Division of Cardiovascular Diseases, Departments of Medicine, Mayo Clinic, Rochester, MN, USA.
J Cardiovasc Transl Res. 2010 Feb;3(1):13-23. doi: 10.1007/s12265-009-9150-5.
Induced pluripotent stem cell (iPS) technology has launched a new platform in regenerative medicine aimed at deriving unlimited replacement tissue from autologous sources through somatic cell reprogramming using stemness factor sets. In this way, authentic cardiomyocytes have been obtained from iPS and recently demonstrated in proof-of-principle studies to repair infarcted heart. Optimizing the cardiogenic potential of iPS progeny would ensure a maximized yield of bioengineered cardiac tissue. Here, we reprogrammed fibroblasts in the presence or absence of c-MYC to determine if the acquired cardiogenicity is sensitive to the method of nuclear reprogramming. Using lentiviral constructs that expressed stemness factors SOX2, OCT4, and KLF4 with or without c-MYC, iPS clones generated through fibroblast reprogramming demonstrated indistinguishable characteristics for 5 days of differentiation with similar cell morphology, growth rates, and chimeric embryo integration. However, 4-factor c-MYC dependent nuclear reprogramming produced iPS progeny that consistently prolonged the expression of pluripotent Oct-4 and Fgf4 genes and repressed cardiac differentiation. In contrast, 3-factor c-MYC-less iPS clones efficiently up-regulated pre-cardiac (CXCR4, Flk-1, and Mesp1/2) and cardiac (Nkx2.5, Mef2c, and Myocardin) gene expression patterns. In fact, 3-factor iPS progeny demonstrated early and robust cardiogenesis during in vitro differentiation with consistent beating activity, sarcomere maturation, and rhythmical intracellular calcium dynamics. Thus, nuclear reprogramming independent of c-MYC enhances production of pluripotent stem cells with innate cardiogenic potential.
诱导多能干细胞(iPS)技术在再生医学领域开创了一个新的平台,旨在通过使用干性因子集对自体来源的体细胞进行重编程,从而获得无限量的替代组织。通过这种方式,已经从 iPS 中获得了真正的心肌细胞,并在最近的原理验证研究中证明可修复梗死的心脏。优化 iPS 后代的成心肌潜能将确保生物工程心脏组织的产量最大化。在这里,我们在存在或不存在 c-MYC 的情况下重编程成纤维细胞,以确定获得的成心肌性是否对核重编程方法敏感。使用表达干性因子 SOX2、OCT4 和 KLF4 的慢病毒构建体,带有或不带有 c-MYC,通过成纤维细胞重编程产生的 iPS 克隆在 5 天的分化过程中表现出相似的细胞形态、生长速度和嵌合胚胎整合,具有不可区分的特征。然而,4 因子 c-MYC 依赖性核重编程产生的 iPS 后代持续延长多能 Oct-4 和 Fgf4 基因的表达,并抑制心脏分化。相比之下,3 因子 c-MYC 缺失的 iPS 克隆有效地上调了前心肌(CXCR4、Flk-1 和 Mesp1/2)和心肌(Nkx2.5、Mef2c 和 Myocardin)基因表达模式。事实上,3 因子 iPS 后代在体外分化过程中表现出早期和强大的成心肌性,具有一致的搏动活性、肌节成熟和节律性细胞内钙动力学。因此,不依赖 c-MYC 的核重编程增强了具有固有成心肌潜能的多能干细胞的产生。
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