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.

Whole-genome CRISPR screening identifies genetic manipulations to reduce immune rejection of stem cell-derived islets.

Human embryonic stem cells (hESCs) provide opportunities for cell replacement therapy of insulin-dependent diabetes. Therapeutic quantities of human stem cell-derived islets (SC-islets) can be produced by directed differentiation. However, preventing allo-rejection and recurring autoimmunity, without the use of encapsulation or systemic immunosuppressants, remains a challenge. An attractive approach is to transplant SC-islets, genetically modified to reduce the impact of immune rejection. To determine the underlying forces that drive immunogenicity of SC-islets in inflammatory environments, we performed single-cell RNA sequencing (scRNA-seq) and whole-genome CRISPR screen of SC-islets under immune interaction with allogeneic peripheral blood mononuclear cells (PBMCs). Data analysis points to "alarmed" populations of SC-islets that upregulate genes in the interferon (IFN) pathway. The CRISPR screen in vivo confirms that targeting IFNγ-induced mediators has beneficial effects on SC-islet survival under immune attack. Manipulating the IFN response by depleting chemokine ligand 10 (CXCL10) in SC-islet grafts confers improved survival against allo-rejection compared with wild-type grafts in humanized mice. These results offer insights into the nature of immune destruction of SC-islets during allogeneic responses and provide targets for gene editing.

A human ESC line for efficient CRISPR editing of pluripotent stem cells.

Human pluripotent stem cells (hPSC) can be directed to differentiate in vitro into insulin-prorducing beta cells (SC-ß). Although these cells accurately respond to glucose and can reverse diabetes in preclinical models improvments in the final cell products are desirable. For example, safety, controlling the cellular compositions and protection against immune rejection may be addressed by genetic modifications of SC-ß pre-transplantation. To screen for gene targets, we have generated a human embryonic stem cell line (hESC) that constitutively express the enhanced specificity Streptococcus pyogenes Cas9 (eSpCas9) gene, knocked-in into the GAPDH locus.

Modeling Type 1 Diabetes In Vitro Using Human Pluripotent Stem Cells.

Understanding the root causes of autoimmune diseases is hampered by the inability to access relevant human tissues and identify the time of disease onset. To examine the interaction of immune cells and their cellular targets in type 1 diabetes, we differentiated human induced pluripotent stem cells into pancreatic endocrine cells, including ß cells. Here, we describe an in vitro platform that models features of human type 1 diabetes using stress-induced patient-derived endocrine cells and autologous immune cells. We demonstrate a cell-type-specific response by autologous immune cells against induced pluripotent stem cell-derived ß cells, along with a reduced effect on α cells. This approach represents a path to developing disease models that use patient-derived cells to predict the outcome of an autoimmune response.

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.

Inhibition of ZEB1 expression induces redifferentiation of adult human ß cells expanded in vitro.

In-vitro expansion of functional adult human ß-cells is an attractive approach for generating insulin-producing cells for transplantation. However, human islet cell expansion in culture results in loss of ß-cell phenotype and epithelial-mesenchymal transition (EMT). This process activates expression of ZEB1 and ZEB2, two members of the zinc-finger homeobox family of E-cadherin repressors, which play key roles in EMT. Downregulation of ZEB1 using shRNA in expanded ß-cell-derived (BCD) cells induced mesenchymal-epithelial transition (MET), ß-cell gene expression, and proliferation attenuation. In addition, inhibition of ZEB1 expression potentiated redifferentiation induced by a combination of soluble factors, as judged by an improved response to glucose stimulation and a 3-fold increase in the fraction of C-peptide-positive cells to 60% of BCD cells. Furthermore, ZEB1 shRNA led to increased insulin secretion in cells transplanted in vivo. Our findings suggest that the effects of ZEB1 inhibition are mediated by attenuation of the miR-200c target genes SOX6 and SOX2. These findings, which were reproducible in cells derived from multiple human donors, emphasize the key role of ZEB1 in EMT in cultured BCD cells and support the value of ZEB1 inhibition for BCD cell redifferentiation and generation of functional human ß-like cells for cell therapy of diabetes.

Redifferentiation of expanded human pancreatic ß-cell-derived cells by inhibition of the NOTCH pathway.

In vitro expansion of ß-cells from adult human pancreatic islets would overcome donor ß-cell shortage for cell replacement therapy for diabetes. Using a ß-cell-specific labeling system we have shown that ß-cell expansion is accompanied by dedifferentiation resembling epithelial-mesenchymal transition and loss of insulin expression. Epigenetic analyses indicate that key ß-cell genes maintain open chromatin structure in expanded ß-cell-derived (BCD) cells, although they are not transcribed. In the developing pancreas important cell-fate decisions are regulated by NOTCH receptors, which signal through the Hairy and Enhancer of Split 1 (HES1) transcription regulator. We have reported that BCD cell dedifferentiation and proliferation in vitro correlate with reactivation of the NOTCH pathway. Inhibition of HES1 expression using shRNA during culture initiation results in reduced ß-cell replication and dedifferentiation, suggesting that HES1 inhibition may also affect BCD cell redifferentiation following expansion. Here, we used HES1 shRNA to down-regulate HES1 expression in expanded human BCD cells, showing that HES1 inhibition is sufficient to induce BCD cell redifferentiation, as manifested by a significant increase in insulin expression. Combined treatment with HES1 shRNA, cell aggregation in serum-free medium, and a mixture of soluble factors further stimulated the redifferentiation of BCD cells. In vivo analyses demonstrated the ability of the redifferentiated cells to replace ß-cell function in hyperglycemic immunodeficient mice. These findings demonstrate the redifferentiation potential of ex vivo expanded BCD cells and the reproducible differentiating effect of HES1 inhibition in these cells.

Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro.

BACKGROUND: Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, attempts at expanding human islet cells in tissue culture result in loss of beta-cell phenotype. Using a lineage-tracing approach we provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain open chromatin structure in expanded BCD cells, although they are not transcribed. Here we investigated whether BCD cells can be redifferentiated into beta-like cells. METHODOLOGY/PRINCIPAL FINDING: Redifferentiation conditions were screened by following activation of an insulin-DsRed2 reporter gene. Redifferentiated cells were characterized for gene expression, insulin content and secretion assays, and presence of secretory vesicles by electron microscopy. BCD cells were induced to redifferentiate by a combination of soluble factors. The redifferentiated cells expressed beta-cell genes, stored insulin in typical secretory vesicles, and released it in response to glucose. The redifferentiation process involved mesenchymal-epithelial transition, as judged by changes in gene expression. Moreover, inhibition of the EMT effector SLUG (SNAI2) using shRNA resulted in stimulation of redifferentiation. Lineage-traced cells also gave rise at a low rate to cells expressing other islet hormones, suggesting transition of BCD cells through an islet progenitor-like stage during redifferentiation. CONCLUSIONS/SIGNIFICANCE: These findings demonstrate for the first time that expanded dedifferentiated beta cells can be induced to redifferentiate in culture. The findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening.