Guided Self-Assembly of ES-Derived Lung Progenitors into Biomimetic Tube Structures That Impact Cell Differentiation.

Chemically directed differentiation of pluripotent stem cells (PSCs) into defined cell types is a potent strategy for creating regenerative tissue models and cell therapies. In vitro observations suggest that physical cues can augment directed differentiation. We recently demonstrated that confining human PSC-derived lung progenitor cells in a tube with a diameter that mimics those observed during lung development results in the alteration of cell differentiation towards SOX2-SOX9+ lung cells. Here we set out to assess the robustness of this geometric confinement effect with respect to different culture parameters in order to explore the corresponding changes in cell morphometry and determine the feasibility of using such an approach to enhance directed differentiation protocols. Culture of progenitor cells in polydimethylsiloxane (PDMS) tubes reliably induced self-organization into tube structures and was insensitive to a variety of extracellular matrix coatings. Cellular morphology and differentiation status were found to be sensitive to the diameter of tube cells that were cultured within but not to seeding density. These data suggest that geometric cues impose constraints on cells, homogenize cellular morphology, and influence fate status.

Human pluripotent stem cell-derived insulin-producing cells: A regenerative medicine perspective.

Tremendous progress has been made over the last two decades in the field of pancreatic beta cell replacement therapy as a curative measure for diabetes. Transplantation studies have demonstrated therapeutic efficacy, and cGMP-grade cell products are currently being deployed for the first time in human clinical trials. In this perspective, we discuss current challenges surrounding the generation, delivery, and engraftment of stem cell-derived islet-like cells, along with strategies to induce durable tolerance to grafted cells, with an eye toward a functional cellular-based therapy enabling insulin independence for patients with diabetes.

Harnessing Proliferation for the Expansion of Stem Cell-Derived Pancreatic Cells: Advantages and Limitations.

Restoring the number of glucose-responsive ß-cells in patients living with diabetes is critical for achieving normoglycemia since functional ß-cells are lost during the progression of both type 1 and 2 diabetes. Stem cell-derived ß-cell replacement therapies offer an unprecedented opportunity to replace the lost ß-cell mass, yet differentiation efficiencies and the final yield of insulin-expressing ß-like cells are low when using established protocols. Driving cellular proliferation at targeted points during stem cell-derived pancreatic progenitor to ß-like cell differentiation can serve as unique means to expand the final cell therapeutic product needed to restore insulin levels. Numerous studies have examined the effects of ß-cell replication upon functionality, using primary islets in vitro and mouse models in vivo, yet studies that focus on proliferation in stem cell-derived pancreatic models are only just emerging in the field. This mini review will discuss the current literature on cell proliferation in pancreatic cells, with a focus on the proliferative state of stem cell-derived pancreatic progenitors and ß-like cells during their differentiation and maturation. The benefits of inducing proliferation to increase the final number of ß-like cells will be compared against limitations associated with driving replication, such as the blunted capacity of proliferating ß-like cells to maintain optimal ß-cell function. Potential strategies that may bypass the challenges induced by the up-regulation of cell cycle-associated factors during ß-cell differentiation will be proposed.

Assembly of lung progenitors into developmentally-inspired geometry drives differentiation via cellular tension.

During organogenesis groups of differentiating cells self-organize into a series of structural intermediates with defined architectural forms. Evidence is emerging that such architectural forms are important in guiding cell fate, yet in vitro methods to guide cell fate have focused primarily on un-patterned exposure of stems cells to developmentally relevant chemical cues. We set out to ask if organizing differentiating lung progenitors into developmentally relevant structures could be used to influence differentiation status. Specifically, we use elastomeric substrates to guide self-assembly of human pluripotent stem cell-derived lung progenitors into developmentally-relevant sized tubes and assess the impact on differentiation. Culture in 100 µm tubes reduced the percentage of SOX2+SOX9+ cells and reduced proximal fate potential compared to culture in 400 µm tubes or on flat surfaces. Cells in 100 µm tubes curved to conform to the tube surface and experienced increased cellular tension and reduced elongation. Pharmacologic disruption of tension through inhibition of ROCK, myosin II activity and actin polymerization in tubes resulted in maintenance of SOX2+SOX9+ populations. Furthermore, this effect required canonical WNT signaling. This data suggests that structural forms, when developmentally relevant, can drive fate choice during directed differentiation via a tension-based canonical WNT dependent mechanism.

I. How can you choose the fate of iPSCs and stem cells, Regeneration or Carcinogenesis? A hypothetical insight.: II. Modelling human beta cell development with pluripotent stem cells.

It is nowadays taken granted that induced pluripotent stem cells (iPSCs) are available for the regeneration therapy since iPSCs differentiate into any kind of phenotypes. If iPSCs can choose their fate in every way of differentiation why they do not choose cancer phenotype. As a body develops for one fertilized egg, embryonic stem cell must choose every phenotype of tissues such as blood, neuron, lung, liver, pancreas and so on depending on the stages. And sometimes the cells get cancer. So do iPSCs because iPSCs are almost equivalent to embryonic cells. Then how can the safety of the regeneration therapy be maintained with iPSCs? When inducing the differentiation of iPSCs it is considered important to choose the proper conditions of culture such as 3D-platform for embryoid, supplement of cytokines and growth factors, inhibition of signaling and so on. On the other hand, several conditions have been reported to induce cancer stem cells. The cancer inducing conditions are possibly summarized as the factors chronically exposed to iPSCs. It is further worthwhile noticing that the conditions do not appear to induce mutations but affecting the epigenetics. Collectively, to secure the safety of regeneration therapy, it appears the best way to avoid the conditions to induce cancer stem cells. Further insights in details will be discussed in the lecture. Type 1 Diabetes (T1D) is an autoimmune disease characterized by destruction of the pancreatic beta cells and loss of insulin. Using the Edmonton protocol, donor-derived islets seeded into the liver successfully restore glycemia in 58% of T1D patients. However, donor scarcity, risks associated with immunosuppressants and poor engraftment limit this therapeutic application to a small number of patients. To overcome these challenges, the developmental potential of human embryonic stem cells and human induced pluripotent stem cells is being harnessed to produce surrogate islets in vitro. We and others have been able to mimic human embryonic development and generate pancreatic progenitors (PP) that have the ability to mature into insulin-producing beta-like cells both in vitro and in vivo. Transplantation of pancreatic progenitors in the kidney capsule of immunodeficient mice leads to formation of islet-like structures that secrete human insulin. However, there are some limitations to the use of pancreatic progenitors for the treatment of T1D. First and foremost, their safety as the PP population can be heterogenous and highly proliferative, which might lead to formation of cellular outgrowth or teratoma after transplantation. Second, while insulin-producing cells develop in vivo 6 weeks after transplantation, restoration of normoglycemia occurs ~5 months later, suggesting that these "early" insulin-producing cells are immature, or poorly connected to the host vasculature. We have been addressing these two limitations and developed approaches to 1) improve safety by identifying markers to purify the PP populations and 2) accelerate functionality by improving vascularization at the time of transplantation.

Stem Cells, Self-Renewal, and Lineage Commitment in the Endocrine System.

The endocrine system coordinates a wide array of body functions mainly through secretion of hormones and their actions on target tissues. Over the last decades, a collective effort between developmental biologists, geneticists, and stem cell biologists has generated a wealth of knowledge related to the contribution of stem/progenitor cells to both organogenesis and self-renewal of endocrine organs. This review provides an up-to-date and comprehensive overview of the role of tissue stem cells in the development and self-renewal of endocrine organs. Pathways governing crucial steps in both development and stemness maintenance, and that are known to be frequently altered in a wide array of endocrine disorders, including cancer, are also described. Crucially, this plethora of information is being channeled into the development of potential new cell-based treatment modalities for endocrine-related illnesses, some of which have made it through clinical trials.

Pluripotent Stem Cell-Derived Pancreatic Progenitors and ß-Like Cells for Type 1 Diabetes Treatment.

In this review, we focus on the processes guiding human pancreas development and provide an update on methods to efficiently generate pancreatic progenitors (PPs) and ß-like cells in vitro from human pluripotent stem cells (hPSCs). Furthermore, we assess the strengths and weaknesses of using PPs and ß-like cell for cell replacement therapy for the treatment of Type 1 diabetes with respect to cell manufacturing, engrafting, functionality, and safety. Finally, we discuss the identification and use of specific cell surface markers to generate safer populations of PPs for clinical translation and to study the development of PPs in vivo and in vitro.

Cell Therapy for Type 1 Diabetes: Current and Future Strategies.

PURPOSE OF THE REVIEW: Type 1 diabetes (T1D) is defined by an autoimmune destruction of insulin producing ß-cells located in the endocrine part of the pancreas, the islets of Langerhans. As exogenous insulin administration fails at preventing severe complications associated with this disease, cell replacement therapies are being considered as a means to treat T1D. The purpose of this manuscript is to review the challenges associated with current strategies and discuss the potential of stem cell therapy for the treatment of T1D. RECENT FINDINGS: The most prominent therapy offered to T1D patients is exogenous insulin administration which, despite formulations improvement, remains a suboptimal treatment, due to the frequency of injections and the issues associated with precise dosing. As immunotherapy approaches have remained unsuccessful, the only cure for T1D is transplantation of donor-derived pancreas or islets. However, donor scarcity, graft loss, and immune response to the foreign tissue are issues challenging this approach and limiting the number of patients who can benefit from such treatments. In this review, we discuss the causes of T1D and the shortcomings of the current treatments. Furthermore, we summarize the cutting edge research that aims to tackle the current challenges in reaching a quality-controlled product with long-term effects, with a focus on regenerative medicine approaches using human pluripotent stem cells.

Generation of polyhormonal and multipotent pancreatic progenitor lineages from human pluripotent stem cells.

Generation of pancreatic ß-cells from human pluripotent stem cells (hPSCs) has enormous importance in type 1 diabetes (T1D), as it is fundamental to a treatment strategy based on cellular therapeutics. Being able to generate ß-cells, as well as other mature pancreatic cells, from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) will also enable the development of platforms that can be used for disease modeling and drug testing for a variety of pancreas-associated diseases, including cystic fibrosis. For this to occur, it is crucial to develop differentiation strategies that are robust and reproducible across cell lines and laboratories. In this article we describe two serum-free differentiation protocols designed to generate specific pancreatic lineages from hPSCs. Our approach employs a variety of cytokines and small molecules to mimic developmental pathways active during pancreatic organogenesis and allows for the in vitro generation of distinct pancreatic populations. The first protocol is designed to give rise to polyhormonal cells that have the potential to differentiate into glucagon-producing cells. The second protocol is geared to generate multipotent pancreatic progenitor cells, which harbor the potential to generate all pancreatic lineages including: monohormonal endocrine cells, acinar, and ductal cells.

Recent advances in cell replacement therapies for the treatment of type 1 diabetes.

Exogenous insulin administration is currently the only treatment available to all type 1 diabetes patients, but it is not a cure. By helping to regulate circulating blood glucose levels, it has significantly improved life expectancy, but there are still long-term complications associated with the disease that may be preventable with a treatment strategy that can provide better glycemic control. Isolated islet (or whole pancreas) transplantation, xenotransplantation, and the transplant of human pluripotent stem cell-derived ß-cells provide the potential to restore endogenous insulin production and reestablish normoglycemia. Here, we discuss the latest advances in these fields, including the use of encapsulation technology, as well as some of the hurdles that still need to be overcome for these alternative therapies to become mainstream and/or progress to clinical development.

Generation of beta cells from human pluripotent stem cells: Potential for regenerative medicine.

The loss of beta cells in Type I diabetes ultimately leads to insulin dependence and major complications that are difficult to manage by insulin injections. Given the complications associated with long-term administration of insulin, cell-replacement therapy is now under consideration as an alternative treatment that may someday provide a cure for this disease. Over the past 10 years, islet transplantation trials have demonstrated that it is possible to replenish beta cell function in Type I diabetes patients and, at least temporarily, eliminate their dependency on insulin. While not yet optimal, the success of these trials has provided proof-of-principle that cell replacement therapy is a viable option for treating diabetes. Limited access to donor islets has launched a search for alternative source of beta cells for cell therapy purposes and focused the efforts of many investigators on the challenge of deriving such cells from human embryonic (hESCs) and induced pluripotent stem cells (hiPSCs). Over the past five years, significant advances have been made in understanding the signaling pathways that control lineage development from human pluripotent stem cells (hPSCs) and as a consequence, it is now possible to routinely generate insulin producing cells from both hESCs and hiPSCs. While these achievements are impressive, significant challenges do still exist, as the majority of insulin producing cells generated under these conditions are polyhormonal and non functional, likely reflecting the emergence of the polyhormonal population that is known to arise in the early embryo during the phase of pancreatic development known as the 'first transition'. Functional beta cells, which arise during the second phase or transition of pancreatic development have been generated from hESCs, however they are detected only following transplantation of progenitor stage cells into immunocompromised mice. With this success, our challenge now is to define the pathways that control the development and maturation of this second transition population from hPSCs, and establish conditions for the generation of functional beta cells in vitro.

Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells.

Directed differentiation of human embryonic stem (hES) cells and human induced pluripotent stem (hiPS) cells captures in vivo developmental pathways for specifying lineages in vitro, thus avoiding perturbation of the genome with exogenous genetic material. Thus far, derivation of endodermal lineages has focused predominantly on hepatocytes, pancreatic endocrine cells and intestinal cells. The ability to differentiate pluripotent cells into anterior foregut endoderm (AFE) derivatives would expand their utility for cell therapy and basic research to tissues important for immune function, such as the thymus; for metabolism, such as thyroid and parathyroid; and for respiratory function, such as trachea and lung. We find that dual inhibition of transforming growth factor (TGF)-ß and bone morphogenic protein (BMP) signaling after specification of definitive endoderm from pluripotent cells results in a highly enriched AFE population that is competent to be patterned along dorsoventral and anteroposterior axes. These findings provide an approach for the generation of AFE derivatives.

Notch induces cell cycle arrest and apoptosis in human erythroleukaemic TF-1 cells.

OBJECTIVE: Notch signalling is known to promote hematopoietic stem cell self-renewal and to influence the lineage commitment decisions of progenitor cells. The purpose of this study was to investigate the mechanism of Notch-induced apoptosis in the erythroleukaemic cell line TF-1, and in primary cord blood CD34+ cells. METHODS: Retroviral constructs containing constitutively active forms of Notch as well as components of the Notch signalling pathway were used to transduce cells and their effect on cell cycle kinetics and apoptosis assayed by immunostaining for the S-phase marker Ki67 and Annexin V. RESULTS: We found that TF-1 cells undergo cell cycle arrest followed by apoptosis in a cytokine-independent manner in response to active Notch. Transduction of TF-1 cells with known targets of Notch signalling, Deltex1, HES1 and HERP2, showed that Notch-induced cell cycle arrest was not mediated by these proteins. However, analysis of cell cycle gene expression revealed that Notch signalling was associated with an up-regulation of IFI16 expression in TF-1 cells and in primary cord blood CD34+ cells. CONCLUSION: These data demonstrate that, in the context of TF-1 cells, Notch signalling can induce cell cycle arrest and apoptosis.

Notch signaling induces apoptosis in primary human CD34+ hematopoietic progenitor cells.

Notch signaling regulates diverse cell fate decisions during development and is reported to promote murine hematopoietic stem cell (HSC) self-renewal. The purpose of this study was to define the functional consequences of activating the Notch signaling pathway on self-renewal in human HSCs. Subsets of human umbilical cord blood CD34(+) cells were retrovirally transduced with the constitutively active human Notch 1 intracellular domain (N1ICD). N1ICD-transduced cells proliferated to a lesser extent in vitro than cells transduced with vector alone, and this was accompanied by a reduction in the percentage and absolute number of CD34(+) cell populations, including CD34(+)Thy(+)Lin(-) HSCs. Ectopic N1ICD expression inhibited cell cycle kinetics concurrent with an upregulation of p21 mRNA expression and induced apoptosis. Transduction of cells with HES-1, a known transcriptional target of Notch signaling and a mediator of Notch function, had no effect on HSC proliferation, indicating that the mechanism of the Notch-induced effect is HES-1-independent. The results of this study show that activation of the Notch signaling pathway has an inhibitory effect on the proliferation and survival of human hematopoietic CD34(+) cells populations. These findings have important implications for strategies aimed at promoting self-renewal of human HSCs.