SUFU and SPOP mediated downregulation of Hh signaling promotes pancreatic beta-cell differentiation through the regulation of organ-specific stromal niche signals

Purpose: Human Embryonic Stem Cell (hESC)-derived insulin-producing beta cells offer a promising cell-based therapy for diabetes. However, efficient hESC to beta cell differentiation has proven difficult, possibly due to the lack of cross-talk with the appropriate mesenchymal niche. To define organ-specific niche signals, we isolated pancreatic and gastrointestinal stromal cells and analyzed their transcriptomes during development. Methods: mRNA profiles from E13.5 mice were generated from mesenchymal cells dissected from wild-type (WT) intestine, stomach, pancreas and mutant Sufu-/- Spop -/- pancreatic mesenchyme, in duplicate, by illumina HiSeq 2500. Reads were aligned to the mm10 assembly and unambiguously mapping reads were analyzed at the gene level for differential expression between tissue types alignment using STAR and differential analysis using DESeq2. Results: Using our workflow, we mapped about 30 million reads per sample to mm10. Differential expression (DE) analysis identified over 900 significantly differentially expressed genes between WT pancreatic mesenchyme and gastrointestinal mesenchymes, and over 1100 genes differentially expressed between WT and mutant pancreatic mesenchyme. Our findings reveal the importance of tightly-regulated Hh signaling in the pancreatic mesenchyme. In vivo inactivation of mesenchymal Hh signaling leads to annular pancreas, and stroma-specific activation of Hh signaling via loss of Hh regulators, Sufu and Spop, impairs pancreatic growth and beta cell genesis. Genetic rescue and transcriptome analyses show that these Sufu and Spop knockout defects occur through GLI2-mediated activation of gastrointestinal stromal signals such as Wnt ligands. Importantly, inhibition of Wnt signaling in organoid and hESC cultures significantly promotes insulin-producing cell generation, revealing the requirement for organ-specific regulation of stromal niche signals. mRNA profiles from E13.5 mice were generated from mesenchymal cells dissected from wild-type (WT) intestine, stomach, pancreas and mutant Sufu-/- Spop -/- pancreatic mesenchyme, in duplicate, by Illumina HiSeq 2500研究目的：人胚胎干细胞（human embryonic stem cell, hESC）诱导获得的胰岛素分泌β细胞为糖尿病提供了极具潜力的细胞治疗方案。然而，高效实现hESC向β细胞的分化仍颇具挑战，其潜在原因可能是缺乏与合适间充质微环境（mesenchymal niche）的交叉对话。为明确器官特异性的微环境信号，我们分离了胰腺与胃肠道基质细胞，并对其发育过程中的转录组（transcriptomes）进行了分析。 实验方法：本研究通过Illumina HiSeq 2500平台，对取自E13.5期小鼠的野生型（wild-type, WT）肠道、胃、胰腺间充质细胞，以及Sufu-/- Spop-/-突变型小鼠胰腺间充质细胞的mRNA表达谱（mRNA profiles）进行双重复测序。测序读段（reads）比对至mm10参考基因组组装版本，使用STAR软件筛选出唯一比对的读段后，通过DESeq2对不同组织间的基因水平差异表达进行分析。 实验结果：采用本研究的分析流程，每个样本约可比对得到3000万条reads至mm10基因组。差异表达分析显示，野生型胰腺间充质与胃肠道间充质间存在超过900个显著差异表达基因，野生型与突变型胰腺间充质间则存在超过1100个差异表达基因。本研究结果揭示了胰腺间充质中严格调控的Hh信号通路（Hh signaling）的重要性：在体内灭活间充质Hh信号会导致环状胰腺；而通过敲除Hh调控因子Sufu与Spop实现基质特异性Hh信号激活，则会损伤胰腺发育与β细胞生成。遗传拯救实验与转录组分析表明，Sufu与Spop敲除的缺陷是通过GLI2介导的胃肠道基质信号（如Wnt配体）激活所引发的。值得注意的是，在类器官与hESC培养体系中抑制Wnt信号可显著促进胰岛素分泌细胞的生成，这证实了器官特异性调控基质微环境信号的必要性。