LSD1多肽抑制剂调控人iPSCs分化为胰岛素分泌细胞的作用及机制研究

Insulin-producing cells (IPCs) derived from human induced pluripotent stem cells (hiPSCs) have been considered as a key area of translational research for the effective treatment of diabetes. Nevertheless, the low efficiency of differentiation limits further application of IPCs in clinical therapy. Previous studies and our results demonstrated that LSD1 directly interacts with p53 in the CoREST complex to repress p53 activation. Interestingly, inhibition of LSD1 revealed a increase in IPCs production and associated p53 and Smad2 activity. However, the mechanism by which LSD1 inactivation promotes the differentiation of IPCs by inducing p53 and Smad2 activation is unclear. Herein, we hypothesize that inhibition of LSD1 activity leading to p53 activation, which is capable of promoting stem cell differentiation by inducing pancreatic beta-cell transcription factors expression in a Smad2-dependent manner. In this study, we designe and synthesize LSD1 peptide inhibitor that block LSD1 activity, in order to improve the differentiation efficiency of IPCs. Further investigation will be carried out to clarify how LSD1 regulates p53-Smad2 signaling pathway, and establish a strategy aimed at generating high numbers of functional pancreatic β cells in vitro, which will contribute to stem cell therapy for diabetes.

人诱导多能干细胞（hiPSCs）来源的胰岛素分泌细胞是治疗糖尿病理想的种子细胞之一，但分化效率低和成熟度差限制了其临床应用。文献报道和课题组前期研究表明，LSD1募集p53锚定在CoREST复合物中，使p53失活；抑制LSD1活性能够促进p53和Smad2信号活化，并提高胰岛素分泌细胞分化效率，但LSD1通过p53-Smad2信号促进hiPSCs向胰岛细胞分化的机制尚不清楚。我们推测抑制LSD1活性促使p53激活，活化后的p53可通过激活Smad2信号促进胰岛β细胞关键转录因子转录，从而促进干细胞分化。本研究设计合成LSD1多肽抑制剂，采用经典四步诱导法筛选出促进胰岛细胞分化最佳的LSD1活性；通过在不同分化阶段加入p53或Smad2抑制剂，明确其在干细胞分化中的作用，揭示LSD1调控p53-Smad2信号促进hiPSCs高效定向分化为胰岛β细胞的分子机制，为干细胞治疗糖尿病奠定理论基础。

人诱导多能干细胞（hiPSCs）来源的胰岛素分泌细胞是治疗糖尿病理想的种子细胞之一，但分化效率低和成熟度差限制了其临床应用。本研究中，①我们首先优化了胰岛素分泌细胞体外诱导分化方案，为开展后续研究工作提供了良好的技术平台；②针对LSD1的9个特异性氨基酸序列，设计并合成了靶向LSD1的多肽化合物，确认了其能在细胞核内与LSD1蛋白共定位，并能以剂量依赖的方式抑制LSD1的去甲基化酶活性；③确认了抑制LSD1对限定性内胚层细胞分化无明显影响，但能够显著促进胰腺祖细胞和胰岛素分泌细胞分化，同时筛选到促进胰岛细胞分化的最适多肽化合物浓度为6.25μM。据此，建立了LSD1多肽抑制剂调控人多能干细胞分化为胰岛素分泌细胞的诱导方案；④阐明了LSD1通过影响组蛋白H3K4甲基化水平和ERK信号活化调控人多能干细胞分化为胰岛细胞的分子机制；⑤筛选到4个可能参与调控胰岛细胞分化的microRNAs，同时阐明了microRNA-181c-5p通过抑制smad7和TGIF2表达，促进smad2/3信号活化，进而促进胰岛细胞分化的分子机制。⑥揭示了IFI16通过与p53相互作用增强p53在基因组上的结合能力，进而促进三胚层分化基因表达、抑制干性基因表达，从而促进干细胞分化的分子机制。综上所述，本研究揭示了LSD1、miR-181c-5p和IFI16调控干细胞分化的分子机制，不仅为体外获得高效分化且功能成熟的胰岛β细胞奠定了理论基础，还是关乎我国大众健康和社会经济发展的重要课题。