Stem Cell Program, Institute for Cell Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Stem Cells. 2010 Apr;28(4):713-20. doi: 10.1002/stem.402.
We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming.
我们在此报告，丁酸是一种天然存在的脂肪酸，通常用作营养补充剂和分化剂，可大大提高诱导多能干细胞（iPS 细胞）从人成体或胎儿成纤维细胞中产生的效率。在用逆转录病毒或猪β类反转录病毒转座子载体表达 4 到 5 个重编程基因进行瞬时丁酸处理后，iPS 细胞的产生效率提高了 15 到 51 倍。在没有 KLF4 或 MYC 转基因的情况下，丁酸刺激在重编程中更为显著（> 100 到 200 倍）。丁酸处理不会对通过 3 个或 4 个逆转录病毒载体或单个猪β类反转录病毒 DNA 转座子载体建立的 iPS 细胞系的特性产生负面影响。这些经过表征的 iPS 细胞系，包括通过猪β类反转录病毒或逆转录病毒载体从镰状细胞病患者中获得的细胞，显示出正常的核型和多能性。为了深入了解丁酸刺激的潜在机制，我们对已建立的 iPS 细胞和重编程过程中中间阶段的细胞进行了全基因组基因表达和启动子 DNA 甲基化微阵列以及其他表观遗传分析。在重编程的第 6 天到第 12 天期间，丁酸处理增强了组蛋白 H3 乙酰化、启动子 DNA 去甲基化以及内源性多能性相关基因的表达，包括 DPPA2，其过表达部分替代了丁酸的刺激作用。因此，丁酸作为一种细胞可渗透的小分子，为进一步研究细胞重编程的分子机制提供了一种简单的工具。此外，丁酸刺激为各种人成体体细胞的重编程提供了一种有效的方法，包括对重编程更具抗性的患者细胞。
