Evert Kroon

Production of pancreatic hormone–expressing endocrine cells from human embryonic stem cells

Of paramount importance for the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function similarly to primary islets. We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor—en route to cells that express endocrine hormones. The hES cell–derived insulin-expressing cells have an insulin content approaching that of adult islets. Similar to fetal β-cells, they release C-peptide in response to multiple secretory stimuli, but only minimally to glucose. Production of these hES cell–derived endocrine cells may represent a critical step in the development of a renewable source of cells for diabetes cell therapy.

Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo

Development of a cell therapy for diabetes would be greatly aided by a renewable supply of human β-cells. Here we show that pancreatic endoderm derived from human embryonic stem (hES) cells efficiently generates glucose-responsive endocrine cells after implantation into mice. Upon glucose stimulation of the implanted mice, human insulin and C-peptide are detected in sera at levels similar to those of mice transplanted with ∼3,000 human islets. Moreover, the insulin-expressing cells generated after engraftment exhibit many properties of functional β-cells, including expression of critical β-cell transcription factors, appropriate processing of proinsulin and the presence of mature endocrine secretory granules. Finally, in a test of therapeutic potential, we demonstrate that implantation of hES cell–derived pancreatic endoderm protects against streptozotocin-induced hyperglycemia. Together, these data provide definitive evidence that hES cells are competent to generate glucose-responsive, insulin-secreting cells.

Efficient differentiation of human embryonic stem cells to definitive endoderm.

The potential of human embryonic stem (hES) cells to differentiate into cell types of a variety of organs has generated much excitement over the possible use of hES cells in therapeutic applications. Of great interest are organs derived from definitive endoderm, such as the pancreas. We have focused on directing hES cells to the definitive endoderm lineage as this step is a prerequisite for efficient differentiation to mature endoderm derivatives. Differentiation of hES cells in the presence of activin A and low serum produced cultures consisting of up to 80% definitive endoderm cells. This population was further enriched to near homogeneity using the cell-surface receptor CXCR4. The process of definitive endoderm formation in differentiating hES cell cultures includes an apparent epithelial-to-mesenchymal transition and a dynamic gene expression profile that are reminiscent of vertebrate gastrulation. These findings may facilitate the use of hES cells for therapeutic purposes and as in vitro models of development.

A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry.

Cell-surface markers for the isolation of pancreatic cell types derived from human embryonic stem cells

Using a flow cytometry–based screen of commercial antibodies, we have identified cell-surface markers for the separation of pancreatic cell types derived from human embryonic stem (hES) cells. We show enrichment of pancreatic endoderm cells using CD142 and of endocrine cells using CD200 and CD318. After transplantation into mice, enriched pancreatic endoderm cells give rise to all the pancreatic lineages, including functional insulin-producing cells, demonstrating that they are pancreatic progenitors. In contrast, implanted, enriched polyhormonal endocrine cells principally give rise to glucagon cells. These antibodies will aid investigations that use pancreatic cells generated from pluripotent stem cells to study diabetes and pancreas biology.

Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells.

Embryonic development is characterized by dynamic changes in gene expression, yet the role of chromatin remodeling in these cellular transitions remains elusive. To address this question, we profiled the transcriptome and select chromatin modifications at defined stages during pancreatic endocrine differentiation of human embryonic stem cells. We identify removal of Polycomb group (PcG)-mediated repression on stage-specific genes as a key mechanism for the induction of developmental regulators. Furthermore, we discover that silencing of transitory genes during lineage progression associates with reinstatement of PcG-dependent repression. Significantly, in vivo- but not in vitro-differentiated endocrine cells exhibit close similarity to primary human islets in regard to transcriptome and chromatin structure. We further demonstrate that endocrine cells produced in vitro do not fully eliminate PcG-mediated repression on endocrine-specific genes, probably contributing to their malfunction. These studies reveal dynamic chromatin remodeling during developmental lineage progression and identify possible strategies for improving cell differentiation in culture.

Insulin-Producing Endocrine Cells Differentiated In Vitro From Human Embryonic Stem Cells Function in Macroencapsulation Devices In Vivo

The PEC‐01 cell population, differentiated from human embryonic stem cells (hESCs), contains pancreatic progenitors (PPs) that, when loaded into macroencapsulation devices (to produce the VC‐01 candidate product) and transplanted into mice, can mature into glucose‐responsive insulin‐secreting cells and other pancreatic endocrine cells involved in glucose metabolism. We modified the protocol for making PEC‐01 cells such that 73%–80% of the cell population consisted of PDX1‐positive (PDX1+) and NKX6.1+ PPs. The PPs were further differentiated to islet‐like cells (ICs) that reproducibly contained 73%–89% endocrine cells, of which approximately 40%–50% expressed insulin. A large fraction of these insulin‐positive cells were single hormone‐positive and expressed the transcription factors PDX1 and NKX6.1. To preclude a significant contribution of progenitors to the in vivo function of ICs, we used a simple enrichment process to remove remaining PPs, yielding aggregates that contained 93%–98% endocrine cells and 1%–3% progenitors. Enriched ICs, when encapsulated and implanted into mice, functioned similarly to the VC‐01 candidate product, demonstrating conclusively that in vitro‐produced hESC‐derived insulin‐producing cells can mature and function in vivo in devices. A scaled version of our suspension culture was used, and the endocrine aggregates could be cryopreserved and retain functionality. Although ICs expressed multiple important β cell genes, the cells contained relatively low levels of several maturity‐associated markers. Correlating with this, the time to function of ICs was similar to PEC‐01 cells, indicating that ICs required cell‐autonomous maturation after delivery in vivo, which would occur concurrently with graft integration into the host.

Tolerance induction and reversal of diabetes in mice transplanted with human embryonic stem cell-derived pancreatic endoderm.

Type 1 diabetes (T1D) is an autoimmune disease caused by T cell-mediated destruction of insulin-producing β cells in the islets of Langerhans. In most cases, reversal of disease would require strategies combining islet cell replacement with immunotherapy that are currently available only for the most severely affected patients. Here, we demonstrate that immunotherapies that target T cell costimulatory pathways block the rejection of xenogeneic human embryonic-stem-cell-derived pancreatic endoderm (hESC-PE) in mice. The therapy allowed for long-term development of hESC-PE into islet-like structures capable of producing human insulin and maintaining normoglycemia. Moreover, short-term costimulation blockade led to robust immune tolerance that could be transferred independently of regulatory T cells. Importantly, costimulation blockade prevented the rejection of allogeneic hESC-PE by human PBMCs in a humanized model in vivo. These results support the clinical development of hESC-derived therapy, combined with tolerogenic treatments, as a sustainable alternative strategy for patients with T1D.

Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts

β-Cells generated from large-scale sources can overcome current shortages in clinical islet cell grafts provided that they adequately respond to metabolic variations. Pancreatic (non)endocrine cells can develop from human embryonic stem (huES) cells following in vitro derivation to pancreatic endoderm (PE) that is subsequently implanted in immune-incompetent mice for further differentiation. Encapsulation of PE increases the proportion of endocrine cells in subcutaneous implants, with enrichment in β-cells when they are placed in TheraCyte-macrodevices and predominantly α-cells when they are alginate-microencapsulated. At posttransplant (PT) weeks 20-30, macroencapsulated huES implants presented higher glucose-responsive plasma C-peptide levels and a lower proinsulin-over-C-peptide ratio than human islet cell implants under the kidney capsule. Their ex vivo analysis showed the presence of single-hormone-positive α- and β-cells that exhibited rapid secretory responses to increasing and decreasing glucose concentrations, similar to isolated human islet cells. However, their insulin secretory amplitude was lower, which was attributed in part to a lower cellular hormone content; it was associated with a lower glucose-induced insulin biosynthesis, but not with lower glucagon-induced stimulation, which together is compatible with an immature functional state of the huES-derived β-cells at PT weeks 20-30. These data support the therapeutic potential of macroencapsulated huES implants but indicate the need for further functional analysis. Their comparison with clinical-grade human islet cell grafts sets references for future development and clinical translation.

Immunogenicity of human embryonic stem cell-derived beta cells

Aims/hypothesisTo overcome the donor shortage in the treatment of advanced type 1 diabetes by islet transplantation, human embryonic stem cells (hESCs) show great potential as an unlimited alternative source of beta cells. hESCs may have immune privileged properties and it is important to determine whether these properties are preserved in hESC-derived cells.MethodsWe comprehensively investigated interactions of both innate and adaptive auto- and allo-immunity with hESC-derived pancreatic progenitor cells and hESC-derived endocrine cells, retrieved after in-vivo differentiation in capsules in the subcutis of mice.ResultsWe found that hESC-derived pancreatic endodermal cells expressed relatively low levels of HLA endorsing protection from specific immune responses. HLA was upregulated when exposed to IFNγ, making these endocrine progenitor cells vulnerable to cytotoxic T cells and alloreactive antibodies. In vivo-differentiated endocrine cells were protected from complement, but expressed more HLA and were targets for alloreactive antibody-dependent cellular cytotoxicity and alloreactive cytotoxic T cells. After HLA compatibility was provided by transduction with HLA-A2, preproinsulin-specific T cells killed insulin-producing cells.Conclusions/interpretationhESC-derived pancreatic progenitors are hypoimmunogenic, while in vivo-differentiated endocrine cells represent mature targets for adaptive immune responses. Our data support the need for immune intervention in transplantation of hESC-derived pancreatic progenitors. Cell-impermeable macro-encapsulation may suffice.