Novak Atara, Shtrichman Ronit, Germanguz Igal, Segev Hanna, Zeevi-Levin Naama, Fishman Bettina, Mandel Ya-El, Barad Lili, Domev Hagit, Kotton Darrell, Mostoslavsky Gustavo, Binah Ofer, Itskovitz-Eldor Joseph
Sohnis and Forman Families Center for Stem Cell and Tissue Regeneration Research, Ruth & Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel.
Cell Reprogram. 2010 Dec;12(6):665-78. doi: 10.1089/cell.2010.0027. Epub 2010 Oct 21.
Induced pluripotent stem cells (iPSCs) represent an ideal cell source for future cell therapy and regenerative medicine. However, most iPSC lines described to date have been isolated from skin fibroblasts or other cell types that require harvesting by surgical intervention. Because it is desirable to avoid such intervention, an alternative cell source that can be readily and noninvasively isolated from patients and efficiently reprogrammed, is required. Here we describe a detailed and reproducible method to derive iPSCs from plucked human hair follicle keratinocytes (HFKTs). HFKTs were isolated from single plucked hair, then expanded and reprogrammed by a single polycistronic excisable lentiviral vector. The reprogrammed HFKTs were found to be very sensitive to human embryonic stem cell (hESC) growth conditions, generating a built-in selection with easily obtainable and very stable iPSCs. All emerging colonies were true iPSCs, with characteristics typical of human embryonic stem cells, differentiated into derivatives of all three germ layers in vitro and in vivo. Spontenaeouly differentiating functional cardiomyocytes (CMs) were successfully derived and characterized from these HFKT-iPSCs. The contracting CMs exhibited well-coordinated intracellular Ca²+ transients and contractions that were readily responsive to β-adrenergic stimulation with isoproterenol. The introduction of Cre-recombinase to HFKT-iPSC clones was able to successfully excise the integrated vector and generate transgene-free HFKT-iPSC clone that could be better differentiated into contracting CMs, thereby revealing the desired cells for modeling human diseases. Thus, HFKTs are easily obtainable, and highly reprogrammed human cell source for all iPSC applications.
诱导多能干细胞（iPSC）是未来细胞治疗和再生医学的理想细胞来源。然而，迄今为止所描述的大多数iPSC系都是从皮肤成纤维细胞或其他需要通过手术干预采集的细胞类型中分离出来的。由于希望避免这种干预，因此需要一种能够从患者身上轻松、无创地分离并有效重编程的替代细胞来源。在此，我们描述了一种从拔除的人毛囊角质形成细胞（HFKT）中获得iPSC的详细且可重复的方法。HFKT是从单根拔除的毛发中分离出来的，然后通过单个多顺反子可切除慢病毒载体进行扩增和重编程。发现重编程的HFKT对人胚胎干细胞（hESC）生长条件非常敏感，从而产生一种易于获得且非常稳定的iPSC的内在选择。所有出现的集落都是真正的iPSC，具有人类胚胎干细胞的典型特征，在体外和体内均可分化为所有三个胚层的衍生物。成功地从这些HFKT-iPSC中获得了自发分化的功能性心肌细胞（CM）并对其进行了表征。收缩的CM表现出协调良好的细胞内Ca²⁺瞬变和收缩，并且对异丙肾上腺素的β-肾上腺素能刺激有反应。将Cre重组酶引入HFKT-iPSC克隆能够成功切除整合的载体并产生无转基因的HFKT-iPSC克隆，该克隆能够更好地分化为收缩的CM，从而为人类疾病建模提供所需的细胞。因此，HFKT是一种易于获得且高度重编程的人类细胞来源，可用于所有iPSC应用。
