” [ 157 ], which causes extrinsic skin damage, characterized by dryness, roughness, irregular pigmentation, lesions, and skin cancers. In intrinsic skin ageing, on the other hand, the loss of elasticity is a characteristic feature. The skin dermis consists of fibroblasts, which are responsible for the synthesis of crucial skin elements, such as procollagen or elastic fibres. These elements form either basic framework extracellular matrix constituents of the skin dermis or play a major role in tissue elasticity. Fibroblast efficiency and abundance decrease with ageing [ 158 ]. Stem cells can promote the proliferation of dermal fibroblasts by secreting cytokines such as platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and basic fibroblast growth factor. Huh et al. [ 159 ] mentioned that a medium of human amniotic fluid-derived stem cells (hAFSC) positively affected skin regeneration after longwave UV-induced (UVA, 315–400 nm) photoageing by increasing the proliferation and migration of dermal fibroblasts. It was discovered that, in addition to the induction of fibroblast physiology, hAFSC transplantation also improved diseases in cases of renal pathology, various cancers, or stroke [ 160 , 161 ].

Oh [ 162 ] also presented another option for the treatment of skin wounds, either caused by physical damage or due to diabetic ulcers. Induced pluripotent stem cell-conditioned medium (iPSC-CM) without any animal-derived components induced dermal fibroblast proliferation and migration.

Natural cutaneous wound healing is divided into three steps: haemostasis/inflammation, proliferation, and remodelling. During the crucial step of proliferation, fibroblasts migrate and increase in number, indicating that it is a critical step in skin repair, and factors such as iPSC-CM that impact it can improve the whole cutaneous wound healing process. Paracrine actions performed by iPSCs are also important for this therapeutic effect [ 163 ]. These actions result in the secretion of cytokines such as TGF-β, interleukin (IL)-6, IL-8, monocyte chemotactic protein-1 (MCP-1), vascular endothelial growth factor (VEGF), platelet-derived growth factor-AA (PDGF-AA), and basic fibroblast growth factor (bFGF). Bae et al. [ 164 ] mentioned that TGF-β induced the migration of keratinocytes. It was also demonstrated that iPSC factors can enhance skin wound healing in vivo and in vitro when Zhou et al. [ 165 ] enhanced wound healing, even after carbon dioxide laser resurfacing in an in vivo study.

Peng et al. [ 166 ] investigated the effects of EVs derived from hESCs on in vitro cultured retinal glial, progenitor Müller cells, which are known to differentiate into retinal neurons. EVs appear heterogeneous in size and can be internalized by cultured Müller cells, and their proteins are involved in the induction and maintenance of stem cell pluripotency. These stem cell-derived vesicles were responsible for the neuronal trans-differentiation of cultured Müller cells exposed to them. However, the research article points out that the procedure was accomplished only on in vitro acquired retina.

Challenges concerning stem cell therapy

Although stem cells appear to be an ideal solution for medicine, there are still many obstacles that need to be overcome in the future. One of the first problems is ethical concern.

The most common pluripotent stem cells are ESCs. Therapies concerning their use at the beginning were, and still are, the source of ethical conflicts. The reason behind it started when, in 1998, scientists discovered the possibility of removing ESCs from human embryos. Stem cell therapy appeared to be very effective in treating many, even previously incurable, diseases. The problem was that when scientists isolated ESCs in the lab, the embryo, which had potential for becoming a human, was destroyed (Fig.  8 ). Because of this, scientists, seeing a large potential in this treatment method, focused their efforts on making it possible to isolate stem cells without endangering their source—the embryo.

Use of inner cell mass pluripotent stem cells and their stimulation to differentiate into desired cell types

For now, while hESCs still remain an ethically debatable source of cells, they are potentially powerful tools to be used for therapeutic applications of tissue regeneration. Because of the complexity of stem cell control systems, there is still much to be learned through observations in vitro. For stem cells to become a popular and widely accessible procedure, tumour risk must be assessed. The second problem is to achieve successful immunological tolerance between stem cells and the patient’s body. For now, one of the best ideas is to use the patient’s own cells and devolve them into their pluripotent stage of development.

New cells need to have the ability to fully replace lost or malfunctioning natural cells. Additionally, there is a concern about the possibility of obtaining stem cells without the risk of morbidity or pain for either the patient or the donor. Uncontrolled proliferation and differentiation of cells after implementation must also be assessed before its use in a wide variety of regenerative procedures on living patients [ 167 ].

One of the arguments that limit the use of iPSCs is their infamous role in tumourigenicity. There is a risk that the expression of oncogenes may increase when cells are being reprogrammed. In 2008, a technique was discovered that allowed scientists to remove oncogenes after a cell achieved pluripotency, although it is not efficient yet and takes a longer amount of time. The process of reprogramming may be enhanced by deletion of the tumour suppressor gene p53, but this gene also acts as a key regulator of cancer, which makes it impossible to remove in order to avoid more mutations in the reprogrammed cell. The low efficiency of the process is another problem, which is progressively becoming reduced with each year. At first, the rate of somatic cell reprogramming in Yamanaka’s study was up to 0.1%. The use of transcription factors creates a risk of genomic insertion and further mutation of the target cell genome. For now, the only ethically acceptable operation is an injection of hESCs into mouse embryos in the case of pluripotency evaluation [ 168 ].

Stem cell obstacles in the future

Pioneering scientific and medical advances always have to be carefully policed in order to make sure they are both ethical and safe. Because stem cell therapy already has a large impact on many aspects of life, it should not be treated differently.

Currently, there are several challenges concerning stem cells. First, the most important one is about fully understanding the mechanism by which stem cells function first in animal models. This step cannot be avoided. For the widespread, global acceptance of the procedure, fear of the unknown is the greatest challenge to overcome.

The efficiency of stem cell-directed differentiation must be improved to make stem cells more reliable and trustworthy for a regular patient. The scale of the procedure is another challenge. Future stem cell therapies may be a significant obstacle. Transplanting new, fully functional organs made by stem cell therapy would require the creation of millions of working and biologically accurate cooperating cells. Bringing such complicated procedures into general, widespread regenerative medicine will require interdisciplinary and international collaboration.

The identification and proper isolation of stem cells from a patient’s tissues is another challenge. Immunological rejection is a major barrier to successful stem cell transplantation. With certain types of stem cells and procedures, the immune system may recognize transplanted cells as foreign bodies, triggering an immune reaction resulting in transplant or cell rejection.

One of the ideas that can make stem cells a “failsafe” is about implementing a self-destruct option if they become dangerous. Further development and versatility of stem cells may cause reduction of treatment costs for people suffering from currently incurable diseases. When facing certain organ failure, instead of undergoing extraordinarily expensive drug treatment, the patient would be able to utilize stem cell therapy. The effect of a successful operation would be immediate, and the patient would avoid chronic pharmacological treatment and its inevitable side effects.

Although these challenges facing stem cell science can be overwhelming, the field is making great advances each day. Stem cell therapy is already available for treating several diseases and conditions. Their impact on future medicine appears to be significant.

After several decades of experiments, stem cell therapy is becoming a magnificent game changer for medicine. With each experiment, the capabilities of stem cells are growing, although there are still many obstacles to overcome. Regardless, the influence of stem cells in regenerative medicine and transplantology is immense. Currently, untreatable neurodegenerative diseases have the possibility of becoming treatable with stem cell therapy. Induced pluripotency enables the use of a patient’s own cells. Tissue banks are becoming increasingly popular, as they gather cells that are the source of regenerative medicine in a struggle against present and future diseases. With stem cell therapy and all its regenerative benefits, we are better able to prolong human life than at any time in history.

Abbreviations

Basic fibroblast growth factor

Bone morphogenic proteins

Dental follicle stem cells

Dental pulp stem cells

Embryonic bodies

Embryonic stem cells

Fibroblast growth factors

Good Manufacturing Practice

Graphene oxide

Human amniotic fluid-derived stem cells

Human embryonic stem cells

Human foreskin fibroblasts

Inner cell mass

Non-coding RNA

Induced pluripotent stem cells

In vitro fertilization

Knockout serum replacement

Leukaemia inhibitory factor

Monocyte chemotactic protein-1

Fibroblasts

Messenger RNA

Mesenchymal stem cells of dental pulp

Myogenic differentiation

Osteoarthritis

Octamer-binding transcription factor 3 and 4

Platelet-derived growth factor

Platelet-derived growth factor-AA

Periodontal ligament stem cells

Rho-associated protein kinase

Stem cells from apical papilla

Stem cells of human exfoliated deciduous teeth

Synthetic Serum Substitute

Trophectoderm

Vascular endothelial growth factor

Transforming growth factors

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Wojciech Zakrzewski, Maria Szymonowicz & Zbigniew Rybak

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WZ is the principal author and was responsible for the first draft of the manuscript. WZ and ZR were responsible for the concept of the review. MS, MD, and ZR were responsible for revising the article and for data acquisition. All authors read and approved the final manuscript.

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Zakrzewski, W., Dobrzyński, M., Szymonowicz, M. et al. Stem cells: past, present, and future. Stem Cell Res Ther 10 , 68 (2019). https://doi.org/10.1186/s13287-019-1165-5

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Stem cells: what they are and what they do.

Stem cells offer promise for new medical treatments. Learn about stem cell types, current and possible uses, and the state of research and practice.

You've heard about stem cells in the news, and perhaps you've wondered if they might help you or a loved one with a serious disease. Here are some answers to frequently asked questions about stem cells.

What are stem cells?

Stem cells: The body's master cells

Stem cells are the body's master cells. All other cells arise from stem cells, including blood cells, nerve cells and other cells.

Stem cells are a special type of cells that have two important properties. They are able to make more cells like themselves. That is, they self-renew. And they can become other cells that do different things in a process known as differentiation. Stem cells are found in almost all tissues of the body. And they are needed for the maintenance of tissue as well as for repair after injury.

Depending on where the stem cells are, they can develop into different tissues. For example, hematopoietic stem cells reside in the bone marrow and can produce all the cells that function in the blood. Stem cells also can become brain cells, heart muscle cells, bone cells or other cell types.

There are various types of stem cells. Embryonic stem cells are the most versatile since they can develop into all the cells of the developing fetus. The majority of stem cells in the body have fewer abilities to give rise to cells and may only help maintain and repair the tissues and organs in which they reside.

No other cell in the body has the natural ability to generate new cell types.

Why is there such an interest in stem cells?

Researchers are studying stem cells to see if they can help to:

• Increase understanding of how diseases occur. By watching stem cells mature into cells in bones, heart muscle, nerves, and other organs and tissue, researchers may better understand how diseases and conditions develop.

Generate healthy cells to replace cells affected by disease (regenerative medicine). Stem cells can be guided into becoming specific cells that can be used in people to regenerate and repair tissues that have been damaged or affected by disease.

People who might benefit from stem cell therapies include those with leukemia, Hodgkin disease, non-Hodgkin lymphoma and some solid tumor cancers. Stem cell therapies also might benefit people who have aplastic anemia, immunodeficiencies and inherited conditions of metabolism.

Stem cells are being studied to treat type 1 diabetes, Parkinson's disease, amyotrophic lateral sclerosis, heart failure, osteoarthritis and other conditions.

Stem cells may have the potential to be grown to become new tissue for use in transplant and regenerative medicine. Researchers continue to advance the knowledge on stem cells and their applications in transplant and regenerative medicine.

Test new drugs for safety and effectiveness. Before giving drugs in development to people, researchers can use some types of stem cells to test the drugs for safety and quality. This type of testing may help assess drugs in development for toxicity to the heart.

New areas of study include the effectiveness of using human stem cells that have been programmed into tissue-specific cells to test new drugs. For the testing of new drugs to be accurate, the cells must be programmed to acquire properties of the type of cells targeted by the drug. Techniques to program cells into specific cells are under study.

Where do stem cells come from?

There are several sources of stem cells:

Embryonic stem cells. These stem cells come from embryos that are 3 to 5 days old. At this stage, an embryo is called a blastocyst and has about 150 cells.

These are pluripotent (ploo-RIP-uh-tunt) stem cells, meaning they can divide into more stem cells or can become any type of cell in the body. This allows embryonic stem cells to be used to regenerate or repair diseased tissue and organs.

• Adult stem cells. These stem cells are found in small numbers in most adult tissues, such as bone marrow or fat. Compared with embryonic stem cells, adult stem cells have a more limited ability to give rise to various cells of the body.

Adult cells altered to have properties of embryonic stem cells. Scientists have transformed regular adult cells into stem cells using genetic reprogramming. By altering the genes in the adult cells, researchers can make the cells act similarly to embryonic stem cells. These cells are called induced pluripotent stem cells (iPSCs).

This new technique may allow use of reprogrammed cells instead of embryonic stem cells and prevent immune system rejection of the new stem cells. However, scientists don't yet know whether using altered adult cells will cause adverse effects in humans.

Researchers have been able to take regular connective tissue cells and reprogram them to become functional heart cells. In studies, animals with heart failure that were injected with new heart cells had better heart function and survival time.

Perinatal stem cells. Researchers have discovered stem cells in amniotic fluid as well as umbilical cord blood. These stem cells can change into specialized cells.

Amniotic fluid fills the sac that surrounds and protects a developing fetus in the uterus. Researchers have identified stem cells in samples of amniotic fluid drawn from pregnant women for testing or treatment — a procedure called amniocentesis.

Why is there controversy about using embryonic stem cells?

The National Institutes of Health created guidelines for human stem cell research in 2009. The guidelines define embryonic stem cells and how they may be used in research and include recommendations for the donation of embryonic stem cells. Also, the guidelines state that embryonic stem cells from embryos created by in vitro fertilization can be used only when the embryo is no longer needed.

Where do these embryos come from?

The embryos being used in embryonic stem cell research come from eggs that were fertilized at in vitro fertilization clinics but never implanted in women's uteruses. The stem cells are donated with informed consent from donors. The stem cells can live and grow in special solutions in test tubes or petri dishes in laboratories.

Why can't researchers use adult stem cells instead?

Progress in cell reprogramming and the formation of iPSCs has greatly enhanced research in this field. However, reprogramming is an inefficient process. When possible, iPSCs are used instead of embryonic stem cells since this avoids the ethical issues about use of embryonic stem cells that may be morally objectionable for some people.

Although research into adult stem cells is promising, adult stem cells may not be as versatile and durable as are embryonic stem cells. Adult stem cells may not be able to be manipulated to produce all cell types, which limits how adult stem cells can be used to treat diseases.

Adult stem cells are also more likely to contain irregularities due to environmental hazards, such as toxins, or from errors acquired by the cells during replication. However, researchers have found that adult stem cells are more adaptable than was first thought.

What are stem cell lines, and why do researchers want to use them?

A stem cell line is a group of cells that all descend from a single original stem cell and are grown in a lab. Cells in a stem cell line keep growing but don't become specialized cells. Ideally, they remain free of genetic defects and continue to create more stem cells. Clusters of cells can be taken from a stem cell line and frozen for storage or shared with other researchers.

What is stem cell therapy (regenerative medicine), and how does it work?

Stem cell therapy, also known as regenerative medicine, promotes the repair response of diseased, dysfunctional or injured tissue using stem cells or their derivatives. It is the next chapter in organ transplantation and uses cells instead of donor organs, which are limited in supply.

Researchers grow stem cells in a lab. These stem cells are manipulated to specialize into specific types of cells, such as heart muscle cells, blood cells or nerve cells.

The specialized cells can then be implanted into a person. For example, if the person has heart disease, the cells could be injected into the heart muscle. The healthy transplanted heart muscle cells could then contribute to repairing the injured heart muscle.

Researchers have already shown that adult bone marrow cells guided to become heart-like cells can repair heart tissue in people, and more research is ongoing.

Have stem cells already been used to treat diseases?

Yes. Doctors have performed stem cell transplants, also known as bone marrow transplants, for many decades. In hematopoietic stem cell transplants, stem cells replace cells damaged by chemotherapy or disease or serve as a way for the donor's immune system to fight some types of cancer and blood-related diseases. Leukemia, lymphoma, neuroblastoma and multiple myeloma often are treated this way. These transplants use adult stem cells or umbilical cord blood.

Researchers are testing adult stem cells to treat other conditions, including some degenerative diseases such as heart failure.

What are the potential problems with using embryonic stem cells in humans?

For embryonic stem cells to be useful, researchers must be certain that the stem cells will differentiate into the specific cell types desired.

Researchers have discovered ways to direct stem cells to become specific types of cells, such as directing embryonic stem cells to become heart cells. Research is ongoing in this area.

Embryonic stem cells also can grow irregularly or specialize in different cell types spontaneously. Researchers are studying how to control the growth and development of embryonic stem cells.

Embryonic stem cells also might trigger an immune response in which the recipient's body attacks the stem cells as foreign invaders, or the stem cells might simply fail to function as expected, with unknown consequences. Researchers continue to study how to avoid these possible complications.

What is therapeutic cloning, and what benefits might it offer?

Therapeutic cloning, also called somatic cell nuclear transfer, is a way to create versatile stem cells independent of fertilized eggs. In this technique, the nucleus is removed from an unfertilized egg. This nucleus contains the genetic material. The nucleus also is removed from the cell of a donor.

This donor nucleus is then injected into the egg, replacing the nucleus that was removed, in a process called nuclear transfer. The egg is allowed to divide and soon forms a blastocyst. This process creates a line of stem cells that is genetically identical to the donor's cells — in essence, a clone.

Some researchers believe that stem cells derived from therapeutic cloning may offer benefits over those from fertilized eggs because cloned cells are less likely to be rejected once transplanted back into the donor. And it may allow researchers to see exactly how a disease develops.

Has therapeutic cloning in people been successful?

No. Researchers haven't been able to successfully perform therapeutic cloning with humans despite success in a number of other species.

Researchers continue to study the potential of therapeutic cloning in people.

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• Stem cell basics. National Institutes of Health. https://stemcells.nih.gov/info/basics/stc-basics/#stc-I. Accessed March 21, 2024.
• Lovell-Badge R, et al. ISSCR guidelines for stem cell research and clinical translation: The 2021 update. Stem Cell Reports. 2021; doi:10.1016/j.stemcr.2021.05.012.
• AskMayoExpert. Hematopoietic stem cell transplant. Mayo Clinic; 2024.
• Stem cell transplants in cancer treatment. National Cancer Institute. https://www.cancer.gov/about-cancer/treatment/types/stem-cell-transplant/. Accessed March 21, 2024.
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• Yen Ling Wang A. Human induced pluripotent stem cell-derived exosomes as a new therapeutic strategy for various diseases. International Journal of Molecular Sciences. 2021; doi:10.3390/ijms22041769.
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• Brown MA, et al. Update on stem cell technologies in congenital heart disease. Journal of Cardiac Surgery. 2020; doi:10.1111/jocs.14312.
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• Augustine R, et al. Stem cell-based approaches in cardiac tissue engineering: Controlling the microenvironment for autologous cells. Biomedical Pharmacotherapy. 2021; doi:10.1016/j.biopha.2021.111425.
• Cloning fact sheet. National Human Genome Research Institute. https://www.genome.gov/about-genomics/fact-sheets/Cloning-Fact-Sheet. Accessed March 21, 2024.
• Dingli D (expert opinion). Mayo Clinic. Nov. 17, 2023.

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Lupus patient Katherine Hammons comforts fellow patient Margaret Laperle, both treated with stem cells from their own bone marrow. Stem cells could launch a new era of regenerative medicine, curing deadly diseases with custom-made tissues and organs.

The Stem Cell Divide

In the beginning, one cell becomes two, and two become four. Being fruitful, they multiply into a ball of many cells, a shimmering sphere of human potential. Scientists have long dreamed of plucking those naive cells from a young human embryo and coaxing them to perform, in sterile isolation, the everyday miracle they perform in wombs: transforming into all the 200 or so kinds of cells that constitute a human body. Liver cells. Brain cells. Skin, bone, and nerve.

The dream is to launch a medical revolution in which ailing organs and tissues might be repaired—not with crude mechanical devices like insulin pumps and titanium joints but with living, homegrown replacements. It would be the dawn of a new era of regenerative medicine, one of the holy grails of modern biology.

Revolutions, alas, are almost always messy. So when James Thomson, a soft-spoken scientist at the University of Wisconsin in Madison, reported in November 1998 that he had succeeded in removing cells from spare embryos at fertility clinics and establishing the world's first human embryonic stem cell line, he and other scientists got a lot more than they bargained for. It was the kind of discovery that under most circumstances would have blossomed into a major federal research enterprise. Instead the discovery was quickly engulfed in the turbulent waters of religion and politics. In church pews, congressional hearing rooms, and finally the Oval Office, people wanted to know: Where were the needed embryos going to come from, and how many would have to be destroyed to treat the millions of patients who might be helped? Before long, countries around the world were embroiled in the debate.

Most alarmed have been people who see embryos as fully vested, vulnerable members of society, and who decry the harvesting of cells from embryos as akin to cannibalism. They warn of a brave new world of "embryo farms" and "cloning mills" for the cultivation of human spare parts. And they argue that scientists can achieve the same results using adult stem cells— immature cells found in bone marrow and other organs in adult human beings, as well as in umbilical cords normally discarded at birth.

Advocates counter that adult stem cells, useful as they may be for some diseases, have thus far proved incapable of producing the full range of cell types that embryonic stem cells can. They point out that fertility clinic freezers worldwide are bulging with thousands of unwanted embryos slated for disposal. Those embryos are each smaller than the period at the end of this sentence. They have no identifying features or hints of a nervous system. If parents agree to donate them, supporters say, it would be unethical not to do so in the quest to cure people of disease.

Few question the medical promise of embryonic stem cells. Consider the biggest United States killer of all: heart disease. Embryonic stem cells can be trained to grow into heart muscle cells that, even in a laboratory dish, clump together and pulse in spooky unison. And when those heart cells have been injected into mice and pigs with heart disease, they've filled in for injured or dead cells and sped recovery. Similar studies have suggested stem cells' potential for conditions such as diabetes and spinal cord injury.

For Hungry Minds

Critics point to worrisome animal research showing that embryonic stem cells sometimes grow into tumors or morph into unwanted kinds of tissues—possibly forming, for example, dangerous bits of bone in those hearts they are supposedly repairing. But supporters respond that such problems are rare and a lot has recently been learned about how to prevent them.

The arguments go back and forth, but policymakers and governments aren't waiting for answers. Some countries, such as Germany, worried about a slippery slope toward unethical human experimentation, have already prohibited some types of stem cell research. Others, like the U.S., have imposed severe limits on government funding but have left the private sector to do what it wants. Still others, such as the U.K., China, Korea, and Singapore, have set out to become the epicenters of stem cell research, providing money as well as ethical oversight to encourage the field within carefully drawn bounds.

In such varied political climates, scientists around the globe are racing to see which techniques will produce treatments soonest. Their approaches vary, but on one point, all seem to agree: How humanity handles its control over the mysteries of embryo development will say a lot about who we are and what we're becoming.

For more than half   of his seven years, Cedric Seldon has been fighting leukemia. Now having run out of options, he is about to become a biomedical pioneer—one of about 600 Americans last year to be treated with an umbilical cord blood transplant.

Cord blood transplants—considered an adult stem cell therapy because the cells come from infants, not embryos—have been performed since 1988. Like bone marrow, which doctors have been transplanting since 1968, cord blood is richly endowed with a kind of stem cell that gives rise to oxygen-carrying red blood cells, disease-fighting white blood cells, and other parts of the blood and immune systems. Unlike a simple blood transfusion, which provides a batch of cells destined to die in a few months, the stem cells found in bone marrow and cord blood can—if all goes well—burrow into a person's bones, settle there for good, and generate fresh blood and immune cells for a lifetime.

Propped on a hospital bed at Duke University Medical Center, Cedric works his thumbs furiously against a pair of joysticks that control a careening vehicle in a Starsky and Hutch video game. "Hang on, Hutch!" older brother Daniel shouts from the bedside, as a nurse, ignoring the screeching tires and gunshots, sorts through a jumble of tubes and hangs a bag of cord blood cells from a chrome pole. Just an hour ago I watched those cells being thawed and spun in a centrifuge—awakening them for the first time since 2001, when they were extracted from the umbilical cord of a newborn and donated by her parents to a cell bank at Duke. The time has come for those cells to prove their reputed mettle.

For days Cedric has endured walloping doses of chemotherapy and radiation in a last-ditch effort to kill every cancer cell in his body. Such powerful therapy has the dangerous side-effect of destroying patients' blood-making stem cells, and so is never applied unless replacement stem cells are available. A search of every bone marrow bank in the country had found no match for Cedric's genetic profile, and it was beginning to look as if he'd run out of time. Then a computer search turned up the frozen cord blood cells at Duke—not a perfect match, but close enough to justify trying.

"Ready?" the nurse asks. Mom and dad, who have spent hours in prayer, nod yes, and a line of crimson wends its way down the tube, bringing the first of about 600 million cells into the boy's body. The video game's sound effects seem to fade behind a muffling curtain of suspense. Although Cedric's balloon-laden room is buoyant with optimism, success is far from certain.

"Grow, cells, grow," Cedric's dad whispers.

His mom's eyes are misty. I ask what she sees when she looks at the cells trickling into her son.

"Life," she says. "It's his rebirth."

It will be a month before tests reveal whether Cedric's new cells have taken root, but in a way he's lucky. All he needs is a new blood supply and immune system, which are relatively easy to re-create. Countless other patients are desperate to regenerate more than that. Diabetics need new insulin-producing cells. Heart attack victims could benefit from new cardiac cells. Paraplegics might even walk again if the nerves in their spinal cords could regrow.

In a brightly lit laboratory halfway across the country from Cedric's hospital room, three teams of scientists at the University of Wisconsin in Madison are learning how to grow the embryonic stem cells that might make such cures possible. Unlike adult stem cells, which appear to have limited repertoires, embryonic stem cells are pluripotent—they can become virtually every kind of human cell. The cells being nurtured here are direct descendants of the ones James Thomson isolated seven years ago.

For years Thomson and his colleagues have been expanding some of those original stem cells into what are called stem cell lines—colonies of millions of pluripotent cells that keep proliferating without differentiating into specific cell types. The scientists have repeatedly moved each cell's offspring to less crowded laboratory dishes, allowing them to divide again and again. And while they worked, the nation struggled to get a handle on the morality of what they were doing.

It took almost two years for President Bill Clinton's administration to devise ethics guidelines and a system for funding the new field. George W. Bush's ascension prevented that plan from going into effect, and all eyes turned to the conservative Texan to see what he would do. On August 9, 2001, Bush announced that federal funds could be used to study embryonic stem cells. But to prevent taxpayers from becoming complicit in the destruction of human embryos, that money could be used only to study the stem cell lines already in the works as of that date—a number that, for practical reasons, has resulted in about two dozen usable lines. Those wishing to work with any of the more than a hundred stem cell lines created after that date can do so only with private funding.

Every month scientists from around the world arrive in Madison to take a three-day course in how to grow those approved cells. To watch what they must go through to keep the cells happy is to appreciate why many feel hobbled by the Bush doctrine. For one thing—and for reasons not fully understood—the surest way to keep these cells alive is to place them on a layer of other cells taken from mouse embryos, a time-consuming requirement. Hunched over lab benches, deftly handling forceps and pipettes with blue latex gloves, each scientist in Madison spends the better half of a day dissecting a pregnant mouse, removing its uterus, and prying loose a string of embryos that look like little red peas in a pod. They then wash them, mash them, tease apart their cells, and get them growing in lab dishes. The result is a hormone-rich carpet of mouse cells upon which a few human embryonic stem cells are finally placed. There they live like pampered pashas.

If their scientist-servants don't feed them fresh liquid nutrients at least once a day, the cells die of starvation. If each colony is not split in half each week, it dies from overcrowding. And if a new layer of mouse cells is not prepared and provided every two weeks, the stem cells grow into weird and useless masses that finally die. By contrast, scientists working with private money have been developing embryonic stem cell lines that are hardier, less demanding, and not dependent on mouse cells. Bypassing the use of mouse cells is not only easier, but it also eliminates the risk that therapeutic stem cells might carry rodent viruses, thereby potentially speeding their approval for testing in humans.

Here in the Madison lab, scientists grumble about how fragile the precious colonies are. "They're hard to get to know," concedes Leann Crandall, one of the course's instructors and a co-author of the 85-page manual on their care and feeding. "But once you get to know them, you love them. You can't help it. They're so great. I see so many good things coming from them."

A few American scientists are finding it is easier to indulge their enthusiasm for stem cells overseas. Scores of new embryonic stem cell lines have now been created outside the U.S., and many countries are aggressively seeking to spur the development of therapies using these cells, raising a delicate question: Can the nation in which embryonic stem cells were discovered maintain its initial research lead?

"I know a lot of people back in the U.S. who would like to move into embryonic stem cell work but who won't because of the political uncertainties," says Stephen Minger, director of the Stem Cell Biology Laboratory at King's College in London, speaking to me in his cramped and cluttered office. "I think the United States is in real danger of being left behind."

Minger could be right. He is one of at least two high-profile stem cell scientists to move from the U.S. to England in the past few years, something less than a brain drain but a signal, perhaps, of bubbling discontent.

The research climate is good here, says Minger. In 2003 his team became the first in the U.K. to grow colonies of human embryonic stem cells, and his nine-person staff is poised to nearly double. He's developing new growth culture systems that won't rely on potentially infectious mouse cells. He's also figuring out how to make stem cells morph into cardiac, neural, pancreatic, and retinal cells and preparing to test those cells in animals. And in stark contrast to how things are done in the U.S., Minger says, he's doing all this with government support—and oversight.

The Human Fertilisation and Embryology Authority (HFEA), the government agency that has long overseen U.K. fertility clinics, is now also regulating the country's embryonic stem cell research. In closed-door meetings a committee of 18 people appointed by the National Health Service considers all requests to conduct research using embryos. The committee includes scientists, ethicists, lawyers, and clergy, but the majority are lay people representing the public.

To an American accustomed to high security and protesters at venues dealing regularly with embryo research, the most striking thing about the HFEA's headquarters in downtown London is its ordinariness. The office, a standard-issue warren of cubicles and metal filing cabinets, is on the second floor of a building that also houses the agency that deals with bankruptcy. I ask Ross Thacker, a research officer at the authority, whether the HFEA is regularly in need of yellow police tape to keep protesters at bay.

"Now that you mention it," he says, "there was a placard holder outside this morning …"

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"… but he was protesting something about the insolvency office."

Thacker politely refrains from criticizing U.S. policy on embryo research, but he clearly takes pride in the orderliness of the British system. The committee has approved about a dozen requests to create stem cell lines in the past 18 months, increasing the number of projects to 35. Most were relatively routine—until a strong-willed fertility doctor named Alison Murdoch decided to ask for permission to do something nobody had done before: create cloned human embryos as sources of stem cells.

As controversial as embryonic stem cell research can be, cloning embryos to produce those stem cells is even thornier. Much of the world became familiar with cloning in 1997, when scientists announced they'd cloned a sheep named Dolly. The process involves creating an animal not from egg and sperm but by placing the nucleus of a cell inside an egg that's had its nucleus removed. It's since been used to replicate mice, rabbits, cats, and cattle, among others.

As in many other countries and a few U.S. states, it's illegal in the U.K. to create cloned human babies (called reproductive cloning), because of concerns that clones may be biologically abnormal and because of ethical issues surrounding the creation of children who would be genetic replicas of their one-and-only "parent."

In 2001 the British Parliament made it legal to create cloned human embryos—as opposed to babies—for use in medical research (called therapeutic cloning). Still, no one on the HFEA was completely comfortable with the idea. The fear was that some rogue scientist would take the work a step further, gestate the embryo in a woman's womb, and make the birth announcement that no one wanted to hear.

But Murdoch, of the University of Newcastle upon Tyne, made a compelling case. If replacement tissues grown from stem cells bore the patient's exact genetic fingerprint, they would be less likely to be rejected by a patient's immune system, she told the committee. And what better way to get such a match than to derive the cells from an embryo cloned from the patient's own DNA? Disease research could also benefit, she said. Imagine an embryo—and its stem cells—cloned from a person with Lou Gehrig's disease, a fatal genetic disorder that affects nerves and muscles. Scientists might learn quite a bit, she argued, by watching how the disease damages nerve and muscle cells grown from those stem cells, and then testing various drugs on them. It's the kind of experiment that could never be done in a person with the disease.

The HFEA deliberated for five months before giving Murdoch permission to make human embryo clones in her lab at the Centre for Life in Newcastle, a sprawling neon-illuminated complex of buildings that strikes a decidedly modern note in the aging industrial hub. But there was a catch: It takes an egg to make a clone. And under the terms of HFEA approval, Murdoch is allowed to use only those eggs being disposed of by the center's fertility clinic after they failed to fertilize when mixed with sperm.

It's not a perfect arrangement, Murdoch says. After all, eggs that have failed to fertilize are almost by definition of poor quality. "They're not brilliant," she says of the eggs. "But the U.K. has decided at the moment that these are the most ethical sort to use. So that's really all we can work with." As of April the group hadn't managed to clone any embryos, despite numerous attempts.

No such obstacle faced Woo-Suk Hwang and his colleagues at Seoul National University in February 2004 when they became the world's first to clone human embryos and extract stem cells from them. The South Korean government allows research on human embryos made from healthy eggs—in this case, donated by 16 women who took egg-ripening hormones.

Cloning is an arduous process that requires great patience and almost always ends in failure as cells burst, tear, or suffer damage to their DNA, but the Koreans are expert cloners, their skills sharpened in the country's state-funded livestock-cloning enterprise. In Hwang's lab alone, technicians produce more than 700 cloned pig or cattle embryos every day, seven days a week, in a quest to produce livestock with precise genetic traits. "There is no holiday in our lab," Hwang told me with a smile.

But there is something else that gives Koreans an edge over other would-be cloners, Hwang says. "As you know, Asian countries use chopsticks, but only the Koreans use steel chopsticks," he explains. "The steel ones are the most difficult to use. Very slippery." I look at him, trying to tell if he's kidding. A lifetime of using steel chopsticks makes Koreans better at manipulating tiny eggs? "This is not simply a joke," he says.

Time will tell whether such skill will be enough to keep Korea in the lead as other countries turn to cloning as a source of stem cells. The competition will be tough. China has pioneered a potentially groundbreaking technique that produces cloned human embryos by mixing human skin cells with the eggs of rabbits, which are more easily obtained than human eggs. A few privately funded researchers in the U.S. are also pursuing therapeutic cloning.

Yet the biggest   competition in the international race to develop stem cell therapies may ultimately come from one of the smallest of countries—a tiny nation committed to becoming a stem cell superpower. To find that place, one need only track the migration patterns of top scientists who've been wooed there from the U.S., Australia, even the U.K. Where they've been landing, it turns out, is Singapore.

Amid the scores of small, botanically rich but barely inhabited islands in the South China Sea, Singapore stands out like a post-modern mirage. The towering laboratory buildings of its Biopolis were created in 2001 to jumpstart Singapore's biotechnology industry. Like a scene from a science fiction story, it features futuristic glass-and-metal buildings with names like Matrix, Proteos, and Chromos, connected by skywalks that facilitate exchanges among researchers.

Academic grants, corporate development money, laws that ban reproductive cloning but allow therapeutic cloning, and a science-savvy workforce are among the lures attracting stem cell researchers and entrepreneurs. Even Alan Colman—the renowned cloning expert who was part of the team that created Dolly, the cloned sheep—has taken leave of his home in the U.K. and become the chief executive of ES Cell International, one of a handful of major stem cell research companies blossoming in Singapore's fertile environs.

"You don't have to fly from New York to San Diego to see what's going on in other labs," says Robert Klupacs, the firm's previous CEO. "You just walk across the street. Because Singapore is small, things can happen quickly. And you don't have to go to Congress at every turn."

The company's team of 36, with 15 nationalities represented, has taken advantage of that milieu. It already owns six stem cell lines made from conventional, noncloned embryos that are approved for U.S. federal funding. Now it is perfecting methods of turning those cells into the kind of pancreatic islet cells that diabetics need, as well as into heart muscle cells that could help heart attack patients. The company is developing new, mouse-free culture systems and sterile production facilities to satisfy regulators such as the U.S. Food and Drug Administration. It hopes to begin clinical tests in humans by 2007.

Despite its research-friendly ethos—and its emphasis on entrepreneurial aspects of stem cell science—Singapore doesn't want to be known as the world's "Wild West" of stem cell research. A panel of scientific and humanitarian representatives spent two years devising ethical guidelines, stresses Hwai-Loong Kong, executive director of Singapore's Biomedical Research Council. Even the public was invited to participate, Kong says—an unusual degree of democratic input for the authoritarian island nation. The country's policies represent a "judicious balance," he says, that has earned widespread public support.

Widespread, perhaps, but not universal. After my conversation with Kong, a government official offered me a ride to my next destination. As we approached her parked car, she saw the surprise on my face as I read the bumper sticker on her left rear window: "Embryos—Let Them Live. You Were Once an Embryo Too!"

"I guess this is not completely settled," I said. "No," she replied, choosing not to elaborate.

That bumper sticker made me feel strangely at home. I am an American, after all. And no country has struggled more with the moral implications of embryonic stem cell research than the U.S., with its high church attendance rates and pockets of skepticism for many things scientific. That struggle promises to grow in the months and years ahead. Many in Congress want to ban the cloning of human embryos, even in those states where it is currently legal and being pursued with private funding. Some states have already passed legislation banning various kinds of embryo research. And federally backed scientists are sure to become increasingly frustrated as the handful of cell colonies they're allowed to work with becomes an ever smaller fraction of what's available.

Yet one thing I've noticed while talking to stem cell experts around the world: Whenever I ask who is the best in the field, the answers are inevitably weighted with the names of Americans. The work of U.S. researchers still fills the pages of the best scientific journals. And while federal policy continues to frustrate them, they are finding some support. Following the lead of California, which has committed 300 million dollars a year for embryonic stem cell research for the next decade, several states are pushing initiatives to fund research, bypassing the federal restrictions in hopes of generating well-paying jobs to boost their economies. Moves like those prompt some observers to predict that when all is said and done, it will be an American team that wins the race to create the first FDA-approved embryonic stem cell therapy.

Tom Okarma certainly believes so, and he intends to be that winner. Okarma is president of Geron, the company in Menlo Park, California, that has been at the center of the embryonic stem cell revolution from the beginning. Geron financed James Thomson's discovery of the cells in Wisconsin and has since developed more than a dozen new colonies. It holds key patents on stem cell processes and products. And now it's laying the groundwork for what the company hopes will be the first controlled clinical trials of treatments derived from embryonic stem cells. Moreover, while others look to stem cells from cloned embryos or newer colonies that haven't come into contact with mouse cells, Okarma is looking no further than the very first colonies of human embryonic stem cells ever grown: the ones Thomson nurtured back in 1998. That may seem surprising, he acknowledges, but after all these years, he knows those cells inside out.

"We've shown they're free of human, pig, cow, and mouse viruses, so they're qualified for use in humans," Okarma says at the company's headquarters. Most important, Geron has perfected a system for growing uniform batches of daughter cells from a master batch that resides, like a precious gem, in a locked freezer. The ability to produce a consistent product, batch after batch, just as drug companies do with their pills is what the FDA wants—and it will be the key to success in the emerging marketplace of stem cell therapies, Okarma says. "Why do you think San Francisco sourdough bread is so successful?" he asks. "They've got a reliable sourdough culture, and they stick with it."

Geron scientists can now make eight different cell types from their embryonic lines, Okarma says, including nerve cells, heart cells, pancreatic islet cells, liver cells, and the kind of brain cells that are lost in Parkinson's disease. But what Geron wants most at this point is to develop a treatment for spinal cord injuries.

Okarma clicks on a laptop and shows me a movie of white rats in a cage. "Pay attention to the tail and the two hind legs," he says. Two months before, the rats were subjected to spinal cord procedures that left their rear legs unable to support their weight and their tails dragging along the floor. "That's a permanent injury," he says. He flips to a different movie: white rats again, also two months after injury. But these rats received injections of a specialized nervous system cell grown from human embryonic stem cells. They have only the slightest shuffle in their gait. They hold their tails high. One even stands upright on its rear legs for a few moments.

"It's not perfect," Okarma says. "It's not like we've made a brand new spinal cord." But tests show the nerves are regrowing, he says. He hopes to get FDA permission to start testing the cells in people with spinal cord injuries in 2006.

Those experiments will surely be followed by many others around the world, as teams in China, the U.K., Singapore, and other nations gain greater control over the remarkable energy of stem cells. With any luck the political and ethical issues may even settle down. Many suspect that with a little more looking, new kinds of stem cells may be found in adults that are as versatile as those in embryos.

At least two candidates have already emerged. Catherine Verfaillie, a blood disease specialist at the University of Minnesota, has discovered a strange new kind of bone marrow cell that seems able to do many, and perhaps even all, the same things human embryonic stem cells can do. Researchers at Tufts University announced in February that they had found similar cells. While some scientists have expressed doubts that either kind of cell will prove as useful as embryonic ones, the discoveries have given birth to new hopes that scientists may yet find the perfect adult stem cell hiding in plain sight.

Maybe Cedric Seldon himself will discover them. The stem cells he got in his cord blood transplant did the trick, it turns out. They took root in his marrow faster than in anyone his doctors have seen. "Everyone's saying, 'Oh my God, you're doing so well,' " his mother says.

That makes Cedric part of the world's first generation of regenerated people, a seamless blend of old and new—and, oddly enough, of male and female. His stem cells, remember, came from a girl, and they've been diligently churning out blood cells with two X chromosomes ever since. It's a detail that will not affect his sexual development, which is under the control of his hormones, not his blood. But it's a quirk that could save him, his mother jokes, if he ever commits a crime and leaves a bit of blood behind. The DNA results would be unambiguous, she notes correctly. "They'll be looking for a girl."

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Stem Cell Research Uncovers Clues to Tissue Repair That Could Help Heal the Uterus and More

Stem cells play a vital role in repairing damaged tissue, whether it’s a scraped knee or a scarred uterus following pregnancy. New stem cell research has identified the molecules that the cells produce to promote the healing process. The finding could pave the way for the development of new, more effective drugs for injuries or various diseases, including conditions related to reproductive health such as Asherman syndrome, a gynecologic condition in which the uterus scars and becomes fibrotic.

Scientists believed in the past that stem cells served as backup cells that repaired tissues by differentiating into new cells that repopulated the site of injury. Now, they have learned that it is rare for stem cells to completely replace injured tissue. But they still don’t fully understand how the cells are able to help damaged areas regenerate.

We found the molecules that stem cells make to help heal and repair tissue, and we hope that understanding this will be potentially useful as a medication in the future. Hugh Taylor, MD

In the uterus, stem cells play a number of roles, including helping it to expand during pregnancy and to regenerate and repair after childbirth. This new study identified several microRNAs (miRNAs) secreted by the stem cells that helped drive the growth and proliferation of cells in uterine tissue. The researchers published their findings in Stem Cell Research & Therapy on May 1.

“We found the molecules that stem cells make to help heal and repair tissue, and we hope that understanding this will be potentially useful as a medication in the future,” says Hugh Taylor, MD , chair and Anita O’Keeffe Young Professor of Obstetrics, Gynecology & Reproductive Sciences at Yale School of Medicine and the study’s principal investigator.

Stem cells secrete miRNAs that support cell proliferation

Exosomes are extracellular vesicles, which contain various bioactive molecules and allow cells to communicate with one another. In their new study, Taylor’s team isolated exosomes secreted by stem cells from human bone marrow. They then used RNA sequencing to characterize all of the miRNA contained in the vesicles and identified those that were most abundant. Then researchers took the most prominent miRNAs and introduced them into human uterine tissue.

The team found that the miRNAs significantly increased the growth and proliferation of the uterine cells. They also studied their effect on the cells’ decidualization in the endometrium. (Decidualization is the differentiation process uterine cells undergo that prepares the uterus to support an embryo.) The study showed that the miRNAs blocked decidualization.

“In a uterus, once a cell becomes differentiated to support pregnancy, it can no longer repair and regenerate. It’s permanently locked in that state and often is shed through menstruation later on,” says Taylor. “By blocking this process, it allows the cells to focus on proliferating and turns on these reparative processes.”

Turning miRNAs into drugs for tissue repair

The study offers insight into how stem cells promote reparative processes without replacing the tissue itself. Taylor hopes that as researchers continue to gain a greater understanding about how miRNAs work, they could one day be used as drugs for repairing various damaged tissue.

Asherman syndrome, for example, typically occurs after pregnancy, when the supply of stem cells may not be adequate to help the organ heal properly, which can hinder fertility in the future. “The idea is that these miRNAs could be used as a medication that is much more readily available and practical,” says Taylor. “We could potentially deliver them to help prepare the uterus in the critical window when it is damaged and may be vulnerable.”

The finding could also have significance beyond the uterus. In future stem cell research, Taylor’s team plans to study how miRNAs respond to other types of traumatic tissue injury in animal models. “We studied the uterus, but the implications are beyond reproduction, potentially including many other conditions where stem cells are involved in repair and regeneration, whether that’s injury due to trauma or degenerative diseases,” says Taylor.

Featured in this article

• Hugh Taylor, MD Anita O'Keeffe Young Professor of Obstetrics, Gynecology, and Reproductive Sciences and Professor of Molecular, Cellular, and Developmental Biology; Chair, Obstetrics, Gynecology & Reproductive Sciences; Chief , Obstetrics and Gynecology, Yale New Haven Hospital

Tackling the hurdle of tumor formation in stem cell therapies

Pluripotent stem cells (PSCs) are a type of stem cells capable of developing into various cell types. Over the past few decades, scientists have been working towards the development of therapies using PSCs. Thanks to their unique ability to self-renew and differentiate (mature) into virtually any given type of tissue, PSCs could be used to repair organs that have been irreversibly damaged by age, trauma, or disease.

However, despite extensive efforts, regenerative therapies involving PSCs still have many hurdles to overcome. One being the formation of tumors (via the process of tumorigenesis) after the transplantation of PSCs. Once the PSCs differentiate into a specific type for stem cell therapy, there is a high probability of tumor formation after differentiated stem cells are introduced to the target organ. For the success of PSC-based therapies, the need of the hour is to minimize the risk of tumorigenesis by identifying potentially problematic cells in cultures, prior to transplantation.

Against this backdrop, a research team led by Atsushi Intoh and Akira Kurisaki from Nara Institute of Science and Technology, Japan, has recently achieved a breakthrough discovery regarding stem cell therapy and tumorigenesis. "Our findings present advancements that could bridge the gap between stem cell research and clinical application," says Intoh, talking about the potential of their findings. Their study was published in Stem Cells Translational Medicine and focuses on a membrane protein called EPHA2, which was previously found to be elevated in PSCs prior to differentiation by the team.

Through several experiments involving both mouse and human stem cell cultures, the researchers gained insights into the role of EPHA2 in preserving the potency of PSCs to develop into several cell types. They found that EPHA2 in stem cells is co-expressed with OCT4 -- a transcription factor protein which controls the expression of genes which are critically involved in the differentiation of embryonic stem cells. Interestingly, when the EPHA2 gene was knocked down from the cells, cultured stem cells spontaneously differentiated. These results suggest that EPHA2 plays a central role in keeping stem cells in an undifferentiated state.

The researchers thus theorized that EPHA2-expressing stem cells, which would fail to differentiate, might be responsible for tumorigenesis upon transplantation into the target organ.

To test this hypothesis, the researchers prepared PSC cultures and artificially induced their differentiation into liver cells. Using a magnetic antibody targeting EPHA2, they extracted EPHA2-positive cells from a group of cultures prior to transplantation into mice. Interestingly, the formation of tumors in mice receiving transplants from cultures from which EPHA2 had been removed was vastly suppressed.

Taken together, these results point to the importance of EPHA2 in emerging stem cell-based therapies. "EPHA2 conclusively emerges as a potential marker for selecting undifferentiated stem cells, providing a valuable method to decrease tumorigenesis risks after stem cell transplantation in regenerative treatments," remarks Kurisaki.

Further in-depth studies on this protein may lead to the development of protocols that make PSCs safer to use. Luckily, however, these findings pave the way towards a future where we will be able to finally restore damaged organs and even overcome degenerative conditions.

• Prostate Cancer
• Skin Cancer
• Brain Tumor
• Medical Topics
• Immune System
• Embryonic stem cell
• Bone marrow transplant
• Adult stem cell
• Stem cell treatments
• Somatic cell nuclear transfer
• Somatic cell
• Monoclonal antibody therapy

Story Source:

Materials provided by Nara Institute of Science and Technology . Note: Content may be edited for style and length.

Journal Reference :

• Atsushi Intoh, Kanako Watanabe-Susaki, Taku Kato, Hibiki Kiritani, Akira Kurisaki. EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells . Stem Cells Translational Medicine , 2024; DOI: 10.1093/stcltm/szae036

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Stem cell research reveals new clues to tissue repair that could help heal the uterus and more

by Isabella Backman, Yale University

Stem cells play a vital role in repairing damaged tissue, whether it's a scraped knee or a scarred uterus following pregnancy. New stem cell research has identified the molecules that the cells produce to promote the healing process.

The finding could pave the way for the development of new, more effective drugs for injuries or various diseases, including conditions related to reproductive health such as Asherman syndrome, a gynecologic condition in which the uterus scars and becomes fibrotic.

Scientists believed in the past that stem cells served as backup cells that repaired tissues by differentiating into new cells that repopulated the site of injury. Now, they have learned that it is rare for stem cells to completely replace injured tissue. But they still don't fully understand how the cells are able to help damaged areas regenerate.

In the uterus, stem cells play a number of roles, including helping it to expand during pregnancy and to regenerate and repair after childbirth. This new study identified several microRNAs (miRNAs) secreted by the stem cells that helped drive the growth and proliferation of cells in uterine tissue. The researchers published their findings in Stem Cell Research & Therapy on May 1.

"We found the molecules that stem cells make to help heal and repair tissue, and we hope that understanding this will be potentially useful as a medication in the future," says Hugh Taylor, MD, chair and Anita O'Keeffe Young Professor of Obstetrics, Gynecology & Reproductive Sciences at Yale School of Medicine and the study's principal investigator.

Stem cells secrete miRNAs that support cell proliferation

Exosomes are extracellular vesicles, which contain various bioactive molecules and allow cells to communicate with one another. In their new study, Taylor's team isolated exosomes secreted by stem cells from human bone marrow. They then used RNA sequencing to characterize all of the miRNA contained in the vesicles and identified those that were most abundant. Then researchers took the most prominent miRNAs and introduced them into human uterine tissue.

The team found that the miRNAs significantly increased the growth and proliferation of the uterine cells. They also studied their effect on the cells' decidualization in the endometrium. (Decidualization is the differentiation process uterine cells undergo that prepares the uterus to support an embryo.) The study showed that the miRNAs blocked decidualization.

"In a uterus, once a cell becomes differentiated to support pregnancy, it can no longer repair and regenerate. It's permanently locked in that state and often is shed through menstruation later on," says Taylor. "By blocking this process, it allows the cells to focus on proliferating and turns on these reparative processes."

Turning miRNAs into drugs for tissue repair

The study offers insight into how stem cells promote reparative processes without replacing the tissue itself. Taylor hopes that as researchers continue to gain a greater understanding about how miRNAs work, they could one day be used as drugs for repairing various damaged tissue.

Asherman syndrome, for example, typically occurs after pregnancy, when the supply of stem cells may not be adequate to help the organ heal properly, which can hinder fertility in the future.

"The idea is that these miRNAs could be used as a medication that is much more readily available and practical," says Taylor. "We could potentially deliver them to help prepare the uterus in the critical window when it is damaged and may be vulnerable."

The finding could also have significance beyond the uterus. In future stem cell research , Taylor's team plans to study how miRNAs respond to other types of traumatic tissue injury in animal models.

"We studied the uterus , but the implications are beyond reproduction, potentially including many other conditions where stem cells are involved in repair and regeneration, whether that's injury due to trauma or degenerative diseases ," says Taylor.

Provided by Yale University

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Guest Essay

The Long-Overlooked Molecule That Will Define a Generation of Science

By Thomas Cech

Dr. Cech is a biochemist and the author of the forthcoming book “The Catalyst: RNA and the Quest to Unlock Life’s Deepest Secrets,” from which this essay is adapted.

From E=mc² to splitting the atom to the invention of the transistor, the first half of the 20th century was dominated by breakthroughs in physics.

Then, in the early 1950s, biology began to nudge physics out of the scientific spotlight — and when I say “biology,” what I really mean is DNA. The momentous discovery of the DNA double helix in 1953 more or less ushered in a new era in science that culminated in the Human Genome Project, completed in 2003, which decoded all of our DNA into a biological blueprint of humankind.

DNA has received an immense amount of attention. And while the double helix was certainly groundbreaking in its time, the current generation of scientific history will be defined by a different (and, until recently, lesser-known) molecule — one that I believe will play an even bigger role in furthering our understanding of human life: RNA.

You may remember learning about RNA (ribonucleic acid) back in your high school biology class as the messenger that carries information stored in DNA to instruct the formation of proteins. Such messenger RNA, mRNA for short, recently entered the mainstream conversation thanks to the role they played in the Covid-19 vaccines. But RNA is much more than a messenger, as critical as that function may be.

Other types of RNA, called “noncoding” RNAs, are a tiny biological powerhouse that can help to treat and cure deadly diseases, unlock the potential of the human genome and solve one of the most enduring mysteries of science: explaining the origins of all life on our planet.

Though it is a linchpin of every living thing on Earth, RNA was misunderstood and underappreciated for decades — often dismissed as nothing more than a biochemical backup singer, slaving away in obscurity in the shadows of the diva, DNA. I know that firsthand: I was slaving away in obscurity on its behalf.

In the early 1980s, when I was much younger and most of the promise of RNA was still unimagined, I set up my lab at the University of Colorado, Boulder. After two years of false leads and frustration, my research group discovered that the RNA we’d been studying had catalytic power. This means that the RNA could cut and join biochemical bonds all by itself — the sort of activity that had been thought to be the sole purview of protein enzymes. This gave us a tantalizing glimpse at our deepest origins: If RNA could both hold information and orchestrate the assembly of molecules, it was very likely that the first living things to spring out of the primordial ooze were RNA-based organisms.

That breakthrough at my lab — along with independent observations of RNA catalysis by Sidney Altman at Yale — was recognized with a Nobel Prize in 1989. The attention generated by the prize helped lead to an efflorescence of research that continued to expand our idea of what RNA could do.

In recent years, our understanding of RNA has begun to advance even more rapidly. Since 2000, RNA-related breakthroughs have led to 11 Nobel Prizes. In the same period, the number of scientific journal articles and patents generated annually by RNA research has quadrupled. There are more than 400 RNA-based drugs in development, beyond the ones that are already in use. And in 2022 alone, more than $1 billion in private equity funds was invested in biotechnology start-ups to explore frontiers in RNA research.

What’s driving the RNA age is this molecule’s dazzling versatility. Yes, RNA can store genetic information, just like DNA. As a case in point, many of the viruses (from influenza to Ebola to SARS-CoV-2) that plague us don’t bother with DNA at all; their genes are made of RNA, which suits them perfectly well. But storing information is only the first chapter in RNA’s playbook.

Unlike DNA, RNA plays numerous active roles in living cells. It acts as an enzyme, splicing and dicing other RNA molecules or assembling proteins — the stuff of which all life is built — from amino acid building blocks. It keeps stem cells active and forestalls aging by building out the DNA at the ends of our chromosomes.

RNA discoveries have led to new therapies, such as the use of antisense RNA to help treat children afflicted with the devastating disease spinal muscular atrophy. The mRNA vaccines, which saved millions of lives during the Covid pandemic, are being reformulated to attack other diseases, including some cancers . RNA research may also be helping us rewrite the future; the genetic scissors that give CRISPR its breathtaking power to edit genes are guided to their sites of action by RNAs.

Although most scientists now agree on RNA's bright promise, we are still only beginning to unlock its potential. Consider, for instance, that some 75 percent of the human genome consists of dark matter that is copied into RNAs of unknown function. While some researchers have dismissed this dark matter as junk or noise, I expect it will be the source of even more exciting breakthroughs.

We don’t know yet how many of these possibilities will prove true. But if the past 40 years of research have taught me anything, it is never to underestimate this little molecule. The age of RNA is just getting started.

Thomas Cech is a biochemist at the University of Colorado, Boulder; a recipient of the Nobel Prize in Chemistry in 1989 for his work with RNA; and the author of “The Catalyst: RNA and the Quest to Unlock Life’s Deepest Secrets,” from which this essay is adapted.

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A new, nano-scale look at how the SARS-CoV-2 virus replicates in cells may offer greater precision in drug development, a Stanford University team reports in Nature Communications . Using advanced microscopy techniques, the researchers produced what might be some of the most crisp images available of the virus’s RNA and replication structures, which they witnessed form spherical shapes around the nucleus of the infected cell.

“We have not seen COVID infecting cells at this high resolution and known what we are looking at before,” said Stanley Qi , Stanford associate professor of bioengineering in the Schools of Engineering and of Medicine and co-senior author of the paper. “Being able to know what you are looking at with this high resolution over time is fundamentally helpful to virology and future virus research, including antiviral drug development.”

Blinking RNA 

The work illuminates molecular-scale details of the virus’ activity inside host cells. In order to spread, viruses essentially take over cells and transform them into virus-producing factories, complete with special replication organelles. Within this factory, the viral RNA needs to duplicate itself over and over until enough genetic material is gathered up to move out and infect new cells and start the process over again.

The Stanford scientists sought to reveal this replication step in the sharpest detail to date. To do so, they first labeled the viral RNA and replication-associated proteins with fluorescent molecules of different colors. But imaging glowing RNA alone would result in fuzzy blobs in a conventional microscope. So they added a chemical that temporarily suppresses the fluorescence. The molecules would then blink back on at random times, and only a few lit up at a time. That made it easier to pinpoint the flashes, revealing the locations of the individual molecules.

Using a setup that included lasers, powerful microscopes, and a camera snapping photos every 10 milliseconds, the researchers gathered snapshots of the blinking molecules. When they combined sets of these images, they were able to create finely detailed photos showing the viral RNA and replication structures in the cells. “We have highly sensitive and specific methods and also high resolution,” said Leonid Andronov, co-lead author and Stanford chemistry postdoctoral scholar. “You can see one viral molecule inside the cell.”

The resulting images, with a resolution of 10 nanometers, reveal what might be the most detailed view yet of how the virus replicates itself inside of a cell. The images show magenta RNA forming clumps around the nucleus of the cell, which accumulate into a large repeating pattern. “We are the first to find that viral genomic RNA forms distinct globular structures at high resolution,” said Mengting Han, co-lead author and Stanford bioengineering postdoctoral scholar.

Video showing the different colored fluorescent labels blinking on and off, revealing more precise locations for individual molecules. | Leonid Andronov, Moerner Laboratory

The clusters help show how the virus evades the cell’s defenses, said W. E. Moerner , the paper’s co-senior author and Harry S. Mosher Professor of Chemistry in the School of Humanities and Sciences. “They’re collected together inside a membrane that sequesters them from the rest of the cell, so that they’re not attacked by the rest of the cell.”

Nanoscale drug testing 

Compared to using an electron microscope, the new imaging technique can allow researchers to know with greater certainty where virus components are in a cell thanks to the blinking fluorescent labels. It also can provide nanoscale details of cell processes that are invisible in medical research conducted through biochemical assays. The conventional techniques “are completely different from these spatial recordings of where the objects actually are in the cell, down to this much higher resolution,” said Moerner. “We have an advantage based on the fluorescent labeling because we know where our light is coming from.” 

Seeing exactly how the virus stages its infection holds promise for medicine. Observing how different viruses take over cells may help answer questions such as why some pathogens produce mild symptoms while others are life-threatening. The super-resolution microscopy can also benefit drug development. “This nanoscale structure of the replication organelles can provide some new therapeutic targets for us,” said Han. “We can use this method to screen different drugs and see its influence on the nanoscale structure.”

Indeed, that’s what the team plans to do. They will repeat the experiment and see how the viral structures shift in the presence of drugs like Paxlovid or remdesivir. If a candidate drug can suppress the viral replication step, that suggests the drug is effective at inhibiting the pathogen and making it easier for the host to fight the infection. 

The researchers also plan to map all 29 proteins that make up SARS-CoV-2 and see what those proteins do across the span of an infection. “We hope that we will be prepared to really use these methods for the next challenge to quickly see what’s going on inside and better understand it,” said Qi.

For more information

Acknowledgements: Additional Stanford co-authors include postdoctoral scholar Yanyu Zhu, PhD student Ashwin Balaji, former PhD student Anish Roy, postdoctoral scholar Andrew Barentine, research specialist Puja Patel, and Jaishree Garhyan, director of the In Vitro Biosafety Level-3 Service Center . Moerner is also a member of Stanford Bio-X and the Wu Tsai Neurosciences Institute, and a faculty fellow of Sarafan ChEM-H . Qi is also a member of Bio-X, the Cardiovascular Institute , the Maternal & Child Health Research Institute (MCHRI), the Stanford Cancer Institute, and the Wu Tsai Neurosciences Institute, an institute scholar at Sarafan ChEM-H , and a Chan Zuckerberg Biohub – San Francisco Investigator.

This research was funded by the National Institute of General Medical Sciences of the National Institutes of Health. We also acknowledge use of the Stanford University Cell Sciences Imaging Core Facility.

Taylor Kubota, Stanford University: [email protected]

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Technology and code article, open and remotely accessible neuroplatform for research in wetware computing.

• FinalSpark, Rue du Clos 12, Vevey, Switzerland

Wetware computing and organoid intelligence is an emerging research field at the intersection of electrophysiology and artificial intelligence. The core concept involves using living neurons to perform computations, similar to how Artificial Neural Networks (ANNs) are used today. However, unlike ANNs, where updating digital tensors (weights) can instantly modify network responses, entirely new methods must be developed for neural networks using biological neurons. Discovering these methods is challenging and requires a system capable of conducting numerous experiments, ideally accessible to researchers worldwide. For this reason, we developed a hardware and software system that allows for electrophysiological experiments on an unmatched scale. The Neuroplatform enables researchers to run experiments on neural organoids with a lifetime of even more than 100 days. To do so, we streamlined the experimental process to quickly produce new organoids, monitor action potentials 24/7, and provide electrical stimulations. We also designed a microfluidic system that allows for fully automated medium flow and change, thus reducing the disruptions by physical interventions in the incubator and ensuring stable environmental conditions. Over the past three years, the Neuroplatform was utilized with over 1,000 brain organoids, enabling the collection of more than 18 terabytes of data. A dedicated Application Programming Interface (API) has been developed to conduct remote research directly via our Python library or using interactive compute such as Jupyter Notebooks. In addition to electrophysiological operations, our API also controls pumps, digital cameras and UV lights for molecule uncaging. This allows for the execution of complex 24/7 experiments, including closed-loop strategies and processing using the latest deep learning or reinforcement learning libraries. Furthermore, the infrastructure supports entirely remote use. Currently in 2024, the system is freely available for research purposes, and numerous research groups have begun using it for their experiments. This article outlines the system’s architecture and provides specific examples of experiments and results.

1 Introduction

The recent rise in wetware computing and consequently, artificial biological neural networks (BNNs), comes at a time when Artificial Neural Networks (ANNs) are more sophisticated than ever.

The latest generation of Large Language Models (LLMs), such as Meta’s Llama 2 or OpenAI’s GPT-4, fundamentally rely on ANNs.

The recent acceleration of ANN use in everyday life, such as in tools like ChatGPT or Perplexity combined with the explosion in complexity in the underlying ANN’s architectures, has had a significant impact on energy consumption. For instance, training a single LLM like GPT-3, a precursor to GPT-4, approximately required 10 GWh, which is about 6,000 times the energy a European citizen uses per year. According to a recent publication the energy consumption projected may increase faster than linearly ( De Vries, 2023 ). At the same time, the human brain operates with approximately 86 billion neurons while consuming only 20 W of power ( Clark and Sokoloff, 1999 ). Given these conditions, the prospect of replacing ANNs running on digital computers with real BNNs is enticing ( Smirnova et al., 2023 ). In addition to the substantial energy demands associated with training LLMs, the inference costs present a similarly pressing concern. Recent disclosures reveal that platforms like OpenAI generate over 100 billion words daily through services such as ChatGPT as reported by Sam Altman, the CEO of OpenAI. When we break down these figures, assuming an average of 1.5 tokens per word—a conservative estimate based on OpenAI’s own tokenizer data—the energy footprint becomes staggering. Preliminary calculations, using the LLaMA 65B model (precursor to Llama 2) as a reference point, suggest energy expenditures ranging from 450 to 600 billion Joules per day for word generation alone ( Samsi et al., 2023 ). While necessary for providing AI-driven insights and interactions to millions of users worldwide, this magnitude of energy use underscores the urgency for more energy-efficient computing paradigms.

Connecting probes to BNNs is not a new idea. In fact, the field of multi-unit electrophysiology has an established state of the art spanning easily over the past 40 years. As a result, there are already well-documented hardware and methods for performing functional electrical interfacing and micro-fluidics needed for nutrient delivery ( Gross et al., 1977 ; Pine, 1980 ; Wagenaar et al., 2005a ; Newman et al., 2013 ). Some systems are also specifically designed for brain organoids ( Yang et al., 2024 ). However, their research is mostly focused on exploring brain biology for biomedical applications (e.g., mechanisms and potential treatments of neurodegenerative diseases). The possibility of using these methods for making new computing hardware has not been extensively explored.

For this reason, there is comparatively less literature on methods that can be used to reliably program those BNNs in order to perform specific input–output functions (as this is essential for wetware computing, not for biomedical applications). To understand what we need for programming of BNNs, it is helpful to look at analogous problem for ANNs.

For ANNs, the programming task involves finding the network parameters, globally denoted as S below, that minimize the difference L computed between expected output E and actual output O , for given inputs I , given the transfer function T of the ANN. This can be written as:

L = f O E , with O = T I S

where f is typically a function that equals 0 when O = E .

The same equation applies to BNNs. However, the key differences compared to ANNs include the fact that the network parameters S cannot be individually adjusted in the case of BNNs, and the transfer function T is both unknown and non-stationary. Therefore, alternative heuristics must be developed, for instance based on spatiotemporal stimulation patterns ( Bakkum et al., 2008 ; Kagan et al., 2022 ; Cai et al., 2023a,b ). Such developments necessitate numerous electrophysiological experiments, including, for instance, complex closed-loop algorithms where stimulation is a function of the network’s prior responses. These experiments can sometimes span days or months.

To facilitate long-term experiments involving a global network of research groups, we designed an open innovation platform. This platform enables researchers to remotely perform experiments on a server interfaced with our hardware. For example, our Neuroplatform enhances the chances of discovering the abovementioned stimulation heuristics. It should be noted that, outside of the field of neuroplasticity, similar open platforms were already proposed in 2023 ( O’Leary et al., 2022 ; Armer et al., 2023 ; Elliott et al., 2023 ; Zhang et al., 2023 ). However, to our knowledge, there are no platforms specifically dedicated to research related to biocomputing.

2 Biological setup

The biological material used in our platform is made of brain spheroids [also called minibrains ( Govindan et al., 2021 ), brain organoids ( Qian et al., 2019 ), or neurospheres ( Brewer and Torricelli, 2007 )] developed from Human iPSC-derived Neural Stem Cells (NSCs), following the protocol of Prof. Roux Lab ( Govindan et al., 2021 ). Based on the recent guidelines to clarify the nomenclature for defining 3D cellular models of the nervous system ( Paşca et al., 2022 ), we can call those brain spheroids “forebrain organoids” (FOs). Generation of brain organoids from NSCs has been already described for both mouse ( Ciarpella et al., 2023 ), and human models ( Lee et al., 2020 ). Our protocol is based on the following steps: expansion phase of the NSCs, induction of the 3D structure, differentiation steps (using GDNF and BDNF), and maturation phase ( Figures 1A , B ). The Figure 1C is an image of the FO obtained using electronic microscope, it shows that it is a compact spheroid. The average shape of FOs obtained with this protocol are spheroids of a diameter around 500 μm ( Govindan et al., 2021 ). Our experiments show that the FOs obtained can be kept alive in an orbital shaker for years, as previously demonstrated ( Govindan et al., 2021 ).

Figure 1 . FO generation and MEA setup. (A) Protocol used for the generation of forebrain organoids (FO). Neural progenitors are first thawed, plated and expanded in T25 flasks. They are then differentiated in P6 dishes on orbital shakers, and finally manually placed on the MEA. (B) Representative images showing various stages of FO formation and differentiation, taken at different time points. The scale bar represents 250 μm. (C) Image of a whole FO taken with scanning electron microscope. The scale bar represents 100 μm. (D) Microscope view of the FO (in white) sitting on the electrodes of the MEA, and the membrane. The hole in the membrane is not visible on the picture since it is hidden by the FO. The scale bar represents 500 μm (E) Overview of the MEA, where the 32 electrodes are visible as 4 sets of 8 electrodes each. An FO is placed atop of each set of 8 electrodes, visible as a darker area. For each FO, the 2 circles correspond to a 2.5 mm circular membrane with a central hole. The scale bar represents 1 mm. (F) Cross-sectional view of the MEA setup, illustrating the air-liquid interface. The medium covering the FO is supplied from the medium chamber through the porous membranes.

Gene expression analysis of mature FOs vs. NSCs showed a marked upregulation of genes characteristic to neurons, oligodendrocytes and astrocytes in FOs compare to NSCs. More precisely, FOs expressed genes typically enriched in the forebrain, such as striatum, sub pallium, and layer 6 of motor cortex ( Govindan et al., 2021 ). Pathway enrichment analysis of FOs vs. NSCs demonstrated activation of biological processes like synaptic activity, neuron differentiation and neurotransmitter release ( Govindan et al., 2021 ).

At the age of 12 weeks, FOs contain a high number of ramified neurons ( Govindan et al., 2021 ), and they are mature enough to be transferred to the electrophysiological measurement system ( Figure 1A ). In this setup, they have a life expectancy of several months, even with 24/7 experiments that include hours of electrical stimulations. This setup has a quick turnaround with occasional downtime – about 1 h – during organoid replacements. Therefore, the platform maintains a high availability for experiments.

3 Hardware architecture

3.1 introduction.

The remotely accessible hardware includes all the systems which are required to preserve homeostasis, monitor environmental parameters and perform electrophysiological experiments. These systems can be controlled interactively using our custom Graphical User Interface (GUI) or via Python scripts. All data is stored in a time-series database (InfluxDB), which can be accessed either via a GUI or via Python scripts. The users typically connect to the system using the Remote Desktop Protocol (RDP).

The platform is composed of several sub-systems, which can be accessed remotely via API calls over the internet, typically through Python.

3.2 Multi-Electrode Array (MEA)

Our current platform features 4 MEAs. The MEAs were designed by Prof. Roux’s Lab form Haute Ecole du Paysage, d’Ingénierie et d’Architecture (HEPIA) and are described in Wertenbroek et al. (2021) . Each MEA can accommodate 4 organoids, with 8 electrodes per organoid ( Figure 1E ).

The MEA setup utilizes an Air-Liquid-Interface (ALI) approach ( Stoppini et al., 1991 ), in which the organoids are directly placed on electrodes located atop of a permeable membrane ( Figure 1D ), with the medium flowing beneath this membrane in a 170 μL chamber. As a result, a thin layer of medium, created by surface tension, separates the upper side of the organoids from the humidified incubator air. This arrangement is further protected by a lid partially covering the MEA ( Figure 1F ). This ALI method enables a higher throughput and higher stability compared to submerged approaches, since no dedicated coating is required, and it is less prone to have the organoids detaching from the electrodes.

3.3 Electrophysiological stimulation and recording system

The electrodes in our system enable both stimulation and recording. The respective digital-to-analog and analog-to-digital conversions are performed by Intan RHS 32 headstages. Stimulations are executed using a current controller that ranges from 10 nA to 2.5 mA, and recordings are obtained by measuring the voltage on each electrode at a 30 kHz sampling frequency with a 16 bits resolution giving an accuracy of 0.15 μV. The headstages are connected to an Intan RHS controller, which in turn is connected to a computer via a USB port. The Figure 2A shows the electrical activity recorded for each of the 32 electrodes. It can be noticed that the recorded activity is different between each electrode. This difference comes from the facts that each set of 8 electrodes records a different FO and that for a given FO, electrodes record at a different location. This display is refreshed in real-time and also available 24/7 on our website at the URL https://finalspark.com/live/ . We compared the recording characteristics of this ALI setup to MCS MEA (60MEA200/30iR-Ti) monitoring a submerged FO, using the exact same Intan system for voltage conversion. The overlays of an action potential recorded, respectively, with the ALI and submerged versions are shown in Figures 2C , D and show similar signal characteristics.

Figure 2 . Recording system and user interface. (A) Electrical activity measured in μV over one second for each of the 32 electrodes. Each set of 8 electrodes records a different FO. (B) Graphical User Interface for manually controlling each of the microfluidic pumps. (C) Overlays of FO action potential recorded by the ALI system of the Neuroplatform. (D) Overlays of FO action potentials recorded with an MCS system. (E) Fluctuations of the flowrate of the medium within the microfluidic system, illustrating the cyclic variations induced by the peristaltic pump operating at 1 round per minute with 10 cams. (F) Temporal variations of the red component of the medium color, triggered by a sudden change in medium acidity, resulting in phenol red color change.

3.4 Micro-fluidics

To sustain the life of the organoids on the MEA, Neuronal Medium (NM) needs to be constantly supplied. Our Neuroplatform is equipped with a closed-loop microfluidic system that allows for a 24/7 medium supply. The medium is circulating at a rate of 15uL/min. The medium flow rate is controlled by a BT-100 2 J peristaltic pump and is continuously adjusted according to needs, for instance during experimental runs. The peristaltic pump is connected to the PC-control software using an RS485 interface, for programmed (i.e., in Python) or manual operations ( Figure 2B ). Additionally, Figure 3A depicts this microfluidic closed-loop circuit.

Figure 3 . Microfluidics. (A) Microfluidic system illustrating the continuously operating primary system, which ensures constant flow in the medium chamber, and the secondary system responsible for medium replacing every 48 h. (B) Side view of the assembly, featuring the camera and the MEA. The entire assembly is enclosed with aluminum foil to ensure the lowest possible noise level. (C) Front view of the assembly, showing the intake and outtake of the microfluidic system, as well as the LED used during image capture.

The microfluidic circuit is made of 0.8 mm (inside diameter, ID) tubing. Continuous monitoring of the microfluidic circuit and flow rate is achieved by using Fluigent flow-rate sensors, which connect to the Neuroplatform control center via USB. Data related to medium flow rate is stored in a database for later access. Figure 2E shows the cyclic variations in flow induced by the cams of the peristaltic pump.

A secondary microfluidic system is used to replace the medium in the closed-loop with fresh medium every 24 h, a process illustrated in Figure 3A . This replacement is fully automated through a Python script and performed in the following consecutive steps:

1. Set the rotary valve to select the path from the reservoir F50 to the syringe pump

2. Pump 2 mL of old medium using the syringe pump

3. Set the rotary valve to select the path from the syringe pump to the waste F50

4. Push 2 mL of old medium to the waste using the syringe pump

5. Set the rotary valve to select the path from the new medium in the F50 in the fridge to the syringe pump

6. Pump 2 mL of fresh medium using the syringe pump

7. Set the rotary valve to select the path from the syringe pump to the reservoir F50

8. Push 2 mL of fresh medium using the syringe pump

3.5 Cameras

Each MEA is equipped with a 12.3-megapixel camera that can be controlled interactively or programmatically (i.e., through a Raspberry Pi) for still image capture or video recording. The camera is positioned below the MEA, while illumination is provided by a remotely controlled LED situated above the MEA. Figures 3B , C illustrate this assembly (the aluminum wrapping is used in order to minimize the noise). This setup is particularly useful for detecting various changes, such as cell necrosis, possible organoid displacement caused by microfluidics, variations in medium acidity (using color analysis since our medium contains Phenol red), contamination, neuromelanin production (which can happen when uncaging dopamine), overflows (where the medium inadvertently fills the chamber above the membrane), or bubbles in the medium. For the latter two events, dedicated algorithms automatically detect these issues and trigger an alert to the on-site operator.

Changes of acidity, for example, can be detected by measuring the average color over a pre-defined window. Figure 2F shows the evolution of the medium’s red color component, with data points recorded hourly. The noticeable sudden drop is attributed to the pumping of medium with a slightly different acidity. This change in acidity results in a color alteration of the phenol red present in the medium.

3.6 UV light controlled uncaging

It is also possible to release molecules at specific timings using a process called uncaging. In this method, a specific wavelength of light is employed to break open a molecular “cage” that contains a neuroactive molecule, such as Glutamate, NMDA or Dopamine. A fiber optic of 1,500 μm core diameter and a numerical aperture of 0.5 is used to direct light in the medium within the MEA chamber. The current system, Prizmatix Silver-LED, operates at 365 nm with an optical power of 260 mW. The uncaging system is fully integrated into the Neuroplatform and can be programmatically controlled during experiment runs via our API (see section 5.3).

3.7 Environmental measurements

The environmental conditions are monitored within two incubators. In both incubators, the following parameters are recorded: CO2, O2 concentrations, humidity, atmospheric pressure and temperature. Door-opening events are also logged since they have a major impact on measurements. The primary purpose of this monitoring is to ensure that experiments are performed in stable and reproducible environmental conditions.

All these parameters are displayed in real-time in a graphic interface showing both instant values as well as variations versus time of noise and flowrates ( Figure 4A ).

Figure 4 . Graphic user interface to monitor critical parameters in the incubators. (A) Graphical User Interface displaying critical environmental conditions for the incubator 1, where electrophysiological experiments are performed, as well as the incubator 2, where FO are maintained on an orbital shaker. (B) The display shows environmental data for incubator 1 for specific time periods, extracted from the database, with door opening events displayed as dashed line. Noise, Temperature, humidity and pressure are indicated by different colored lines. The units of each measurement are normalized between 0 and 1 for the selected time interval.

Incubator 1 houses the MEAs and the organoids used for electrophysiological experiments. In addition to the mentioned parameters, flowmeters are also utilized to report the actual flow rate of the microfluidic for each MEA, as depicted in the graph labelled “Pump” in Figure 4A . The system’s state is indirectly monitored through the noise level of each MEA, as shown in the graph labelled “Noise Intan” in Figure 4A . The noise level is calculated based on the standard deviation of the electrical signals recorded by the electrodes over a 30 ms period.

Incubator 2 houses the organoids which are kept in orbital shakers. Piezoelectric gyroscopes are used to measure the actual rotation speed of the orbital shakers.

Since all the data is logged in the database, it is also possible to access the historical measurements through a dedicated GUI ( Figure 4B ).

4.1 General architecture

The core of the system relies on a computational notebook which provides access to 3 resources ( Figure 5A ):

1. A database where all the information regarding the Neuroplatform system is stored

2. The Intan software running on a dedicated PC, which is used for:

• Recording the number of detected spikes in a 200 ms time window

• Setting stimulation parameters

3. A Raspberry Pi for triggering current stimulation according to stimulation parameters

Figure 5 . Software setup and electrical stimulation. (A) General architecture of the Neuroplatform. The Jupyter Notebook serves as the main controller, enabling initiation and reading of spikes, configuration stimulation signals and access to database via, e.g., Python (B) Parameters of the stimulation current: settings optimally these parameters can elicit spikes. Through the Python API, parameters that can be adjusted for the bi-phasic stimulation signals include the duration (D1) and amplitude (A1) of the positive current phase, and, respectively, D2 and A2 for the negative current phase. Additionally, the polarity of the biphasic signal can be reversed to start with a negative current.

4.2 Database

The Neuroplatform records monitored data 24/7 using InfluxDB, a database designed for time series. Other options are also available.

This database contains all the data coming from the hardware listed in Section 3.

The electrical activity of the neurons is also recorded 24/7 at a sampling rate of 30 kHz. To minimize the volume of stored data, we designed a dedicated process that focuses on significant events, such as threshold crossings that are likely to be due to action potentials (spikes). The following pseudo code illustrates the implemented approach:

- Each 1-min write buffer to database

- Each 33 μs

- For each electrode

- If, at time t , the voltage exceeds a threshold T

- Store (in buffer) 3 ms of data [ t -1 ms, t  + 2 ms]

- Each 3 s update T

Additionally, a timestamp corresponding to each detected event is also stored in the database, along with the maximum value of voltage during the 3 ms spike waveform recording.

The threshold T is computed directly from voltage values sampled each 33 μs, according to the following formula:

Where σ i is the standard deviation computed over a set i of 30 ms consecutive voltage values, and M d n represents the median function computed over 101 consecutive σ i values. The use of the median reduces the sensitivity to outliers, which is typically caused by action potentials. In our current setup, a multiplier of 6 on the median has proven to be a good compromise for achieving reliable spike detection.

Besides electric tension data, the number spikes recorded per minute is also computed and stored in the database every minute by a batch process.

4.3 Recording electrical activity

As previously discussed, the communication among neurons is captured by the MEA and converted into a voltage signal sampled at 30 kHz. The Neuroplatform offers two basic access modes to the recorded neural activity:

1. Raw: raw sampling values.

2. Optimized: waveforms of the raw signal near neuronal spikes, available directly from the database.

In addition to the aforementioned features, the Neuroplatform offers even more advanced methods. For instance, it includes counting spikes over a fixed time period of 200 ms following stimulation, with a 10 ms delay suppressing the stimulation artifact.

From a technical perspective, accessing the number of spikes can be accomplished in two different ways:

- Retrieving the number of spikes per minute from the database

- Through direct communication with the PC managing the Intan controller for spike count

The second approach is required when the stimulation protocol demands real-time responsiveness. This is typically the case for certain closed-loop strategies. For instance, closed-loop stimulation strategies have been deployed in primary cortical cultures for effective burst control ( Wagenaar et al., 2005a , b ) and for goal-directed learning ( Samsi et al., 2023 ).

4.4 Syntax for stimulations

Programmatically stimulating the FO on the Neuroplatform is accomplished by sending an electrical current to the MEA electrodes. The electrical current profile can be parameterized in a variety of ways, which is partly shown in Figure 5B . These parameters and controls include:

- Basic shape of stimulation signal:

o Bi-phasic

o Bi-phasic with interphase delay

o Tri-phasic

- Stimulation duration and intensity:

o Positive (A1) and negative (A2) electrical current intensity (typical 1uA, ranging from 0.1uA to 20uA)

o Duration of positive (D1) and negative (D2) stimulation currents

- Stimulation triggers

o Single start

o Table with collection of start triggers

o Pulse trains

- MEA electrodes

send_stim_param (electrodes, params)

5 Examples of electrophysiological experiments

To demonstrate the effectiveness of the Neuroplatform, the following sections will provide an overview of several experiments conducted on the Neuroplatform at FinalSpark’s Laboratories in Vevey, Switzerland.

5.1 Modification of spontaneous activity

The spontaneous electrical activity of the FO can be represented by the concept of “Center of Activity” (CA) ( Bakkum et al., 2008 ) which is defined as a virtual position C on the MEA described by:

Where X k Y k define the spatial position of the 8 electrodes and F k is the number of spontaneous spikes detected. The interest of the concept of CA is that its position provides statistical information about the average location of the activity over the surface of the FO. The ability to change the position of the CA is interesting because it also shows the ability to memorize information in the state of the FO.

The coordinates of the CA can be modified using a high frequency stimulation. In the following experiment we use the following protocol:

1) Compute the CA using the number of detected spikes over 500 ms

2) Goto 1,100x

3) Perform a 20 Hz stimulation during 500 ms using a bi-phasic current (negative first) of 2 μA of 200 μS, for both phases, on one electrode

4) Wait 1 s

5) Goto 5,100x

Figure 6A displays the 100 measured positions of the CA corresponding to the spontaneous activity before the 20 Hz stimulation in blue, and after the high-frequency stimulation in red (the average position is indicated by a cross). A close-up is shown in Figure 6B . The timestamps of the spontaneous activity, before and after stimulation, are presented in Figures 6C , D , respectively. Each graph shows one example of the 100 records of 500 ms used to compute the CA location (showing a decrease of spontaneous firing activity of electrodes 3, 4 and 6). A noticeable shift in the average position (shown by a cross) of the CA can be observed before and after the high-frequency stimulation (as seen in Figure 6A ), indicating a change of state of the biological network. A classifier based on a simple logistic regression is employed to predict if the network has received the 20 Hz stimulation. In this particular experiment, the classification accuracy, computed from the confusion matrix, is 95.5%.

Figure 6 . Center of activity modification. (A) Graph showing the 2D layout of the 8 electrodes, the X and Y axis are normalized units showing the spatial coordinates of the electrodes. All electrodes can be used for both stimulation and reading. A 20 Hz stimulation signal is applied to electrode 6. The 100 blue circles represent the positions of the Center of Activity (CA) before 20 Hz stimulation, while the 100 red circles indicate the positions after the stimulation. The cross mark the average position. (B) A closer look at the two groups of CA. (C) Timestamps depicting the spontaneous activity over 500 ms for each of the 8 electrodes before the high-frequency stimulation. (D) Spontaneous activity observed after the high-frequency stimulation, showing a lower activity of electrodes 6, 4 and 3, compared to (C) .

The Neuroplatform allows users to perform both the experimental part (including stimulation and reading operations) and the visualization of the CA displacement within the same Python source code. The 500 ms 20 Hz signal is generated directly by the Python source code shown below. The first trigger.send instruction sends the trigger for the stimulation on a specific electrode and time.sleep pauses the execution for 50 ms.

Despite the common perception of Python as being less than ideal for real-time signal processing due to its inherent latency, our empirical data reveals a time accuracy of under 1 ms (on an Intel Xeon CPU E5-2690 v2 @ 3.00GHz), a level of precision that is satisfactory for the generation of tetanic signals.

5.2 Optimization of stimulation parameters

In this example, the objective is to identify the set of stimulation parameters that can elicit the maximum number of action potentials within 200 ms after a stimulation.

Depending on the FOs, their composition, and maturity, only specific combinations of electrodes and parameters can elicit spikes. In our experiment, we use an 8-electrode MEA and cycle through several stimulation signal parameters as shown in Figure 7A . Consequently, we need to test a total of 342 different parameter-electrode combinations. The following pseudo code illustrates the Python script used in this experiment.

1) For each set of stimulation parameters

2) For each stimulation electrode

3) For each recording electrode

4) During 15 s, every 250 ms

5) Decide between stimulating, or recording spontaneous activity, with a 50% probability

6) Record number of spikes during 200 ms

Figure 7 . Neural activity stimulation and dopamine uncaging. (A) Graph depicting the number of spikes recorded over 250 ms. The spike counts in orange were measured following a stimulation, while those in blue were measured during periods without stimulation. For clarity in visualization, a small bar is displayed even when no spikes are detected. (B) Diagram illustrating the different steps involved in the closed-loop uncaging process of dopamine, which is repeated 240 times. (C) Timestamps of action potentials from the 8 electrodes before and after stimulation (shown as red line), showcasing the elicited spikes. (D) Graph displaying the number of elicited spikes over the 240 steps of the closed-loop (in blue) alongside the activation events of the UV light source (red).

The aim of probabilistic stimulation and no stimulation in step 5 is to evaluate the difference between elicited and spontaneous spikes in a way that ensures there is no bias.

The bar chart in Figure 7A displays a segment of the experimental results. It shows a 15-s recording from a single electrode, corresponding to one execution of step 4 in the pseudo code above. Each bar represents the spike count during a 200 ms period, repeated every 250 ms. The orange bars in this plot are the result of the parameters selected in step 1 of the pseudo code. The blue bars represent no-stimulation periods, thus corresponding to the spontaneous activity of the neurons.

From Figure 7A , we can see that this particular combination of electrode and parameters reliably elicits responses.

In practice, the Python script can also be used to automatically display the 342 graphs similar to Figure 7A , allowing the operator to select the optimal set of parameters. Additionally, it can compute a scalar metric to characterize the “efficiency” of the parameters, and automatically identify the optimal parameters.

An example of a parameter maximization metric is given in the equation below. Let us denote μ r and μ s the average number of spikes recorded spontaneously or after a stimulation, respectively, and σ r and σ s as their standard deviations. The following metric is used:

The set of parameters that maximize this metric can then be utilized to perform other experiments requiring elicited spikes, such as investigating the effect of pharmacological agents on a biological network’s ability to react quickly to stimulation.

5.3 UV light-induced uncaging of molecules

‘Uncaging’ is a pivotal technique in cellular biology, enabling the precise control of molecular interactions within cells ( Gienger et al., 2020 ). It involves the use of photolabile caged compounds that are activated by specific light wavelengths, releasing bioactive molecules in a targeted and timely manner. This method is particularly valuable for studying dynamic processes in neural networks and intracellular signaling, offering real-time insights into complex biological mechanisms.

Our Neuroplatform is equipped with all necessary components to perform uncaging. In this example, we investigate closed-loop stimulation, where dopamine is used to reward the network when more spikes are elicited by the same stimulation. The release of the dopamine is achieved through the uncaging of CNV-dopamine using the UV system described in section 3.6.

Figure 7B shows the flow chart of the closed-loop uncaging process. The optimal stimulation parameters are first found using the technique shown in 5.2 (in this case, a current of 4uA, biphasic with 100uS per phase), which is sent successively to each of the 8 electrodes with a delay of 10 ms between each electrode.

Figure 7C shows the response timestamps of the 8 electrodes for a period of 1,200 ms, 600 ms before and after the stimulation. The stimulation event is indicated by the vertical red line. It is interesting to observe that in this particular case, most of the elicited spikes originate from 2 electrodes, specifically electrode 112 and electrode 119.

The Python source code implementing the closed-loop process illustrated in Figure 7B is provided below. We would like to highlight here how concise the code is. With only 13 lines of code, the entire closed-loop process has been implemented.

The graph in Figure 7D shows the variation in the number of spikes elicited during the execution of the script above across 5 h. A general increase in the number of elicited spikes can be observed. However, it is obviously not possible to establish causality between the closed-loop strategy and the observed increase with this single experiment alone. The primary purpose of this closed-loop experiment is to demonstrate the flexibility offered by the Neuroplatform.

6 External users of the Neuroplatform

Access to the Neuroplatform is freely available for research purposes. For researchers lacking lab infrastructure, the Neuroplatform provides the capability to conduct real-time experiments on biological networks. Additionally, it allows others to replicate results obtained in their own lab. The database is shared between all research groups, however the Python scripts and Jupyter Notebooks are in private sections.

In 2023, 36 academic groups proposed research projects, of which 8 were selected. At the time of writing, 4 of these have already yielded some results:

• University Côte d’Azure, CNRS, NeuroMod Institute and Laboratoire JA Dieudonné: investigates the functional connectivity of FO and how electrical stimulation can modify it.

• University of Michigan, investigates stimulation protocols that induce global changes in electrical activity of a FO.

• Free University of Berlin, investigates stimulation protocols that induce changes in the electrical activity of a FO. Additionally, this research employs machine learning tools to extract information from neural firing patterns and to develop well-conditioned responses. Moreover, it utilizes both shallow and deep reinforcement learning techniques to identify optimal training strategies, aiming to elicit reproducible behaviors in the FO.

• University of Exeter, Department of Mathematics and Statistics, Living Systems Institute, investigates storing and retrieving of spatiotemporal spiking patterns, using closed-loop experiments that combine mathematical models of synaptic communication with the Neuroplatform.

• Lancaster University Leipzig and University of York: characterizes computational properties of FOs under the reservoir computing model, with a view to building low-power environmental sensors.

• Oxford Brookes University, School of Engineering, Computing and Mathematics: investigating the properties of emerging dynamics and criticality within neural organizations using the FOs.

• University of Bath, ART-AI, IAH: using the free energy principle and active inference to study the learning capabilities of neurons, embodied in a virtual environment.

• University of Bristol: stimulating of FOs based on data gathered from an artificial tactile sensor. Use machine learning techniques to interpret the FO’s output, investigating their ability to process real-world data.

7 Discussion and conclusion

The Neuroplatform has now been operational 24/7 for the past 4 years. During this time, the organoids on the MEA have been replaced over 250 times. Considering that we place at least 4 organoids per MEA, and change all the organoids simultaneously, this amounts to testing over 1,000 organoids. Initially, their lifetime was only a few hours, but various improvements, especially related to the microfluidics setup, have extended this to up to 100 days in best cases. It is important to note that the spontaneous activity of the organoids can vary over their lifetime, a factor that must be taken into consideration when conducting experiments ( Wagenaar et al., 2006 ). Additionally, we observed that the minimum current required to elicit spikes, computed using the method described in section 5.2, is increasing over the lifetime of the organoid. This phenomenon may be linked to an impedance increase caused by glial encapsulation ( Salatino et al., 2017 ).

The 24/7 recording strategy as described in section 4.2, results in the constant growth of the database. As of this writing, its size has reached 18 terabytes. This volume encompasses the recording of over 20 billion individual action potentials, each sampled at a 30 kHz resolution for 3 ms. This extensive dataset is significant not only due to its size but also because it was all recorded in a similar in-vitro environment, as described in section 3.2. We are eager to share this data with any interested research group.

8 Future extensions

In the future, we plan to extend the capabilities of our platform to manage a broader range of experimental protocols relevant to wetware computing. For example, we aim to enable a remote control over the injection of specific molecules into the medium, facilitating remote experiments that involve pharmacological manipulation of neuronal activity. This expansion will provide additional degrees of freedom for the automatic optimization of parameters influencing neuroplasticity.

Currently, as detailed in Chapter 2, only one differentiation protocol is used for generating organoids. We plan to introduce additional types of organoid generation protocols soon, with the aim of exploring a broader range of possibilities.

Although 32 research groups requested to access to the Neuroplatform, our current infrastructure only allows us to accommodate 7 groups, considering our own research needs as well. We are in the process of scaling-up the AC/DC hardware system to support more users simultaneously. Additionally, we are currently limited to executing close-loop algorithms for neuroplasticity on one single FO, as these algorithms require sending in real-time adapted simulation signals to each FO. Our software is being updated to run closed-loops in parallel on up to 32 FO.

9.1 Brain organoid generation

Human forebrain organoids were originated as described in Govindan et al. (2021) . Briefly, Human Neural Stem Cells derived from the human induced pluripotent stem (hiPS) cell line (ThermoFisher), were plated in flasks coated with CellStart (Fisher Scientific) and amplified in Stempro NSC SFM kit (ThermoFischer) complete medium: KnockOut D-MEM/F12, 2 mM of GlutaMAX, 2% of StemPro Neural supplement, 20 ng/mL of Human FGF-basic (FGF-2/bFGF) Recombinant Protein, and 20 ng/mL of EGF Recombinant Human Protein (Fisher Scientific). Cells were then detached with StemPro ™ Accutase (Gibco) and plated in p6 at the concentration of 250,000 cells/well. The plates were sealed with breathable adhesive paper and leads, placed on an orbital shaker at 80 rpm, and culture for 7 days at 37°C 5% CO2. After one week the newly formed spheroids were put in differentiation medium I (Diff I), containing DMEM/F-12, GlutaMAX ™ supplement (Gibco), 2% BSA, 1X of Stempro® hESC Supplement, 20 ng/mL of BDNF Recombinant Human Protein (Invitrogen), 20 ng/mL of GDNF Recombinant Human Protein (Gibco), 100 mM of N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt, and 20 mM of 2-Phospho-L-ascorbic acid trisodium salt. After one week, brain spheroids were put in differentiation medium II (Diff II) made of 50% of Diff I and 50% of Neurobasal Plus (Invitrogen). After 3 weeks of culture in Diff II, brain organoids were plated in Neurobasal Plus and kept in the orbital shaker until the transfer on the MEA. Medium was change once per week.

9.2 Electron microscopy analysis of FOs

Mature FOs were fixed in 2.5% Glutaraldehyde in 0.1 M phosphate buffer pH 7.4, at RT. After 24 h the samples were processed as described in Cakir et al. (2019) at the Electron Microscopy Facility of University of Lausanne. The whole FO images were acquired with Quanta FEG 250 Scanning Electron Microscope.

9.3 Transfer of FOs on MEA

MEA connected with the microfluid system was moved from the incubator and placed on a 12.3-megapixel camera system (with an optical lens of 16 mm of focal, giving a magnification power of 21x) inside the cell culture hood. The lid was removed to access the top of the liquid/air interface. Sterile Hydrophilic PTFE MEMBRANE Hole ‘confetti’ (diameter 2.5 mm, diameter of the hole 0.7 mm) (HEPIA) were positioned on top of each electrode and left there 2 min to absorb the medium. FOs were collected from the plate using wide bore pipette tips (Axygen) and placed in the middle of confetti, in a 10 μL drop of medium. The position of the organoids was adjusted with the help of sterile forceps. After all the organoids were put on place, the chamber was covered with the plate sealer Greiner Bio-One ™ BREATHseal ™ Sealer (Fisher Scientific), and with the MEA lid. MEA containing the organoids were placed immediately back in the cell incubator and were ready to be used for recording and stimulation. A similar procedure was used for the positioning of organoids on MCS MEA (60MEA200/30iR-Ti). In this case the Hydrophilic PTFE MEMBRANE was not used and organoids were directly laid on the electrodes in a 30 μL drop of medium. Recording of organoid activity was performed immediately afterwards.

9.4 System design and assembly

Cell culture media was stored in a 50 mL Falcon tube with a multi-port delivery cap (ElveFlow) and stored at 4°C. Each reservoir delivery cap contained a single 0.8 mm ID × 1.6 mm OD PTFE tubing (Darwin Microfluidics), sealed by a two-piece PFA Fittings and ferrule threaded adapter (IDEX), extending from the bottom of the reservoir to an inlet port on the 4-port valve head of the RVM Rotary Valve (Advance Microfluidics SA). Sterile air is permitted to refill the reservoir through a 0.22-μm filter (Milian) fixed to the cap to compensate for syringe pump medium withdrawal. A similar PTFE tubing and PFA Fittings and adapters were used to connect the syringe pump to the 4-port valve head of the RVM Rotary Valve (Advance Microfluidics SA). Each PTFE tubing coming from the distribution valve connects with a 50 mL falcon tube inside the cell culture incubator (Binder) and to a borosilicate glass bottle (Milian) to collect discarded cell culture medium.

A secondary microfluid system made of 0.8 mm ID × 1.6 mm OD PTFE tubing, were used to connect each 50 mL falcon tube inside the cell culture incubator with its own MEA (HEPIA). The connection was through a precise peristaltic pump BT100-2 J (Darwin Microfluidics) containing 10 rollers. A compute module (Raspberry Pi 4) controlled the peristaltic pump and the Rotary Valve, through a custom application program interface (API), using RS485 interface and RS-232 interface, respectively. A Fluigent flow-rate sensor connected via USB to the Raspberry Pi 4 allowed the monitoring of the flow rate inside the microfluidic system between the peristaltic pump and the MEA. Python was used to develop the software required to carry out automation protocols.

9.5 Uncaging of dopamine

Carboxynitroveratryl (CNV)-caged dopamine (Tocris Bioscience) was dissolved in Neurobasal Plus at the concentration of 1 mM, and injected in the fluidic system. After 3 h from the injection, the uncaging experiment started as described in paragraph 5.3. UV Silver-LED fiber-coupled LED (Prizmatix) was used to uncage the dopamine at the wavelength of 365 nm for 800 ms each time.

Data availability statement

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.

Author contributions

FJ: Writing – original draft, Writing – review & editing. MK: Writing – original draft, Writing – review & editing. J-MC: Writing – original draft, Writing – review & editing. FB: Writing – original draft, Writing – review & editing. EK: Writing – original draft, Writing – review & editing.

The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article.

Acknowledgments

We thank Steve M. Potter and Daniel Burger for their multiple advices and editing, as well as Mathias Reusser for the figures.

Conflict of interest

FJ, MK, J-MC, FB, and EK are employed at FinalSpark, Switzerland.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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Keywords: wetware computing, organoid intelligence, biocomputing, synthetic biology, AI, biological neural network, hybrot

Citation: Jordan FD, Kutter M, Comby J-M, Brozzi F and Kurtys E (2024) Open and remotely accessible Neuroplatform for research in wetware computing. Front. Artif. Intell . 7:1376042. doi: 10.3389/frai.2024.1376042

Received: 24 January 2024; Accepted: 11 March 2024; Published: 02 May 2024.

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Copyright © 2024 Jordan, Kutter, Comby, Brozzi and Kurtys. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Fred D. Jordan, [email protected]

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Intersection between the biological and digital: Synthetic Biological Intelligence and Organoid Intelligence

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Advances in stem cell research and therapeutic development

• Michele De Luca   ORCID: orcid.org/0000-0002-0850-8445 1   na1 ,
• Alessandro Aiuti 2 , 3   na1 ,
• Giulio Cossu 4   na1 ,
• Malin Parmar   ORCID: orcid.org/0000-0001-5002-4199 5 , 6   na1 ,
• Graziella Pellegrini 7   na1 &
• Pamela Gehron Robey 8   na1  

Nature Cell Biology volume  21 ,  pages 801–811 ( 2019 ) Cite this article

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Despite many reports of putative stem-cell-based treatments in genetic and degenerative disorders or severe injuries, the number of proven stem cell therapies has remained small. In this Review, we survey advances in stem cell research and describe the cell types that are currently being used in the clinic or are close to clinical trials. Finally, we analyse the scientific rationale, experimental approaches, caveats and results underpinning the clinical use of such stem cells.

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Acknowledgements

The authors would to thank the following parties, from whose work elements of our figures were modified; F. Aiuti (Fig. 2a ), A. De Luca (Fig. 3a ), and J. Drouin-Ouellet (Fig. 4 ). This work was partially supported by Regione Emilia-Romagna, Asse 1 POR-FESR 2007-13 to M.D.L. and G.P.; Italian Telethon Foundation to A.A.; Division of Intramural Research, National Institute of Dental Research, a part of the Intramural Research Program, the National Institutes of Health, Department of Health and Humman Services (ZIA DE000380 to P.G.R.), the Wellcome Trust (ME070401A1), the MRC (MR/P016006/1) the GOSH-SPARKS charity (V4618) to G.C.

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These authors contributed equally: Michele De Luca, Alessandro Aiuti, Giulio Cossu, Malin Parmar, Graziella Pellegrini, Pamela Gehron Robey.

Authors and Affiliations

Center for Regenerative Medicine “Stefano Ferrari”, Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy

Michele De Luca

San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) and Pediatric Immunohematology and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy

Alessandro Aiuti

Vita-Salute San Raffaele University, Milan, Italy

Division of Cell Matrix Biology and Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK

Giulio Cossu

Developmental and Regenerative Neurobiology, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund, Sweden

Malin Parmar

Lund Stem Cell Center, Lund University, Lund, Sweden

Center for Regenerative Medicine “Stefano Ferrari”, Department of Surgery, Medicine, Dentistry and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy

Graziella Pellegrini

National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA

Pamela Gehron Robey

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Corresponding author

Correspondence to Michele De Luca .

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Competing interests.

M.D.L. and G.P. are members of the Board of Directors of Holostem Terapie Avanzate Srl and consultant at J-TEC Ltd, Japan Tissue Engineering. A.A. is the principal investigator of clinical trials of HSC-GT for ADA-SCID, MLD and Wiskott–Aldrich, sponsored by Orchard Therapeutics. Orchard Therapeutic is the marketing authorization holder of Strimvelis in the European Union. M.P. is the owner of Parmar Cells AB and co-inventor of the US patent application 15/093,927 owned by Biolamina AB and EP17181588 owned by Miltenyi Biotec. M.P. is a New York Stem Cell Foundation Robertson Investigator.

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De Luca, M., Aiuti, A., Cossu, G. et al. Advances in stem cell research and therapeutic development. Nat Cell Biol 21 , 801–811 (2019). https://doi.org/10.1038/s41556-019-0344-z

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Accepted : 09 May 2019

Published : 17 June 2019

Issue Date : July 2019

DOI : https://doi.org/10.1038/s41556-019-0344-z

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How to make money from medical research and donations

• You can make thousands of dollars by donating some time or body parts to science.
• But these procedures are not all painless, and not everyone can participate.
• Below is a short list, though be warned: these strategies aren't all easy money.

Get dysentery. Play cards with someone who has the flu . Or, spend 45 days trapped in a tiny apartment with three total strangers.

These are just a few of the many ways you can get paid for helping out with scientific research. If you want to aid the science community and potentially save some lives while making a little extra cash, there are some unconventional options to consider.

Below is a short list, though be warned: these strategies aren't all exactly easy money.

Sell your blood plasma

Payout (per donation): around $50

Plasma is the largest component in human blood. It's a protein-rich liquid that contains mostly water but is also filled with enzymes, antibodies, and salts. This gooey, sticky yellow-ish stuff can be used to create therapies that treat people with blood clotting disorders, autoimmune diseases, and even burn victims. Donating plasma is often called "the gift of life" since treatments for some conditions can't be made synthetically and require this kind of human contribution.

During plasma donation, blood is drawn and an automated machine separates the plasma from other blood components, which are returned to the donor. Plasma donation pay varies from site to site, but the average payout is typically around $50 per donation. You can donate safely roughly once a month, according to the American Red Cross , and a typical session takes less than two hours. Find a licensed and certified plasma center near you.

Donate your sex cells

Payout for eggs (per donation): usually $10,000 to $12,000;

Payout for sperm (per donation): typically $35-$150

Egg and sperm donation can allow couples who have trouble conceiving naturally to become parents by using a donor's sex cells. But the time commitment and risk involved in a woman's egg donation is far steeper than what a man goes through donating his sperm.

In the United States, egg donors generally net around $10,000-$12,000. Weill Cornell outlines the standard steps for egg donation , which requires about a four-week time commitment.

During the egg donation cycle, patients are injected with fertility drugs so that the ovaries make more mature eggs than normal. (Eligible women are generally between the ages of 21 and 30). The egg retrieval procedure takes about 20 minutes but may require several days of recovery. Donors should be aware of the risks involved (largely related to the hormones used) before signing up.

Related stories

Men are generally paid anywhere from $35 to $150 per sperm donation, according to The Sperm Bank of California , but sperm donation can really start to add up if you regularly donate samples (many programs require a six-month or one-year donation commitment).

Donors should bear in mind that even if they choose to donate anonymously, sperm and egg donation is never really 100% incognito. Your DNA always knows who you are.

Spend 45 days on a fake spaceship

Payout: $160/day

NASA will pay you to spend 45 days traveling in space. Well, sort of.

You'll actually be on the ground the whole time in Houston, Texas, but you'll be locked inside a model space capsule (650 square feet) along with three strangers. This simulation is designed to study what being cooped up for a very long time inside a spaceship might do to a person, both physically and mentally. NASA wants to check this out thoroughly before they start sending astronauts on missions to Mars, or to explore faraway asteroids.

Participants in NASA's human research program share a capsule with each other that includes some workspace for doing lab experiments, a little kitchen table for eating meals that are just like what's served aboard the International Space Station, plus an exercise bike and some free weights. There's no internet, but you do get your own little cozy sleeping pod on the top floor.

The fake astronauts "on board" the capsule in May and June of 2024 include an aerospace engineering professor, a US Air Force Reserve member, a commercial pilot, and a biomedical engineer.

And that mission is nothing compared to NASA's CHAPEA Mars simulation , which keeps recruits in a simulated habitat of the red planet for 378 days. (NASA declined to comment on how much CHAPEA pays).

Take part in a clinical trial

Payout: Varies by program

The National Institutes of Health run a searchable database, ClincalTrials.gov , that rounds up human clinical studies ongoing around the world. Participants may be guinea pigs for new medical products, like drugs to treat high blood pressure, or they take part in observational research, like a study that records the effects of different lifestyles on heart health.

Subjects are generally paid to participate in such clinical trials, and most of the time, the bigger the risk, the bigger the reward. For example, a participant in one study in which participants were exposed to dysentery-causing bacteria was paid over $7,000, while a single blood draw or lab visit for a more straightforward study may only be worth $100 or so.

If you do decide to enroll in a study, choose wisely and carefully because not all of the studies on the site are regulated or evaluated for safety by the US Food and Drug Administration.

Enroll in a psychological study

Paid psychological studies, such as those that examine human behavior and brain function, may not generate as high of a return as clinical trials, but they are generally lower risk and require a shorter time commitment.

Most research universities keep an online database of studies so people can easily sign up. For example, here's a list of the most recent paid research studies offered by New York University . At NYU, you can make $12 an hour playing video games, and receive a $50 bonus if you're good at it.

Give your dead body to science

Payout: free cremation

This last idea is sort of morbid, but if you're worried about being a bother when you're dead, you can donate your body to science . This helps with various types of research and education.

Places like BioGift and Science Care will cover the costs of cremation, which can run upwards of $2,000.

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