These cells provide scientists with laboratory models of diseases such as Down syndrome and muscular dystrophy, and will help them find innovative ways to understand, prevent and treat such diseases. This work was recognized at the end of as contributing to the Breakthrough of the Year in Science magazine.
In the video above, George Daley is interviewed for a Science magazine video introducing cell reprogramming as its Breakthrough of the Year. Similar to the pioneering work of Andrew Lassar and Harold Weintraub from , this experiment shows it is possible to reprogram one type of adult cell into another type of adult cell, skipping the intermediary step of creating iPS cells.
This was the first FDA approval of a clinical trial for a therapy based on human embryonic stem cells. March 1, — Scientists in Toronto report the creation of iPS cells in their lab in a manner that is safer than previously used methods. These researchers are able to remove the genes necessary to reprogram an adult cell into a stem cell after the reprogramming step is complete.
March 9, — President Barack Obama signs Executive Order to repeal some of the restrictions on human embryonic stem cell research funds placed by the previous administration. The order requires the National Institutes of Health to draft new guidelines for federal funding policies within days. July 7, — The NIH issues the revised guidelines on federal funding for stem cell research. Included are strict provisions for informed donor consent and the ethical procurement of leftover embryos from in vitro fertilization.
These trials are being conducted in leukemia and lymphoma patients who have been implanted with blood stem cells from donated umbilical cords. If the trials are successful, single doses of umbilical cord blood stem cells , combined with PGE2, may be a viable source for blood stem cells for adult patients who cannot receive a bone marrow transplant.
Any scientist wanting to conduct research on any of these cell lines can now apply for federal funding. Read more in this blog post. Stem cells hold great promise and potential in the field of medicine, whether doctors inject them into patients to replace diseased bone marrow , or lab scientists scrutinize them under a microscope to see how lung cancer develops.
The road to innovation is long and full of obstacles, and there are plenty of questions left unanswered. But progress is ongoing and in many cases startling. How could one circumvent that procedure? Sir John Gurdon showed in the early s that, contrary to the prevalent belief back then, cells are not locked in their differentiation state and can be reverted to a more primitive state with a higher developmental potential. He demonstrated this principle by injecting the nucleus of a differentiated frog cell into an egg cell from which the nucleus had been removed.
This is commonly known as reproductive cloning, which was used to generate Dolly the Sheep. When allowed to develop, this egg gave rise to a fertile adult frog, proving that differentiated cells retain the information required to give rise to all cell types in the body. More than forty years later, Shinya Yamanaka and colleagues shocked the world when they were able to convert skin cells called fibroblasts into pluripotent stem cells by altering the expression of just four genes [].
This represented the birth of induced pluripotent stem cells, or iPS cells see Figure 1, right column. The enormous importance of these findings is hard to overstate, and is perhaps best illustrated by the fact that, merely six years later, Gurdon and Yamanaka shared the Nobel Prize in Physiology or Medicine [].
Today, these cells are the hope of personalized medicine, as they allow one to capture the unique genome of each individual in a cell type that can be used to generate, in principle, all cell types in our body, as illustrated on the right panel of Figure 1. The replacement of diseased tissues or organs without facing the barrier of immune rejection due to donor incompatibility thus becomes approachable in this era of iPS cells and is the object of intense research [].
The first proof-of-principle study showing that iPS cells can potentially be used to correct genetic diseases was carried out in the laboratory of Rudolf Jaenisch. In brief, tail tip cells from mice with a mutation causing sickle cell anemia were harvested and reprogrammed into iPS cells. The mutation was then corrected in these iPS cells, which were then differentiated into blood progenitor cells and transplanted back into the original mice, curing them []. Even though iPS cells have been found not to completely match ES cells in some instances, detailed studies have failed to find consistent differences between iPS and ES cells [].
This similarity, together with the constant improvements in the efficiency and robustness of generating iPS cells, provides bright prospects for the future of stem cell research and stem cell-based treatments for degenerative diseases unapproachable with more conventional methods. Leonardo M. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature The distribution of colony-forming cells among spleen colonies. J Cell Comp Physiol , 62 3 : Establishment in culture of pluripotential stem cells from mouse embryos.
Nature , — Nature Education , 1 1 Embryonic stem cell lines derived from human blastocysts. Science , : The hope that stem cells could provide potential therapies in regenerative medicine prompted further research toward the directed differentiation of hESCs toward specialized cell types Trounson, Derivation reports showed that suboptimal culture conditions led to spontaneous differentiation of hESCs Reubinoff et al. Subsequently, Schuldiner et al. This work provided a valuable framework for the directed derivation of a number of specialized cell types from hESCs, including mature neurons Reubinoff et al.
These early reports were fundamental for further in vitro manipulations of pluripotent stem cells PSCs , setting the stage for future clinical applications. The profound impact of iPSC technology on the study of cell biology, and especially nuclear reprogramming, was recognized when Dr S. Yamanaka, along with Dr J. Interestingly, when asked about the name iPSC, S. The initial methods for iPSC derivation used retroviral or lentiviral vectors to deliver the four reprogramming factors Takahashi et al.
To overcome this issue, various non-integrating methods for human iPSC generation have been developed, including episomal DNA plasmids Okita et al. Several cell sources and combination of fewer, or different, factors have also been successfully used for reprogramming, making it safer and more efficient in the process reviewed in Takahashi and Yamanaka, One of the main advantages of human iPSCs is their potential to model disease in vitro. Already in , the derivation of patient-specific iPSCs from a child with spinal muscular atrophy showed disease-related deficits in the motor neurons generated in vitro Ebert et al.
Since then, an increasing number of disease models with iPSCs have been generated, especially for monogenic diseases, such as Rett syndrome Marchetto et al.
In addition, iPSCs hold great promise for developing personalized treatments. Furthermore, iPSCs may be used in regenerative medicine either as an autologous cell source or as a HLA-matched allogeneic cell source for transplantation, minimizing the risk of rejection and the use of long-term immunosuppression.
In , the first clinical study using hiPSC-derived cells was initiated Fig. Somatic cell nuclear transfer SCNT , colloquially known as cloning, is the process of transferring the nuclear DNA of a donor somatic cell into an enucleated oocyte, followed by embryo development Fig.
When the SCNT embryo is transferred to a surrogate recipient with the aim to achieve a live birth, the process is defined as reproductive cloning. The first success in mammals was achieved with the birth of Dolly the sheep in Wilmut et al.
This successful attempt proved that it is possible to revert the differentiated status of the somatic nucleus to totipotency reprogramming Wilmut et al. However, when pluripotent SCNT stem cells are harvested from the reconstructed SCNT embryo, the process is called therapeutic cloning, aiming at deriving pluripotent stem cells for future cell therapy and research purposes Fig.
The advantage of therapeutic cloning over ESCs is that SCNT stem cells, like iPSCs, are genetically identical to the somatic cell they are derived from, thereby overcoming immune rejection, inherently valuable for future clinical applications.
Somatic cell nuclear transfer was first attempted in amphibians due to the comparatively large size of the eggs, enabling easier micromanipulation coupled with the possibility of using considerable numbers of eggs and embryos. Tadpoles developed following transfer of nuclei from early cleavage stage embryos to enucleated eggs Briggs and King, Subsequently, the group of Dr J. Gurdon Gurdon et al. This was the first experiment to show that differentiated cells could be set back to an embryonic state.
Since Dolly, several attempts have been made to generate SCNT-ESCs in several mammalian species, due to their potential benefits in biomedical applications such as allo-transplantation and personalized drug selection Matoba and Zhang, These attempts have further enabled optimization of the SCNT process, including cell cycle synchronization between donor cells and recipient oocytes, erasure of epigenetic marks by using donor cells of varying ages and from different tissues, as well as the addition of small molecules and the modification of culture conditions Akagi et al.
The first primate SCNT-ESCs were derived in the rhesus macaque from adult skin fibroblasts, partly owing to the non-invasive removal of the spindle-chromosome complex by polarized microscopy Byrne et al. Although very successful in all species tested, pseudoblastocyst development following human SCNT was not achieved, with most SCNT embryos arresting at the stage of embryonic genome activation Heindryckx et al. The key to success was minor SCNT technological adjustments and the use of in vivo matured oocytes from young donors.
Subsequently, Noggle and collaborators adjusted the conventional SCNT approach by transferring the somatic nucleus into a non-enucleated recipient oocyte. The group of Dr S. Mitalipov Tachibana et al. We then used such skin-derived oocytes to develop into blastocysts and ESCs.
The biggest hurdle for human SCNT applications remains the scarcity of human oocytes. However, most of these lines are not suitable for clinical use as they have been derived and maintained in complex and poorly defined culture systems containing several xenogeneic components.
Conversely, clinical-grade hPSC need to be generated and maintained in fully defined, xeno-free culture conditions, in compliance with current good manufacturing practices GMPs. The importance of clinical-grade cells has been recognized for several years now, as expressed in the words of pioneer Dr O.
Hence, we decided to develop better derivation systems. In fact, as mentioned by Dr O. Hovatta, the first methods described for the derivation of hESC entailed the use of mouse feeder cell layers and medium containing fetal bovine serum FBS , two factors that may contain animal pathogens and immunogens. FBS can be replaced by Knock-Out Serum Replacement SR , which contains several amino acids, vitamins, antioxidants and trace elements, but also proteins including lipid-rich albumin which seems to play an important role in hPSC self-renewal Garcia-Gonzalo and Izpisua Belmonte, Although more defined than FBS, SR is still xenogeneic and therefore maintains the risk for pathogenic contamination of hPSCs, which may be transmitted to patients upon transplantation Martin et al.
The use of human cells, mainly foreskin fibroblast cells, as feeder layers was the first important step toward xeno-free hPSC cultures Richards et al.
However, if used for clinical-grade hPSC derivation and culture, the human feeder cells must also be produced under current GMP conditions Prathalingam et al. The first feeder-free hESC derivation was reported in , using extracellular matrix extracted from mouse embryonic fibroblasts Klimanskaya et al. They applied a serum-free medium with high concentrations of basic fibroblast growth factor bFGF to support hESC growth in the absence of fibroblasts.
The formulation of the medium was created by testing the effect on hESC marker expression after the systematic addition of growth factors. Ludwig and colleagues also generated an artificial human extracellular matrix with a combination of human collagen IV, laminin, fibronectin and vitronectin to support hESC derivation and long-term culture. These conditions, for the first time, completely eliminated the use of animal products Ludwig et al. Further progress in the study of the interactions between different media components led to the formulation of a completely chemically-defined albumin-free medium, composed of eight factors: Essential 8 medium E8.
The first surface coatings used to substitute feeder cells were often protein mixtures obtained from cell cultures from which the exact composition can vary significantly from lot to lot Klimanskaya et al. During their search for more defined coatings, several groups identified extracellular matrix proteins with primary roles in supporting hPSC self-renewal and pluripotency. The recombinant forms of these proteins were then used for derivation and maintenance of hPSC lines.
Known examples are vitronectin, laminin and fibronectin Braam et al. In , Rodin et al. Finally, physical methods have also been used for surface optimization. One of the most crucial points of improvement for the future will be the upscaling of hPSC cultures through 3D systems.
Large-scale hPSC production will be essential for their successful application in regenerative medicine. The number of cells required for effective cell therapy treatment varies according to the therapeutic goal but is expected to range between millions and billions of cells Serra et al.
While the 2D culture systems remain time-consuming and labor-intensive, 3D automated culture systems, e. To avoid hydrodynamic stress and agglomeration of growing spherical aggregates, researchers have attempted to encapsulate hPSCs in hydrogel scaffolds Li et al.
With this last approach, systems can be set up that are simple, scalable, highly efficient, defined and GMP-compatible. An important aspect to keep in mind is that culture conditions may affect the genetic and epigenetic stability of the hPSC cells. An elegant study by Jacobs et al. Highly recurrent chromosomal abnormalities over culture passages have been reported by various labs worldwide, reflecting the progressive adaptation of hPSCs to culture conditions and the culture advantage conferred to the cells by these genetic changes Spits et al.
An example is the duplication of the long arm of chromosome Similarly, also at the epigenetic level, the loss of DNA methylation and specific histone modifications has been linked to suboptimal culture conditions Nazor et al.
In the past two decades, insights into early embryo development have broadened our perception of pluripotency. As such, pluripotency is no longer viewed as a fixed state but rather a highly dynamic, malleable signaling network Wu and Izpisua Belmonte, ; Weinberger et al. Unraveling the complete potency spectrum and its transitions will remain central to our understanding of lineage commitment. Derived from the ICM of mouse blastocysts, mESCs demonstrated characteristic features of pluripotency, including long-term self-renewal, ability to differentiate toward all germ layers, high single-cell clonogenicity and efficient contribution to chimeras Evans and Kaufman, ; Martin, Fig.
It soon became evident that hESCs rely on different signaling pathways to maintain pluripotency Vallier et al. States of pluripotency: primed vs naive.
Some years later, mouse epiblast stem cells mEpiSCs were isolated from post-implantation embryos and were found to share many similarities with hESCs Brons et al. Their transcriptome was similar to that of the post-implantation epiblast Brons et al. Subsequently, two states of pluripotency were proposed: naive and primed Nichols and Smith, Fig.
Accordingly, mESCs exist in a naive state, which constitutes the functional in vitro equivalent of the preimplantation epiblast, while hESCs are in a primed state. The naive or ground state of pluripotency is characterized by a seemingly unbiased differentiation potential, low variability in pluripotency linked gene expression, global DNA hypo-methylation and two active X-chromosomes in female cells Nichols and Smith, ; Hackett and Surani, ; Davidson et al.
Conversely, cells in the primed state display distinct pluripotency associated gene patterns, DNA hyper-methylation, X-chromosome inactivation and inefficiency in forming chimeras; this state corresponds to the transition of naive epiblast cells toward a more committed state in vivo Nichols and Smith, ; Hackett and Surani, Fig. The notion that pluripotency exists in at least two distinct forms prompted further research toward the identification of in vitro culture conditions that stabilize the naive state in humans.
However, the resulting naive hESCs required continued expression of integrated transgenes for long-term self-renewal. To address this limitation, several groups attempted to modify the cell culture conditions in order to induce naive characteristics in hESCs Xu et al. Soon after, optimized chemically defined conditions were established, with the naive human stem cell medium NHSM allowing rapid conversion of primed hESCs to the naive state Gafni et al. In a more systematic approach, Theunissen et al.
However, several hESC lines presented with an abnormal karyotype, leading to the notion that naive hPSCs may be more prone to genomic instability in culture. Takashima et al. To date, several other conversion protocols have been established Chan et al.
The generation of naive hESCs allowed for their molecular signature to be described in detail. However, the methods used to generate naive hESCs vary considerably, restricting the primed state at different molecular levels. In the embryo, the transition to lineage commitment is highly efficient Smith, , yet during in vitro culture, altered conditions may modify this progression, contributing to inconsistent and inefficient directed differentiation Warrier et al.
The possibility to obtain different types of stem cells illustrates that pluripotency is highly determined by the synergistic interplay between embryo developmental stage and the micro-environment.
Considering future therapeutic applications, unraveling the full potency spectrum of hESCs may enhance efficiency and control over directed differentiation. Exploring cellular plasticity during embryogenesis and recapitulating this potential in vitro will undoubtedly have profound effects on both the reproductive and stem cell fields. To date, hPSCs have been differentiated to many cell types, either by directed differentiation to a specific cell type of interest, for example cardiomyocytes, dopaminergic neurons or pancreatic beta-cells or by undirected differentiation for example in embryoid bodies.
For some purposes, the generation of a single-cell type may be desirable e. However, the initial population of PSCs is usually rather heterogeneous, hence differentiation protocols need constant optimization to increase their efficiency. Scientists are still attempting to identify factors that may be used to mature the cells further. One such initiative is KeyGenes, a platform that includes transcriptomics data of human fetal and adult organs and compares the transcriptional profile of differentiated cells to in vivo organs at different developmental stages Roost et al.
For example, this platform was used to demonstrate the fidelity of differentiation of kidney-organoids to the kidney, providing fast and reliable quantification of the differentiation protocol used and revealing that after 16 days of differentiation the organoids resembled first trimester kidneys Takasato et al.
Another initiative that will surely contribute to increase our understanding of specific cell types and signals produced at every stage of maturation to help improve the quality and efficiency of differentiation protocols is the Human Cell Atlas project Rozenblatt-Rosen et al. More recently, there has been a significant effort to generate human organoids or mini-organs by taking advantage of patient-specific PSCs for drug testing and disease modeling.
Organoids are typically free-floating hollow spheres made of cells, with self-organizing and self-patterning properties that recapitulate some aspects of development and organ morphogenesis. Typically, they have epithelial or epithelial and mesenchymal architectural compartments, with the apical part of the epithelial compartment on the inside bordering a fluid-filled cavity. Organoids are usually maintained long-term in defined culture medium on Matrigel-scaffolds.
However, Matrigel is an undefined product, making organoids unsuitable for regenerative medicine applications. One of the advantages of organoids is the potential for scaling up at an industrial level for translational applications. With the current screening technologies, organoid systems could be exploited to model patient-specific drug response to tumorigenesis Arai et al. In addition to organoids, efforts are also underway to generate fully grown organs in interspecies-chimeras organism composed of a mix of at least two genetically distinct cells , including human—animal chimeras, broadening the application of patient-specific PSCs.
This successful application was taken a step further and the technology to generate pig blastocyst mutants for PDX1 was subsequently developed Matsunari et al. Next steps include investigating whether human PSCs have the potential to integrate into mutant pig blastocysts, such that a human pancreas can be developed in the pig. However, recent work introducing rat and human PSCs in porcine and bovine blastocysts has demonstrated limited integration and development of chimeric pig embryos until mid-gestation Wu et al.
Producing gametes in vitro by directed differentiation of hPSCs represent a complex goal, which may be used in the future to provide for an unlimited source of in vitro -produced gametes for toxicology and pharmacology studies, but possibly also for granting certain infertile couples the possibility to have genetic children. Germ cell development is a highly orchestrated process, controlled by a unique set of genetic and epigenetic regulators, taking into account important variables like primordial germ cell PGC state, meiosis and gonadal somatic cells with important differences between mouse and human models Eguizabal et al.
Thus far, researchers have been successful in deriving in vitro germ cells from mouse PSCs, however many obstacles remain to be overcome for the robust generation of mature gametes in humans Yamashiro et al. Also the PGC state can be reached from different pluripotency states primed and naive Mitsunaga et al. The second step for male germ cell differentiation is reaching the post-meiotic stage of spermatozoa.
Unfortunately, differentiation past the PGC stage has proven challenging in human. One of the strategies employed includes supplementing the differentiation medium with growth factors such as bone morphogenetic protein BMP -4 Tilgner et al. Recently, hiPSCs from azoospermic and normospermic males were directly transplanted into mouse testis, partially colonizing the testicular niche and showing signs of early spermatogenesis Ramathal et al.
In a mouse model, it has been demonstrated that transplanting in vitro -produced PGC-like cells from PSCs into mouse testis resulted in functional sperm, capable of producing healthy and fertile offspring Hayashi et al.
So far, the need of a natural testicular niche to obtain mature functional spermatozoa is still present, and in vitro production of spermatozoa is currently not possible in humans Zhou et al. In , Dr H. Several years later, in , Hayashi et al. PGC-derived immature oocytes were obtained and were subsequently matured and fertilized in vitro and the resulting embryos were transplanted into foster mothers Hayashi et al.
Later, Hikabe et al. Hayashi's earlier results Hikabe et al. Recently, Jung et al. Jung et al. The follicle-like cells derived resembled in vivo primordial follicle, as proven by transplantation experiments. At present, robust in vitro production of functional human oocytes from pluripotent cells remains a distant prospect. Over the past decades, several gene editing strategies have been employed in hESCs and hiPSCs, each with benefits and limitations.
The first efforts to manipulate the hESC genome involved untargeted transgenic approaches, used to monitor cellular differentiation Eiges et al. Bacterial plasmids or viral vectors were used to randomly integrate reporter genes, using cell-specific promoters to drive expression Eiges et al. Similarly, a number of fluorescent reporter lines were generated to monitor differentiation toward specific lineages, allowing for the identification of a variety of hESC derivatives Lavon et al.
Homologous recombination HR -based gene editing allowed integration of a nucleotide sequence into a specific site within the hESC genome Mansour et al. HR-mediated methods utilize the innate DNA repair machinery of the cell to alter or replace a specific nucleotide sequence by a homologous one Leavitt and Hamlett, ; Brookhouser et al.
Although these reports demonstrated the viability of HR-based gene editing they also underscored the technical challenges involved. Poor single-cell survival resulted in considerable cell death and low transfection efficiencies in hESCs Zwaka and Thomson, ; Urbach et al.
Moreover, as recombination activity is heavily dependent on cell type and cell cycle, applications of this method were relatively limited Eid and Mahfouz, ; Chandrasekaran et al. There have been several reports demonstrating the efficacy and value of all three approaches for editing hPSC genomes. ZFN applications range from the introduction of reporter genes to monitor pluripotency and differentiation Hockemeyer et al.
Unlike ZFNs and TALENs, limited by their high cost and poorer specificity, Cas nucleases rapidly became the preferred enzymes for genome editing due to their higher efficiency and versatility Plaza Reyes and Lanner, To explore underlying mechanisms of gene regulation, Liao et al. The HDR-based approach may also be useful in cases where generating patient-specific hiPSCs is unfeasible, creating opportunities for studying a wide range of genetic pathologies Zhang et al.
Along with its success, several limitations remain to be overcome prior to clinical applications of gene editing in hPSCs. Off target effects, resulting from the random integration of nucleotides, still persist, while exploring novel safe delivery strategies is also necessary Zhang et al. Moreover, the safety and efficacy of the edited cells require evaluation prior to implementation in a clinical setting. Elucidating the full extent of off target effects and improving editing efficiencies in hPSCs will require constant innovation in both gene editing and stem cell research.
Nevertheless, the complementary nature of these two fields has certainly allowed remarkable progress. Manipulating the hPSC genome to unravel gene function and underlying processes of human development will continue to enhance stem cell technologies.
Concurrently, disease modeling and therapeutic approaches will further foster the ultimate vision of clinical applications through personalized regenerative medicine. Cell therapies with hPSC are emerging as a possible solution to degenerative diseases. As Dr P. Clinical trials with cells derived from human embryonic stem cell and human-induced pluripotent stem cells.
Most of the information regarding the results of clinical trials with PSCs in this section have been obtained from sponsor company press releases or press articles. Whenever the results arise from a scientific publication, the reference is provided. The first clinical trial with cells from hPSCs was launched in for patients with spinal cord injury Fig. Geron corp. One year later, the company canceled the trial for economic reasons, reporting that no significant side effects were observed in any of the five patients treated.
Preliminary data reported by the company at the ISCCR meeting in June showed cavitation, improved myelin coating, neovascularization and the production of neuronal growth stimulating factors, in addition to the absence of relevant side effects and good tolerance for the immunosuppressive treatment.
ACT has published preliminary Schwartz et al. One year post-treatment, the best corrected visual acuity BCVA improved from 10 to 39 and from 8 to 29 letters in Patients 1 and 2, respectively.
In an article published recently in Ophtalmology Mehat et al. Borderline improvements in BCVA in four participants either were unsustained or were matched by a similar improvement in the untreated contralateral eye.
Microperimetry demonstrated no evidence of benefit at 12 months in the 12 participants. In the Chinese Clinical Trial Register www. The preliminary results of the first treated patient were published by Menasche et al.
This device, implanted subcutaneously, allows the entry of oxygen and nutrients, as well as the release of insulin and other hormones, while protecting the cells from the autoimmune reaction that causes type 1 diabetes. The company announced in August two more trials with a modified device VC that allows direct vascularization into itself. After 6 months of treatment, a reduction of the off period was observed in the treated patients, as well as an improvement in motor and cognitive abilities.
The patient did not present any side effects after the treatment. The trial was suspended when attempting to treat a second patient as mutations were detected in the generated hiPSCs Mandai et al. Due to these findings and to the fact that the generation of patient-specific iPSCs is time-consuming and expensive, Riken opted for the use of tested and safe allogenic hiPSCs.
A new clinical trial has been initiated involving the generation of a hiPSC bank from peripheral blood samples with the most frequent homozygous HLA haplotypes that could match a sufficient proportion of the general population Okita et al. To date, no results have been published so far. Clinical trials registered in Clinical Trials. Lead author Dr H. Obokata was accused of misconduct and in August the papers were retracted.
This purported breakthrough was published in Science , however both papers were soon retracted based on suspicions of fraud.
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