Junior Research Group Stem Cell Biology, Cluster of Excellence REBIRTH, Hannover Medical School, 30625 Hannover, Germany.
Cytometry A. 2011 Jun;79(6):426-35. doi: 10.1002/cyto.a.21072. Epub 2011 May 4.
Somatic cells can be reprogrammed toward pluripotency by overexpression of a set of transcription factors, yielding induced pluripotent stem cells (iPSCs) with features similar to embryonic stem cells. Little is known to date about stoichiometric requirements of the individual reprogramming factors (RFs) for efficient reprogramming and especially about whether stoichiometry also influences the quality of derived iPSCs. To address this important issue, we chose bicistronic lentiviral vectors coexpressing fluorescent reporters (eGFP, dTomato, Cerulean, or Venus) along with the canonical RFs to transduce a bulk of murine embryonic fibroblasts (MEFs). Using a flow cytometric approach, we were able to independently and proportionally quantify all fluorophores in multiple-infected MEFs and more importantly could sort these cells into all 16 stoichiometric combinations of high or moderate expression of the four factors. On average, we obtained about 600 alkaline phosphatase-expressing colonies from 20,000 seeded cells. Interestingly, only seven different stoichiometric ratios gave rise to any colonies at all. The by far most colonies were obtained from those fractions, where Oct4 was in excess over the other three factors (2,386 colonies/20,000 cells), or where both Oct4 and c-Myc were in excess over Sox2 and Klf4 (1,593 colonies/20,000 cells). Our findings suggest that increased Oct4 levels opposite to modest ones for Sox2 and Klf4 are required for satisfying reprogramming efficiencies and that these stoichiometries are also highly beneficial for achieving a stable pluripotent state independent of ectopic RF expression. Finally, the eligible Oct4(high) , Sox2(low) , and Klf4(low) subpopulation only resembles a small fraction of cells targeted by equal vector amounts, suggesting the necessity to address stoichiometry also in alternative approaches for iPSC generation or between different experimental systems.
体细胞可以通过过表达一组转录因子重编程为多能性，从而产生具有类似于胚胎干细胞特征的诱导多能干细胞（iPSC）。目前，人们对单个重编程因子（RF）的化学计量要求知之甚少，对于有效重编程，特别是对于衍生的 iPSC 的质量是否也受到化学计量的影响。为了解决这个重要问题，我们选择了双顺反子慢病毒载体，共同表达荧光报告基因（eGFP、dTomato、Cerulean 或 Venus）以及经典的 RF，以转导大量的小鼠胚胎成纤维细胞（MEF）。使用流式细胞术方法，我们能够独立且成比例地量化多个感染的 MEF 中的所有荧光团，更重要的是，我们可以将这些细胞按四个因素的高或中表达的 16 种不同化学计量比进行分选。平均而言，我们从 20,000 个接种细胞中获得了约 600 个碱性磷酸酶阳性集落。有趣的是，只有七种不同的化学计量比会产生任何集落。到目前为止，获得的集落最多的是那些 Oct4 相对于其他三个因子过量的部分（2,386 个集落/20,000 个细胞），或者 Oct4 和 c-Myc 相对于 Sox2 和 Klf4 过量的部分（1,593 个集落/20,000 个细胞）。我们的研究结果表明，增加 Oct4 水平，同时适度增加 Sox2 和 Klf4 的水平，对于满足重编程效率是必要的，并且这些化学计量对于获得独立于异位 RF 表达的稳定多能状态也非常有益。最后，合格的 Oct4(高)、Sox2(低)和 Klf4(低)亚群仅类似于用等量载体靶向的一小部分细胞，这表明在 iPSC 生成的替代方法或不同的实验系统之间也需要解决化学计量问题。
