Engineering human stem cell-derived islets to evade immune rejection and promote localized immune tolerance.

Immunological protection of transplanted stem cell-derived islet (SC-islet) cells is yet to be achieved without chronic immunosuppression or encapsulation. Existing genetic engineering approaches to produce immune-evasive SC-islet cells have so far shown variable results. Here, we show that targeting human leukocyte antigens (HLAs) and PD-L1 alone does not sufficiently protect SC-islet cells from xenograft (xeno)- or allograft (allo)-rejection. As an addition to these approaches, we genetically engineer SC-islet cells to secrete the cytokines interleukin-10 (IL-10), transforming growth factor ß (TGF-ß), and modified IL-2 such that they promote a tolerogenic local microenvironment by recruiting regulatory T cells (Tregs) to the islet grafts. Cytokine-secreting human SC-ß cells resist xeno-rejection and correct diabetes for up to 8 weeks post-transplantation in non-obese diabetic (NOD) mice. Thus, genetically engineering human embryonic SCs (hESCs) to induce a tolerogenic local microenvironment represents a promising approach to provide SC-islet cells as a cell replacement therapy for diabetes without the requirement for encapsulation or immunosuppression.

Retrieval and purification of human ß cells from stem cell-derived islet engrafted mice.

Stem cell-derived beta cells (SC-ß-cells) engrafted into mice serve as a pre-clinical model of diabetes. It is helpful to recover viable ß cells following transplantation to perform tests on the graft. We developed a protocol to retrieve and purify a sufficient number of live ß cells from mice following long-term human SC-ß-cell engraftment. The protocol enables examination of SC-ß-cells undergoing developmental and metabolic changes in vivo and may facilitate the understanding of metabolic demand on SC-ß-cells.

Charting cellular identity during human in vitro ß-cell differentiation.

In vitro differentiation of human stem cells can produce pancreatic ß-cells; the loss of this insulin-secreting cell type underlies type 1 diabetes. Here, as a step towards understanding this differentiation process, we report the transcriptional profiling of more than 100,000 human cells undergoing in vitro ß-cell differentiation, and describe the cells that emerged. We resolve populations that correspond to ß-cells, α-like poly-hormonal cells, non-endocrine cells that resemble pancreatic exocrine cells and a previously unreported population that resembles enterochromaffin cells. We show that endocrine cells maintain their identity in culture in the absence of exogenous growth factors, and that changes in gene expression associated with in vivo ß-cell maturation are recapitulated in vitro. We implement a scalable re-aggregation technique to deplete non-endocrine cells and identify CD49a (also known as ITGA1) as a surface marker of the ß-cell population, which allows magnetic sorting to a purity of 80%. Finally, we use a high-resolution sequencing time course to characterize gene-expression dynamics during the induction of human pancreatic endocrine cells, from which we develop a lineage model of in vitro ß-cell differentiation. This study provides a perspective on human stem-cell differentiation, and will guide future endeavours that focus on the differentiation of pancreatic islet cells, and their applications in regenerative medicine.

Generation of hypoimmunogenic human pluripotent stem cells.

Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage "don't-eat me" signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.