Abnormal exocrine-endocrine cell cross-talk promotes ß-cell dysfunction and loss in MODY8.

MODY8 (maturity-onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes associated with mutations in the carboxyl ester lipase (CEL) gene expressed by pancreatic acinar cells. MODY8 patients develop childhood-onset exocrine pancreas dysfunction followed by diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes. In the present study, we report the transfer of CEL proteins from acinar cells to ß-cells as a form of cross-talk between exocrine and endocrine cells. Human ß-cells show a relatively higher propensity for internalizing the mutant versus the wild-type CEL protein. After internalization, the mutant protein forms stable intracellular aggregates leading to ß-cell secretory dysfunction. Analysis of pancreas sections from a MODY8 patient reveals the presence of CEL protein in the few extant ß-cells. The present study provides compelling evidence for the mechanism by which a mutant gene expressed specifically in acinar cells promotes dysfunction and loss of ß-cells to cause diabetes.

Genome-scale screens identify JNK-JUN signaling as a barrier for pluripotency exit and endoderm differentiation.

Human embryonic stem cells (ESCs) and human induced pluripotent stem cells hold great promise for cell-based therapies and drug discovery. However, homogeneous differentiation remains a major challenge, highlighting the need for understanding developmental mechanisms. We performed genome-scale CRISPR screens to uncover regulators of definitive endoderm (DE) differentiation, which unexpectedly uncovered five Jun N-terminal kinase (JNK)-JUN family genes as key barriers of DE differentiation. The JNK-JUN pathway does not act through directly inhibiting the DE enhancers. Instead, JUN co-occupies ESC enhancers with OCT4, NANOG, SMAD2 and SMAD3, and specifically inhibits the exit from the pluripotent state by impeding the decommissioning of ESC enhancers and inhibiting the reconfiguration of SMAD2 and SMAD3 chromatin binding from ESC to DE enhancers. Therefore, the JNK-JUN pathway safeguards pluripotency from precocious DE differentiation. Direct pharmacological inhibition of JNK significantly improves the efficiencies of generating DE and DE-derived pancreatic and lung progenitor cells, highlighting the potential of harnessing the knowledge from developmental studies for regenerative medicine.

Genome Editing and Directed Differentiation of hPSCs for Interrogating Lineage Determinants in Human Pancreatic Development.

Interrogating gene function in self-renewing or differentiating human pluripotent stem cells (hPSCs) offers a valuable platform towards understanding human development and dissecting disease mechanisms in a dish. To capitalize on this potential application requires efficient genome-editing tools to generate hPSC mutants in disease-associated genes, as well as in vitro hPSC differentiation protocols to produce disease-relevant cell types that closely recapitulate their in vivo counterparts. An efficient genome-editing platform for hPSCs named iCRISPR has been developed through the TALEN-mediated targeting of a Cas9 expression cassette in the AAVS1 locus. Here, the protocols for the generation of inducible Cas9 hPSC lines using cells cultured in a chemically defined medium and a feeder-free condition are described. Detailed procedures for using the iCRISPR system for gene knockout or precise genetic alterations in hPSCs, either through non-homologous end joining (NHEJ) or via precise nucleotide alterations using a homology-directed repair (HDR) template, respectively, are included. These technical procedures include descriptions of the design, production, and transfection of CRISPR guide RNAs (gRNAs); the measurement of the CRISPR mutation rate by T7E1 or RFLP assays; and the establishment and validation of clonal mutant lines. Finally, we chronicle procedures for hPSC differentiation into glucose-responsive pancreatic ß-like cells by mimicking in vivo pancreatic embryonic development. Combining iCRISPR technology with directed hPSC differentiation enables the systematic examination of gene function to further our understanding of pancreatic development and diabetes disease mechanisms.

Genome Editing in hPSCs Reveals GATA6 Haploinsufficiency and a Genetic Interaction with GATA4 in Human Pancreatic Development.

Human disease phenotypes associated with haploinsufficient gene requirements are often not recapitulated well in animal models. Here, we have investigated the association between human GATA6 haploinsufficiency and a wide range of clinical phenotypes that include neonatal and adult-onset diabetes using CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome editing coupled with human pluripotent stem cell (hPSC) directed differentiation. We found that loss of one GATA6 allele specifically affects the differentiation of human pancreatic progenitors from the early PDX1+ stage to the more mature PDX1+NKX6.1+ stage, leading to impaired formation of glucose-responsive ß-like cells. In addition to this GATA6 haploinsufficiency, we also identified dosage-sensitive requirements for GATA6 and GATA4 in the formation of both definitive endoderm and pancreatic progenitor cells. Our work expands the application of hPSCs from studying the impact of individual gene loci to investigation of multigenic human traits, and it establishes an approach for identifying genetic modifiers of human disease.

The p53 Family Coordinates Wnt and Nodal Inputs in Mesendodermal Differentiation of Embryonic Stem Cells.

In this study, we outline a regulatory network that involves the p53 tumor suppressor family and the Wnt pathway acting together with the TGF-ß pathway in mesendodermal differentiation of mouse and human embryonic stem cells. Knockout of all three members, p53, p63, and p73, shows that the p53 family is essential for mesendoderm specification during exit from pluripotency in embryos and in culture. Wnt3 and its receptor Fzd1 are direct p53 family target genes in this context, and induction of Wnt signaling by p53 is critical for activation of mesendodermal differentiation genes. Globally, Wnt3-activated Tcf3 and nodal-activated Smad2/3 transcription factors depend on each other for co-occupancy of target enhancers associated with key differentiation loci. Our results therefore highlight an unanticipated role for p53 family proteins in a regulatory network that integrates essential Wnt-Tcf and nodal-Smad inputs in a selective and interdependent way to drive mesendodermal differentiation of pluripotent cells.

CRISPR/Cas9-Mediated Mutagenesis of Human Pluripotent Stem Cells in Defined Xeno-Free E8 Medium.

The recent advent of engineered nucleases including the CRISPR/Cas9 system has greatly facilitated genome manipulation in human pluripotent stem cells (hPSCs). In addition to facilitating hPSC-based disease studies, the application of genome engineering in hPSCs has also opened up new avenues for cell replacement therapy. To improve consistency and reproducibility of hPSC-based studies, and to meet the safety and regulatory requirements for clinical translation, it is necessary to use a defined, xeno-free cell culture system. This chapter describes protocols for CRISPR/Cas9 genome editing in an inducible Cas9 hPSC-based system, using cells cultured in chemically defined, xeno-free E8 Medium on a recombinant human vitronectin substrate. We detail procedures for the design and transfection of CRISPR guide RNAs, colony selection, and the expansion and validation of clonal mutant lines, all within this fully defined culture condition. These methods may be applied to a wide range of genome-engineering applications in hPSCs, including those that utilize different types of site-specific nucleases such as zinc finger nucleases (ZFNs) and TALENs, and form a closer step towards clinical utility of these cells.

Genome Editing of Lineage Determinants in Human Pluripotent Stem Cells Reveals Mechanisms of Pancreatic Development and Diabetes.

Directed differentiation of human pluripotent stem cells (hPSCs) into somatic counterparts is a valuable tool for studying disease. However, examination of developmental mechanisms in hPSCs remains challenging given complex multi-factorial actions at different stages. Here, we used TALEN and CRISPR/Cas-mediated gene editing and hPSC-directed differentiation for a systematic analysis of the roles of eight pancreatic transcription factors (PDX1, RFX6, PTF1A, GLIS3, MNX1, NGN3, HES1, and ARX). Our analysis not only verified conserved gene requirements between mice and humans but also revealed a number of previously unsuspected developmental mechanisms with implications for type 2 diabetes. These include a role of RFX6 in regulating the number of pancreatic progenitors, a haploinsufficient requirement for PDX1 in pancreatic ß cell differentiation, and a potentially divergent role of NGN3 in humans and mice. Our findings support use of systematic genome editing in hPSCs as a strategy for understanding mechanisms underlying congenital disorders.

FOXN1 (GFP/w) reporter hESCs enable identification of integrin-ß4, HLA-DR, and EpCAM as markers of human PSC-derived FOXN1(+) thymic epithelial progenitors.

Thymic epithelial cells (TECs) play a critical role in T cell maturation and tolerance induction. The generation of TECs from in vitro differentiation of human pluripotent stem cells (PSCs) provides a platform on which to study the mechanisms of this interaction and has implications for immune reconstitution. To facilitate analysis of PSC-derived TECs, we generated hESC reporter lines in which sequences encoding GFP were targeted to FOXN1, a gene required for TEC development. Using this FOXN1 (GFP/w) line as a readout, we developed a reproducible protocol for generating FOXN1-GFP(+) thymic endoderm cells. Transcriptional profiling and flow cytometry identified integrin-ß4 (ITGB4, CD104) and HLA-DR as markers that could be used in combination with EpCAM to selectively purify FOXN1(+) TEC progenitors from differentiating cultures of unmanipulated PSCs. Human FOXN1(+) TEC progenitors generated from PSCs facilitate the study of thymus biology and are a valuable resource for future applications in regenerative medicine.

Rewiring immunity: generating a functional thymus from hESCs are we there yet?

Two papers in this issue of Cell Stem Cell have made a significant advance in solving one of the great challenges of modern immunology-resurrecting thymus function through the induction of thymus epithelial cells (TECs) by directed differentiation of human embryonic stem cells (hESCs).

A targeted NKX2.1 human embryonic stem cell reporter line enables identification of human basal forebrain derivatives.

We have used homologous recombination in human embryonic stem cells (hESCs) to insert sequences encoding green fluorescent protein (GFP) into the NKX2.1 locus, a gene required for normal development of the basal forebrain. Generation of NKX2.1-GFP(+) cells was dependent on the concentration, timing, and duration of retinoic acid treatment during differentiation. NKX2.1-GFP(+) progenitors expressed genes characteristic of the basal forebrain, including SHH, DLX1, LHX6, and OLIG2. Time course analysis revealed that NKX2.1-GFP(+) cells could upregulate FOXG1 expression, implying the existence of a novel pathway for the generation of telencephalic neural derivatives. Further maturation of NKX2.1-GFP(+) cells gave rise to γ-aminobutyric acid-, tyrosine hydroxylase-, and somatostatin-expressing neurons as well as to platelet-derived growth factor receptor α-positive oligodendrocyte precursors. These studies highlight the diversity of cell types that can be generated from human NKX2.1(+) progenitors and demonstrate the utility of NKX2.1(GFP/w) hESCs for investigating human forebrain development and neuronal differentiation.