Supplementary MaterialsSupplementary Information 41467_2018_4918_MOESM1_ESM

Supplementary MaterialsSupplementary Information 41467_2018_4918_MOESM1_ESM. mutant causes -cell loss of life, by activating the?TGF pathway. This study establishes an optimized directed differentiation protocol for modeling human being -cell disease and identifies a drug candidate L,L-Dityrosine hydrochloride for treating a broad range of is the only one (other than insulin) associated with T1D1C3, T2D4C7, and, in addition, neonatal diabetes (ND)8. During mouse development, PDX1+ pancreatic progenitors appear around L,L-Dityrosine hydrochloride embryonic day (E) E8.5; at E11.5, a small subset gives rise to mostly poly-hormonal endocrine cells commonly referred to as primary transition endocrine cells that likely do not contribute to the mature -cell pool9. At E14.5, the secondary transition begins with extensive differentiation and emergence of mono-hormonal -cells10. begins to be expressed only in the secondary transition stage, L,L-Dityrosine hydrochloride is continually expressed in pancreatic -cells and ductal cells11, and plays a critical role in endocrine development12. In is also essential for compensatory -cell proliferation in adult mice15. The absence or decreased expression of predisposes the mice to T2D15,16. In addition mutations in non-obese diabetic (NOD) mice have been shown to underlie -cell fragility and susceptibility to T1D17. However, the role of GLIS3 in human pancreatic development and human -cells remains unclear. Human pluripotent stem cells (hPSCs) have provided robust platforms to recapitulate pancreatic -cell defects in diabetes, including maturity-onset diabetes of the young18 and neonatal diabetes19C21. Recently, we used an isogenic hESC differentiation platform to evaluate the role of T2D-associated genes in L,L-Dityrosine hydrochloride pancreatic -cell function and survival in disease conditions22. However, our initial attempt using isogenic hESCs failed to recapitulate the defects observed in mice20. This raised the question whether GLIS3 plays different roles in mouse and human or whether the current differentiation strategy is not optimal to model GLIS3-related pancreatic -cell defects. To distinguish between these possibilities, we monitored mRNA in hESC-derived pancreatic progenitors and INS+ cells and found that the expression mRNA is undetectable, suggesting that the previous protocol23 failed to efficiently generate the disease-relevant cells20. Here, we describe an optimized strategy to efficiently derive GLIS3+ late-stage pancreatic progenitors (PP2), which give rise to mono-hormonal pancreatic -cells (PP2- cells). We use this platform to determine the role of GLIS3 in human pancreatic -cell generation and survival, and to identify a lead hit drug candidate for treating the broad range of human patients who suffer from HES3 hESCs were differentiated to the early-stage pancreatic progenitors (PP1 at day 9/D9, Fig.?1a, Supplementary Table?1), giving rise to a pool that contains around 75C90% PDX1+ cells (Supplementary Fig.?1a, b). The PP1 cells can differentiate into INS+ (PP1-) cells when cultured for seven additional days in basal differentiation medium (DMEM B27, Fig.?1a). However, the derived PP1- cells are mostly poly-hormonal (comprising a population of 60C70% poly-hormonal and 30C40% mono-hormonal INS+ cells), which represent the cells from older protocols23,24(Fig.?1dCf). The INS-GFP+ PP1- cells do not express detectable levels of by RT-PCR (Supplementary Fig.?1d). Poly-hormonal INS-GFP+ PP1- cells were previously shown to differentiate mostly to -cells when transplanted in vivo25, which suggests that their identity is closer to the primary transition cells in mouse development. We performed a pilot screen to establish a strategy to promote the generation of PP2 cells that provide rise to mono-hormonal INS+ cells. Among 14 different tradition conditions, we found one which generates the best percentage of PP2-derived insulin+/glucagon consistently?somatostatin? (INS+/GCG?SST?) cells for the full total INS+ inhabitants (Supplementary Fig.?1c). This is accomplished with PP expansion medium including 2?M RA, 200?nM LDN193189, 0.25?M SANT1, 10?ng/mL EGF, and 10?ng/mL FGF2. After 2 weeks of tradition (from day time 9 to day time 23) in PP expansion medium, a lot more than 90% from the cells indicated PDX1 at day time 23/D23_L (Supplementary Fig.?1b). Weighed against PP1, PP2 cells communicate higher degrees of past due trunk PP markers, including so that as indicated by qRT-PCR assays (Fig.?1b, Supplementary Desk?2) and RNA-seq profiling (Fig.?1c). Moreover, after seven days of differentiation, 85C95% of INS+ cells produced from PP2 are mono-hormonal, expressing insulin, however, not glucagon (Fig.?1dCf), somatostatin, or ghrelin (Supplementary Fig.?1gCj). On the other hand, just 30C40% of INS+ PP1- cells are mono-hormonal (Fig.?1dCf and Supplementary Fig.?1h, j). Furthermore, PP2- cells co-express mature -cell markers (Fig.?1g, h), including PDX1 Rabbit Polyclonal to KLF11 (97.3%), NKX2.2 (98.8%), PAX6 (86.0%), ISL1 (91.8%), and NKX6.1 (50.8%), plus they also express UCN3 (63.6%), an adult -cell marker26 that had not been reported as expressed using some of three previously published protocols27C29..

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