Maherali Nimet, Ahfeldt Tim, Rigamonti Alessandra, Utikal Jochen, Cowan Chad, Hochedlinger Konrad
Department of Stem Cell and Regenerative Biology, Harvard Stem Cell Institute, Massachusetts General Hospital Center for Regenerative Medicine, Massachusetts General Hospital Cancer Center, Boston, MA 02114, USA.
Cell Stem Cell. 2008 Sep 11;3(3):340-5. doi: 10.1016/j.stem.2008.08.003.
Direct reprogramming of human fibroblasts to a pluripotent state has been achieved through ectopic expression of the transcription factors OCT4, SOX2, and either cMYC and KLF4 or NANOG and LIN28. Little is known, however, about the mechanisms by which reprogramming occurs, which is in part limited by the low efficiency of conversion. To this end, we sought to create a doxycycline-inducible lentiviral system to convert primary human fibroblasts and keratinocytes into human induced pluripotent stem cells (hiPSCs). hiPSCs generated with this system were molecularly and functionally similar to human embryonic stem cells (hESCs), demonstrated by gene expression profiles, DNA methylation status, and differentiation potential. While expression of the viral transgenes was required for several weeks in fibroblasts, we found that 10 days was sufficient for the reprogramming of keratinocytes. Using our inducible system, we developed a strategy to induce hiPSC formation at high frequency. Upon addition of doxycycline to hiPSC-derived differentiated cells, we obtained "secondary" hiPSCs at a frequency at least 100-fold greater than the initial conversion. The ability to reprogram cells at high efficiency provides a unique platform to dissect the underlying molecular and biochemical processes that accompany nuclear reprogramming.
通过异位表达转录因子OCT4、SOX2以及cMYC和KLF4或NANOG和LIN28,已实现将人类成纤维细胞直接重编程为多能状态。然而,对于重编程发生的机制知之甚少,这在一定程度上受到转化效率低下的限制。为此,我们试图创建一种强力霉素诱导的慢病毒系统,将原代人类成纤维细胞和角质形成细胞转化为人类诱导多能干细胞(hiPSC)。用该系统产生的hiPSC在分子和功能上与人类胚胎干细胞(hESC)相似,这通过基因表达谱、DNA甲基化状态和分化潜能得以证明。虽然在成纤维细胞中病毒转基因的表达需要数周时间,但我们发现10天足以使角质形成细胞重编程。利用我们的诱导系统,我们开发了一种在高频下诱导hiPSC形成的策略。在向hiPSC衍生的分化细胞中添加强力霉素后,我们获得了“二级”hiPSC,其频率比初始转化至少高100倍。高效重编程细胞的能力为剖析伴随核重编程的潜在分子和生化过程提供了一个独特的平台。