Cell Replacement Therapy For Type Research Articles

Brown Adipose Tissue as a Unique Niche for Islet Organoid Transplantation: Insights From In Vivo Imaging.

Transplantation of human-induced pluripotent stem cell (hiPSC)-derived islet organoids is a promising cell replacement therapy for type 1 diabetes (T1D). It is important to improve the efficacy of islet organoids transplantation by identifying new transplantation sites with high vascularization and sufficient accommodation to support graft survival with a high capacity for oxygen delivery. A human-induced pluripotent stem cell line (hiPSCs-L1) was generated constitutively expressing luciferase. Luciferase-expressing hiPSCs were differentiated into islet organoids. The islet organoids were transplanted into the scapular brown adipose tissue (BAT) of nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice as the BAT group and under the left kidney capsule (KC) of NOD/SCID mice as a control group, respectively. Bioluminescence imaging (BLI) of the organoid grafts was performed on days 1, 7, 14, 28, 35, 42, 49, 56, and 63 posttransplantation. BLI signals were detected in all recipients, including both the BAT and control groups. The BLI signal gradually decreased in both BAT and KC groups. However, the graft BLI signal intensity under the left KC decreased substantially faster than that of the BAT. Furthermore, our data show that islet organoids transplanted into streptozotocin-induced diabetic mice restored normoglycemia. Positron emission tomography/MRI verified that the islet organoids were transplanted at the intended location in these diabetic mice. Immunofluorescence staining revealed the presence of functional organoid grafts, as confirmed by insulin and glucagon staining. Our results demonstrate that BAT is a potentially desirable site for islet organoid transplantation for T1D therapy.

Pig Xenotransplantation in Beta Cell Replacement: Addressing Challenges and Harnessing Potential for Type 1 Diabetes Therapy.

This opinion paper evaluates the potential of porcine islets as a promising alternative in beta cell replacement therapy for Type 1 Diabetes (T1D), juxtaposed with the current limitations of human donor islets. It analyzes the compatibility of pig islets with human glucose metabolism, their prospects as a limitless and high-quality source of beta cells, and the unique immunogenic challenges they present in xenotransplantation. Additionally, the paper discusses the regulatory and ethical considerations pertinent to the use of porcine islets. By synthesizing current research and expert perspectives, the paper highlights both the opportunities and significant barriers that need addressing to advance pig islets as a viable therapeutic option. The findings advocate for a balanced and forward-looking approach to the integration of pig islets in T1D treatment, underscoring the need for continued research and dialogue in this evolving field.

Cell Replacement Therapy for Type 1 Diabetes Patients: Potential Mechanisms Leading to Stem-Cell-Derived Pancreatic β-Cell Loss upon Transplant.

Cell replacement therapy using stem-cell-derived insulin-producing β-like cells (sBCs) has been proposed as a practical cure for patients with type one diabetes (T1D). sBCs can correct diabetes in preclinical animal models, demonstrating the promise of this stem cell-based approach. However, in vivo studies have demonstrated that most sBCs, similarly to cadaveric human islets, are lost upon transplantation due to ischemia and other unknown mechanisms. Hence, there is a critical knowledge gap in the current field concerning the fate of sBCs upon engraftment. Here we review, discuss effects, and propose additional potential mechanisms that could contribute toward β-cell loss in vivo. We summarize and highlight some of the literature on phenotypic loss in β-cells under both steady, stressed, and diseased diabetic conditions. Specifically, we focus on β-cell death, dedifferentiation into progenitors, trans-differentiation into other hormone-expressing cells, and/or interconversion into less functional β-cell subtypes as potential mechanisms. While current cell replacement therapy efforts employing sBCs carry great promise as an abundant cell source, addressing the somewhat neglected aspect of β-cell loss in vivo will further accelerate sBC transplantation as a promising therapeutic modality that could significantly enhance the life quality of T1D patients.

β-cell replacement therapy for type 1 diabetes: closer and closer.

β-cell replacement therapy for type 1 diabetes: closer and closer.

Generation of a heterozygous GAPDH-Luciferase human ESC line (HVRDe008-A-1) for in vivo monitoring of stem cells and their differentiated progeny

Human stem cell-derived beta (SC-β) cells are a candidate for cell replacement therapy for type 1 diabetes. Whilst refinements to the differentiation protocol have resulted in the production of SC-β cells that resemble adult beta cells, the unsolved challenge to protect transplanted SC-β cells from the host immune system remains. To monitor the survival of SC-β cells in vivo, we knocked-in the Firefly luciferase gene into the GAPDH locus of the HUES8 human embryonic stem cell (hESC) line, such that differentiated islet cells constitutively express luciferase.

Early-Phase Luciferase Signals of Islet Grafts Predicts Successful Subcutaneous Site Transplantation in Rats.

The transplantation of pancreatic islets is a promising cell replacement therapy for type 1 diabetes. Subcutaneous islet transplantation is currently under investigation as a means to circumvent problems associated with standard intra-hepatic islet transplantation. As modifications are being developed to improve the efficacy of subcutaneous islet transplantation, it is important to have robust methods to assess engraftment. Experimentally, ATP-dependent bioluminescence imaging using luciferase reporter genes has been effective for non-invasively tracking engraftment. However, it was heretofore unknown if the bioluminescence of subcutaneously transplanted luciferase-expressing islet grafts correlates with diabetes reversal, a primary outcome of transplantation. A retrospective analysis was conducted using data obtained from subcutaneous islet transplantations in Lewis rats. The analysis included transplantations from our laboratory in which islet donors were transgenic rats ubiquitously expressing luciferase and recipients were wild type, streptozotocin-induced diabetic rats. Data from 79 bioluminescence scans were obtained from 27 islet transplantations during the post-transplant observation period (up to 6 weeks). The bioluminescence intensity of the subcutaneously transplanted grafts, captured after the intravenous administration of luciferin, was correlated with diabetes reversal. After subcutaneous transplantation, islet bioluminescence decreased over time, dropping > 50 % from 1 to 3 weeks post-transplant. Bioluminescence intensity in the early post-transplant phase (1-2 weeks) correlated with the subsequent reversal of diabetes; based on optimized bioluminescence cutoff values, the bioluminescence intensity of islets at 1 and 2 weeks predicted successful transplantations. However, intensity in the late post-transplant phase (≥ 4 weeks) did not reflect transplantation outcomes. Early-phase bioluminescence imaging of luciferase-expressing islets could serve as a useful tool to predict the success of subcutaneous islet transplantations by preceding changes in glucose homeostasis.

Pancreatic islet transplantation: toward definitive treatment for diabetes mellitus.

Since the late 20th century, advances in pancreatic islet transplantation have targeted improved glycemic control and fewer hypoglycemic events in patients with type 1 diabetes, and some important milestones have been reached. Following the Edmonton group's success in achieving insulin independence in all transplanted patients with type 1 diabetes, clinical islet transplantation is now performed worldwide. β cell replacement therapy for type 1 diabetes was established based on the favorable outcomes of a phase 3, prospective, open-label, single-arm, clinical study conducted at 8 centers in North America, in which 42 of 48 patients who underwent islet transplantation from 2008 to 2011 achieved HbA1c < 7.0% (53 mmol/mol) at day 365, which was maintained at 2 years in 34 patients. In Japan, a phase 2 multicenter clinical trial of islet transplantation for type 1 diabetes patients is currently ongoing and will end soon, but the interim results have already led to positive changes, with allogeneic islet transplantation being covered by the national health insurance system since April 2020. Current efforts are being made to solve the problem of donor shortage by studying alternative donor sources, such as porcine islets and pancreatic progenitor cells derived from pluripotent stem cells. The results of clinical trials in this area are eagerly awaited. It is hoped that they will contribute to establishing alternative sources for insulin-producing β cells in the near future.

MicroRNA-181c-5p promotes the formation of insulin-producing cells from human induced pluripotent stem cells by targeting smad7 and TGIF2

Generating insulin-producing cells (IPCs) from human pluripotent stem cells is a promising method for studying the molecular mechanism underlying pancreas development and a potential treatment source for type 1 diabetes. Previous studies have shown that miR-181c-5p is highly enriched in adult islets; however, its role in pancreatic β cell differentiation is poorly understood. In this study, we differentiated human induced pluripotent stem cells (hiPSCs) into IPCs in a stepwise process that recapitulated pancreas organogenesis and observed that miR-181c-5p continuously accumulated throughout the entire differentiation process. hiPSCs were transduced with lentiviral vectors containing human miR-181c-5p precursor, which significantly increased the endodermal markers SOX17, FOXA2, CXCR4 and GATA4 and pancreatic endocrine-specific gene expression, including PDX1, NKX6.1, MAFA and Insulin. miR-181c-5p overexpression exerted little effect on the efficiency of definitive endoderm, whereas it promoted the differentiation of pancreatic progenitors and IPCs, especially for NKX6.1-positive and insulin-positive cells differentiation. Transplanted these cells exhibit glucose-stimulated C-peptide secretion in vivo and protect mice from chemically induced diabetes. It was found that miR-181c-5p directly targets the 3′UTR of smad7 and TGIF2 mRNA, which are known to be endogenous repressors of TGF-β-smad2/3 signaling, to decrease their mRNA and protein levels. Furthermore, overexpressed miR-181c-5p led to an elevation of the smad2/3 phosphorylation levels in hiPSC-derived cells, while treatment with smad2/3 inhibitors following miR-181c-5p overexpression had opposite effects on IPC formation. These results suggest that miR-181c-5p is critically involved in pancreatic lineage commitment through direct repression of smad7 and TGIF2 and that it modulates TGF-β-smad2/3 signaling activation and increases the feasibility of using patient-specific hiPSCs for β cell replacement therapy for type 1 diabetes.

Understanding and Modulating Immunity With Cell Reprogramming.

Cell reprogramming concepts have been classically developed in the fields of developmental and stem cell biology and are currently being explored for regenerative medicine, given its potential to generate desired cell types for replacement therapy. Cell fate can be experimentally reversed or modified by enforced expression of lineage specific transcription factors leading to pluripotency or attainment of another somatic cell type identity. The possibility to reprogram fibroblasts into induced dendritic cells (DC) competent for antigen presentation creates a paradigm shift for understanding and modulating the immune system with direct cell reprogramming. PU.1, IRF8, and BATF3 were identified as sufficient and necessary to impose DC fate in unrelated cell types, taking advantage of Clec9a, a C-type lectin receptor with restricted expression in conventional DC type 1. The identification of such minimal gene regulatory networks helps to elucidate the molecular mechanisms governing development and lineage heterogeneity along the hematopoietic hierarchy. Furthermore, the generation of patient-tailored reprogrammed immune cells provides new and exciting tools for the expanding field of cancer immunotherapy. Here, we summarize cell reprogramming concepts and experimental approaches, review current knowledge at the intersection of cell reprogramming with hematopoiesis, and propose how cell fate engineering can be merged to immunology, opening new opportunities to understand the immune system in health and disease.

Reprogramming human gallbladder cells into insulin-producing β-like cells.

The gallbladder and cystic duct (GBCs) are parts of the extrahepatic biliary tree and share a common developmental origin with the ventral pancreas. Here, we report on the very first genetic reprogramming of patient-derived human GBCs to β-like cells for potential autologous cell replacement therapy for type 1 diabetes. We developed a robust method for large-scale expansion of human GBCs ex vivo. GBCs were reprogrammed into insulin-producing pancreatic β-like cells by a combined adenoviral-mediated expression of hallmark pancreatic endocrine transcription factors PDX1, MAFA, NEUROG3, and PAX6 and differentiation culture in vitro. The reprogrammed GBCs (rGBCs) strongly induced the production of insulin and pancreatic endocrine genes and these responded to glucose stimulation in vitro. rGBCs also expressed an islet-specific surface marker, which was used to enrich for the most highly reprogrammed cells. More importantly, global mRNA and microRNA expression profiles and protein immunostaining indicated that rGBCs adopted an overall β-like state and these rGBCs engrafted in immunodeficient mice. Furthermore, comparative global expression analyses identified putative regulators of human biliary to β cell fate conversion. In summary, we have developed, for the first time, a reliable and robust genetic reprogramming and culture expansion of primary human GBCs—derived from multiple unrelated donors—into pancreatic β-like cells ex vivo, thus showing that human gallbladder is a potentially rich source of reprogrammable cells for autologous cell therapy in diabetes.

Erythropoietin facilitates definitive endodermal differentiation of mouse embryonic stem cells via activation of ERK signaling.

Artificially generated pancreatic β-cells from pluripotent stem cells are expected for cell replacement therapy for type 1 diabetes. Several strategies are adopted to direct pluripotent stem cells toward pancreatic differentiation. However, a standard differentiation method for clinical application has not been established. It is important to develop more effective and safer methods for generating pancreatic β-cells without toxic or mutagenic chemicals. In the present study, we screened several endogenous factors involved in organ development to identify the factor, which induced the efficiency of pancreatic differentiation and found that treatment with erythropoietin (EPO) facilitated the differentiation of mouse embryonic stem cells (ESCs) into definitive endoderm. At an early stage of differentiation, EPO treatment significantly increased Sox17 gene expression, as a marker of the definitive endoderm. Contrary to the canonical function of EPO, it did not affect the levels of phosphorylated JAK2 and STAT5, but stimulated the phosphorylation of ERK1/2 and Akt. The MEK inhibitor U0126 significantly inhibited EPO-induced Sox17 expression. The differentiation of ESCs into definitive endoderm is an important step for the differentiation into pancreatic and other endodermal lineages. This study suggests a possible role of EPO in embryonic endodermal development and a new agent for directing the differentiation into endodermal lineages like pancreatic β-cells.

Adult Murine Pancreatic Progenitors Require Epidermal Growth Factor and Nicotinamide for Self-Renewal and Differentiation in a Serum- and Conditioned Medium-Free Culture.

Adult pancreatic stem and progenitor cells may serve as an alternative source of insulin-secreting endocrine cells in cell replacement therapy for type 1 diabetes, but much remained unknown about these cells. We previously identified adult murine pancreatic progenitor-like cells that displayed in vitro self-renewal and tri-lineage differentiation activities in a three-dimensional colony/organoid assay containing 1% methylcellulose and 5% Matrigel. However, the presence of other undefined culture components, such as serum and conditioned medium, has prevented a complete understanding of the signals required for progenitor cell growth. Here, we have established a serum-free, conditioned medium-free colony assay with the inclusion of seven defined factors: epidermal growth factor (EGF), R-Spondin 1 (RSPO1), Noggin, nicotinamide, exendin-4, activin B, and vascular endothelial growth factor (VEGF)-A. The requirements for colony growth were characterized and we found that EGF and nicotinamide were necessary and sufficient for the colony growth and long-term self-renewal of these progenitors. However, the seven factor (7F) culture medium better induced colony size and self-renewal in long-term culture than EGF plus nicotinamide alone. Individual 3-week-old colonies grown in the 7F culture medium expressed ductal, acinar, and endocrine lineage markers, suggesting that tri-lineage differentiation of the tri-potent progenitors was occurring without genetic manipulation. A delayed inhibition of Notch signaling using small molecules in 2-week-old cultures enhanced endocrine gene expression in 3-week-old colonies. This better-defined colony assay system will enable our and other laboratories for in-depth mechanistic studies on the biology of these progenitor cells.

Efficient generation of pancreatic β-like cells from the mouse gallbladder

Direct reprogramming is a promising approach for the replacement of β cells in diabetes. Reprogramming of cells originating from the endodermal lineage, such as acinar cells in the pancreas, liver cells and gallbladder cells has been of particular interest because of their developmental proximity to β cells. Our previous work showed that mouse gallbladder epithelium can be partially reprogrammed in vitro to generate islet-like cells (rGBC1). Here, the reprogramming protocol was substantially improved, yielding cells (rGBC2) closer to functional β cells than the 1st generation method with higher conversion efficiency and insulin expression. In addition to insulin synthesis and processing, rGBC2 presented many hallmark features of β cells, including insulin secretion in response to high glucose stimulation. Gene expression analysis indicated that rGBC2 clustered closer with β cells and had a metabolic gene expression profile resembling neonatal β cells. When transplanted into immune-deficient animals, rGBC2 were stable for at least 5months and further matured in vivo. Taken together, this approach provides further understanding of endodermal lineage conversion and potential for development of cell replacement therapy for type 1 diabetes patients.

Transplantable bioartificial pancreas devices: current status and future prospects.

Islet transplantation has become a valuable therapy for patients with diabetes mellitus type 1.However, only selected patients with exhausted insulin therapy characterized by instable metabolic control and repeated severe hypoglycemia are transplant candidates. This strict indication is mainly due to the requirement for lifelong immunosuppression and the critical shortage for donor organs. Therefore, numerous research activities address these issues in order to provide beta cell replacement therapy to a broader cohort of patients with diabetes. The encapsulation of pancreatic islets within mainly alginate-based macro- or microcapsules withvarious physical configurations may allow protecting the islet graft without the need for immunosuppressive agents and moreover expanding the donor pool to animal tissue and novel insulin-producing cells. Despite major advances in encapsulation technology, a significant translation into clinical application is not evident. There are still issues that need to be resolved associated with graft oxygenation, immunprotection, inflammatory response, material biocompatibility, and transplantation site to list some of them. The recent advances in xenotransplantation and particularly in the field of stem cell-derived beta cells have generated a renewed scientific interest in encapsulation. This review aims to provide an overview on current encapsulation technologies as a treatment modality in cell replacement therapy for type 1 diabetes.

MiRNA-375 promotes beta pancreatic differentiation in human induced pluripotent stem (hiPS) cells

Islet transplantation is considered as an ultimate option for the treatment of type I diabetes. Human induced pluripotent stem cells (hiPSCs) have raised the possibility that patient-specific insulin-secreting cells might be derived from somatic cells through cell fate reprogramming. However, current protocols mostly rely on the use of several cytokines and inhibitors for directing differentiation towards pancreatic fate. Given the high manufacturing cost of these recombinant proteins, this approach is prohibitive for clinical applications. Knowing that microRNAs (miRNAs) are key players in various stages of pancreatic development, we present a novel and cost-effective strategy in which over-expression of miR-375 promotes pancreatic differentiation in hiPSCs in the absence of any other stimulator. We used a polycistronic viral vector expressing Sox2, Klf4, c-Myc, and Oct4 to drive hiPSCs from human foreskin fibroblasts. The established hiPSCs are similar to human embryonic stem cells in many aspects including morphology, passaging, surface and pluripotency markers, and gene expression. For differentiation induction, miR-375 was lentivirally overexpressed in these hiPSCs. Morphological assessment, immunocytochemistry, and expression analysis of islet marker genes confirmed that islet like cells were obtained in miR-375 transduced cells compared to controls. Our differentiated clusters secreted insulin in a glucose-dependant manner, showing in vitro functionality. We demonstrated for the first time that miRNAs might be ideal substitutes to induce pancreatic differentiation in hiPSCs. This work provides a new approach to study the role of miRNAs in pancreatic specification and increase the feasibility of using patient-specific iPSCs for beta cell replacement therapy for type I diabetes.

Development of a novel scale to assess the quality of life in type 1 diabetic patients for beta cell replacement therapy

The main purpose of beta cell replacement therapy for type 1 diabetic patients is to improve their health-related quality of life (HR-QoL). Previous HR-QoL surveys for beta cell replacement therapy on the point of familial support and public understanding have not been studied adequately. We hypothesize that familial support and public understanding have a significant impact on HR-QoL of type 1 diabetic patients, and low HR-QoL assessed with a new scale is associated with seeking a new therapy. The initial questionnaire with 26 items was created after content validation based on 778 inquiries from patients or associates. One hundred five type 1 diabetic patients answered the questionnaire as well as separate questions about their opinions on beta cell replacement therapy. Finally, the new scale, named the Japanese Insulin-Dependent Diabetic Patient QoL (JAPID-QoL) scale, consisted of 19 items with the following three subscales: diabetes status, familial support and social acceptance. Cronbach’s α for overall scale was 0.878, and those for three subscales were over 0.70. All items were satisfied with convergent validity. Lower scores on the JAPID-QoL scale were associated with higher interest in and acceptance of beta cell replacement therapy (Spearman’s coefficients; r = −0.25, p < 0.001 and r = −0.33, p < 0.001, respectively) and also correlated with the necessity of psychosomatic care (r = −0.49, p < 0.001). Our newly proposed JAPID-QoL scale showed enough reliability and validity to assess HR-QoL of type 1 diabetic patients. The patients who had a low HR-QoL on the JAPID-QoL scale desired to receive beta cell replacement therapy and psychosomatic care.

Reprogramming to pluripotency: stepwise resetting of the epigenetic landscape

In 2006, the "wall came down" that limited the experimental conversion of differentiated cells into the pluripotent state. In a landmark report, Shinya Yamanaka's group described that a handful of transcription factors (Oct4, Sox2, Klf4 and c-Myc) can convert a differentiated cell back to pluripotency over the course of a few weeks, thus reprograming them into induced pluripotent stem (iPS) cells. The birth of iPS cells started off a rush among researchers to increase the efficiency of the reprogramming process, to reveal the underlying mechanistic events, and allowed the generation of patient- and disease-specific human iPS cells, which have the potential to be converted into relevant specialized cell types for replacement therapies and disease modeling. This review addresses the steps involved in resetting the epigenetic landscape during reprogramming. Apparently, defined events occur during the course of the reprogramming process. Immediately, upon expression of the reprogramming factors, some cells start to divide faster and quickly begin to lose their differentiated cell characteristics with robust downregulation of somatic genes. Only a subset of cells continue to upregulate the embryonic expression program, and finally, pluripotency genes are upregulated establishing an embryonic stem cell-like transcriptome and epigenome with pluripotent capabilities. Understanding reprogramming to pluripotency will inform mechanistic studies of lineage switching, in which differentiated cells from one lineage can be directly reprogrammed into another without going through a pluripotent intermediate.

Pancreatic Islet-Like Clusters from Bone Marrow Mesenchymal Stem Cells of Human First-Trimester Abortus Can Cure Streptozocin-Induced Mouse Diabetes

Bone marrow mesenchymal stem cells (BMSCs) have been reported to possess low immunogenicity and cause immunosuppression of recipients when allografted. They can differentiate into insulin-producing cells and may be a valuable source for islet formation. However, the extremely low differentiating rate of adult BMSCs toward insulin-producing cells and the insufficient insulin secretion of the differentiated BMSCs in vitro prevent their clinical use in diabetes treatment. Little is known about the potential of cell replacement therapy with human BMSCs. Previously, we isolated and identified human first-trimester fetal BMSCs (hfBMSCs). Under a novel four-step induction procedure established in this study, the hfBMSCs effectively differentiated into functional pancreatic islet-like cell clusters that contained 62 ± 14% insulin-producing cells, expressed a broad gene profile related to pancreatic islet β-cell development, and released high levels of insulin (2.245 ± 0.222 pmol/100 clusters per 30 min) and C-peptide (2.200 ± 0.468 pmol/100 clusters per 30 min) in response to 25 mmol/L glucose stimulus in vitro. The pancreatic islet-like cell clusters normalized the blood glucose level of diabetic model mice for at least 9 weeks when xenografted; blood glucose levels in these mice rose abnormally again when the grafts were removed. Examination of the grafts indicated that the transplanted cells survived in recipients and produced human insulin and C-peptide in situ. These results demonstrate that hfBMSCs derived from a human first-trimester abortus can differentiate into pancreatic islet-like cell clusters following an established four-step induction. The insulin-producing clusters present advantages in cell replacement therapy of type 1 diabetic model mice.

Do Stem Cell-Derived Islets Represent a Commercially Viable Treatment for Type 1 and 2 Diabetes?

The potential of stem cell therapies for the treatment of diabetes has been the cornerstone for successful political action, fundraising and academic research programs. How can these lofty goals be translated? Clinical translation of stem cell therapies for diabetes requires unraveling the underlying biology of pancreatic development and islet function, and then recapitulating development in a defined, scalable and commercially viable manner. The b-cell or surrogate b-cell product must be safe, and must secrete insulin in a physiologically appropriate response to glucose. Xenotransplantation of pig islets would address the shortage of human cadaveric donor islets and would be an alternative to b-cells derived from human pluripotent stem cells. Stem cell-derived b-cells represent a cell replacement therapy for Type 1 diabetes (T1D) and for severe Type 2 diabetes (T2D).

Pluripotent stem cells: private obsession and public expectation.

In the 1960s it was shown that a rare type of tumour called a teratocarcinoma contains cells that are both pluripotent and self‐renewing (Kleinsmith & Pierce, 1964). Pluripotent means the capacity of an individual cell to give rise to all other cell types of the body and the germline. This property is normally restricted to a brief window in early development. Self‐renewal is the production of identical daughter cells while retaining the ability for differentiation. It is the defining feature of a stem cell. The study of teratocarcinoma stem cells led to particular culture conditions that allowed them to be propagated ex vivo without differentiation. In 1981 Martin Evans, Matt Kaufman and Gail Martin found that cells from early mouse embryos exposed to the same culture environment can suspend developmental progression and continue to multiply while remaining pluripotent (Evans & Kaufman, 1981; Martin, 1981). These are embryonic stem (ES) cells. I was completing my undergraduate studies in Oxford at that time and was fortunate to learn about this rather esoteric research from a wonderful teacher, Chris Graham. Chris also talked about parallel discoveries of growth factors and the provocative idea that they might regulate cell fate. I was captivated by the idea of understanding and controlling the pluripotent state » I was captivated by the idea of understanding and controlling the pluripotent state. « and that has been my obsession ever since. I went to Edinburgh to do a PhD on teratocarcinoma stem cells with Martin Hooper. There I was struck by an observation by a previous PhD student that the requirement of the stem cells for co‐culture with a feeder layer could be replaced for a short time by conditioned medium. To me this meant only one thing; a growth factor that could block differentiation. This was not …