Insulin-producing cells generated from dedifferentiated human pancreatic beta cells expanded in vitro

Expansion of beta cells from the limited number of adult human islet donors is an attractive prospect for increasing cell availability for cell therapy of diabetes. However, while evidence supports the replicative capacity of adult beta cells in vivo, attempts at expanding human islet cells in tissue culture resulted in loss of beta-cell phenotype. Using a genetic lineage-tracing approach we have provided evidence for massive proliferation of beta-cell-derived (BCD) cells within these cultures. Expansion involves dedifferentiation resembling epithelial-mesenchymal transition (EMT). Epigenetic analyses indicate that key beta-cell genes maintain a partially open chromatin structure in expanded BCD cells, although they are not transcribed. Here we report that BCD cells can be induced to redifferentiate by a combination of soluble factors. The redifferentiated cells express beta-cell genes, store insulin in typical secretory vesicles, and release it in response to glucose. The redifferentiation process involves mesenchymal-epithelial transition, as judged from changes in gene expression. Moreover, inhibition of the EMT effector SLUG using shRNA results in stimulation of redifferentiation. BCD cells also give rise at a low rate to cells expressing other islet hormones, suggesting transition through an islet progenitor-like stage during redifferentiation. These findings suggest that ex-vivo expansion of adult human islet cells is a promising approach for generation of insulin-producing cells for transplantation, as well as basic research, toxicology studies, and drug screening. Gene expression was studied in unexpanded islets (4 donors), expanded and dedifferentiated islet cells (4 donors), and re-differentiated islet cells (3 donors). The experiment was performed in 3 batches (see Date in the description table below).

从有限的成人人类胰岛供体中扩增β细胞，是提升糖尿病细胞治疗可用细胞资源的极具吸引力的研究方向。然而，尽管已有证据证实成人β细胞在体内具备复制潜能，但在组织培养体系中扩增人类胰岛细胞的尝试往往会导致β细胞表型丢失。本研究采用遗传谱系示踪（genetic lineage-tracing）技术，证实了上述培养体系中β细胞来源（beta-cell-derived, BCD）细胞的大规模增殖现象。该扩增过程涉及类似上皮间质转化（epithelial-mesenchymal transition, EMT）的去分化过程。表观遗传分析显示，尽管关键β细胞基因未发生转录，但在扩增后的BCD细胞中，这些基因的染色质仍维持部分开放状态。本研究报道，可通过可溶性因子组合诱导BCD细胞发生再分化。再分化后的细胞可表达β细胞相关基因，在典型分泌囊泡中储存胰岛素，并可响应葡萄糖刺激释放胰岛素。从基因表达变化来看，该再分化过程涉及间质上皮转化（mesenchymal-epithelial transition, MET）。此外，采用短发夹RNA（short hairpin RNA, shRNA）抑制EMT效应因子SLUG，可促进再分化过程。BCD细胞还可低效率分化为表达其他胰岛激素的细胞，提示再分化过程中存在类胰岛祖细胞阶段的过渡。上述研究结果表明，成人人类胰岛细胞的离体扩增，是获取可用于移植的胰岛素分泌细胞的极具前景的策略，同时也可应用于基础研究、毒理学研究及药物筛选领域。本研究对未扩增胰岛（4名供体）、扩增并去分化的胰岛细胞（4名供体）以及再分化胰岛细胞（3名供体）进行了基因表达分析。实验共设置3个生物学重复批次（详见下文描述表格中的日期信息）。