Panciera Tito, Azzolin Luca, Di Biagio Daniele, Totaro Antonio, Cordenonsi Michelangelo, Piccolo Stefano
Department of Molecular Medicine, University of Padua School of Medicine.
Department of Molecular Medicine, University of Padua School of Medicine;
J Vis Exp. 2018 May 7(135):57462. doi: 10.3791/57462.
Here we present protocols to isolate primary differentiated cells and turn them into stem/progenitor cells (SCs) of the same lineage by transient expression of the transcription factor YAP. With this method, luminal differentiated (LD) cells of the mouse mammary gland are converted into cells that exhibit molecular and functional properties of mammary SCs. YAP also turns fully differentiated pancreatic exocrine cells into pancreatic duct-like progenitors. Similarly, to endogenous, natural SCs, YAP-induced stem-like cells ("ySCs") can be eventually expanded as organoid cultures long term in vitro, without further need of ectopic YAP/TAZ, as ySCs are endowed with a heritable self-renewing SC-like state. The reprogramming procedure presented here offers the possibility to generate and expand in vitro progenitor cells of various tissue sources starting from differentiated cells. The straightforward expansion of somatic cells ex vivo has implications for regenerative medicine, for understanding mechanisms of tumor initiation and, more in general, for cell and developmental biology studies.
在此，我们展示了分离原代分化细胞并通过瞬时表达转录因子YAP将其转化为同谱系的干细胞/祖细胞（SCs）的方案。通过这种方法，小鼠乳腺的腔面分化（LD）细胞被转化为具有乳腺SCs分子和功能特性的细胞。YAP还能将完全分化的胰腺外分泌细胞转化为胰腺导管样祖细胞。同样，与内源性天然SCs一样，YAP诱导的干细胞样细胞（“ySCs”）最终可作为类器官培养物在体外长期扩增，无需进一步异位表达YAP/TAZ，因为ySCs具有可遗传的类似干细胞的自我更新状态。本文介绍的重编程程序为从分化细胞开始体外生成和扩增各种组织来源的祖细胞提供了可能性。体细胞在体外的直接扩增对再生医学、理解肿瘤起始机制以及更广泛地对细胞和发育生物学研究都有影响。
