scGOF-Seq using ND islets

Type 2 diabetes is associated with defective insulin secretion and reduced ß-cell mass. Available treatments provide a temporary reprieve, but secondary failure rates are high, making insulin supplementation necessary. Reversibility of b-cell failure is a key translational question. Here, we reverse-engineered and interrogated pancreatic islet-specific regulatory networks to discover T2D-specific subpopulations characterized by metabolic-inflexibility and endocrine-progenitor/stem cell features. Single-cell gain- and loss-of-function and glucose-induced Ca++ flux analyses of top candidate MR in islet cells validated transcription factor BACH2 and associated epigenetic effectors as a key driver of T2D cell states. BACH2 knockout in T2D islets reversed cellular features of the disease, restoring a non-diabetic phenotype. BACH2-immunoreactive islet cells increased ~4-fold in diabetic patients, confirming the algorithmic prediction of clinically relevant subpopulations. Treatment with a BACH inhibitor lowered glycemia and increased plasma insulin levels in diabetic mice, and restored insulin secretion in diabetic mice and human islets. The findings suggest that T2D-specific populations of failing b-cells can be reversed and indicate pathways for pharmacological intervention, including via BACH2 inhibition. Overall design: Adenovirus encoding candidate TF with its corresponding guide-barcode was infected in human nondiabetic islets which are subjected to scRNA-seq.

2型糖尿病（Type 2 diabetes, T2D）与胰岛素分泌缺陷及β细胞（β-cell）质量减少密切相关。现有治疗手段仅能提供暂时缓解，但二次失效比例极高，最终需依赖胰岛素补充治疗。β细胞衰竭的可逆性是转化医学领域的核心研究问题。本研究通过逆向工程解析胰腺胰岛特异性调控网络，发现了以代谢灵活性丧失及内分泌祖细胞/干细胞特征为标志的2型糖尿病特异性细胞亚群。随后，我们对胰岛细胞中的核心候选调控因子开展单细胞功能获得与功能缺失实验，并进行葡萄糖诱导钙离子流分析，验证了转录因子（Transcription Factor, TF）BACH2及其关联的表观遗传效应因子是驱动2型糖尿病细胞状态的关键调控因子。在2型糖尿病胰岛中敲除BACH2可逆转疾病相关细胞特征，恢复非糖尿病表型。糖尿病患者体内BACH2免疫阳性胰岛细胞数量较健康人群增加约4倍，印证了算法预测的临床相关细胞亚群的存在。使用BACH抑制剂治疗可降低糖尿病小鼠的血糖水平并升高血浆胰岛素浓度，同时可恢复糖尿病小鼠及人胰岛的胰岛素分泌功能。本研究结果表明，衰竭的2型糖尿病特异性β细胞群体可被逆转，同时为包括BACH2抑制在内的药物干预途径提供了理论依据。实验整体设计：将携带对应引导条形码的候选转录因子（Transcription Factor, TF）的腺病毒感染人非糖尿病胰岛，随后对其进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。