Characterization of the role of major histocompatibility complex in type 1 diabetes recurrence after islet transplantation.

BACKGROUND: Major histocompatibility complex (MHC) molecules are essential determinants of beta-cell destruction in type 1 diabetes (T1D). MHC class I- or class II-null nonobese diabetic (NOD) mice do not spontaneously develop autoimmune diabetes and are resistant to adoptive transfer of disease. Both CD4+ and CD8+ T cells are associated with graft destruction after syngeneic islet transplantation. MHC molecules within the graft (i.e., on beta-cells or donor lymphocytes) may influence the interactions between antigen presenting cells and effector T cells and, therefore, the survival outcome of the graft. METHODS: Donor islets from NOD mice deficient in one or both of beta2-microglobulin and class II transactivator genes were transplanted into diabetic NOD mice. Immunohistochemistry was performed to identify the phenotype of infiltrating cells and to assess graft insulin production. The presence of cytokines in the grafts was assayed by reverse transcription polymerase chain reaction. RESULTS: MHC class II-null islets demonstrated rates of rejection comparable with control wild-type (wt) islets. In contrast, MHC class I- and II-null islets demonstrated indefinite survival (over 100 days). Infiltrates of both failed and surviving grafts were comprised of cytotoxic lymphocytes (CTL), helper T cells, and macrophages. Grafts also showed the presence of both Th1- and Th2-type cytokines (interleukin [IL]-2, IL-4, IL-10, and interferon-gamma), independent of graft status. CONCLUSIONS: These results demonstrate the primary importance of MHC class I molecules in the pathogenesis of diabetes recurrence postislet transplantation. Conversely, MHC class II expression is not a necessary mechanistic component of transplant destruction. In addition, these results implicate MHC class I-restricted CTLs but not MHC class II-restricted T cells in disease recurrence.

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.