Laboratory of Biomedical Embryology, UniStem, Center for Stem Cell Research, Università degli Studi di Milano, Italy.
Proc Natl Acad Sci U S A. 2013 May 28;110(22):8948-53. doi: 10.1073/pnas.1220637110. Epub 2013 May 21.
The differentiated state of mature cells of adult organisms is achieved and maintained through the epigenetic regulation of gene expression, which consists of several mechanisms including DNA methylation. The advent of induced pluripotent stem cell technology enabled the conversion of adult cells into any other cell type passing through a stable pluripotency state. However, indefinite pluripotency is unphysiological, inherently labile, and makes cells prone to culture-induced alterations. The direct conversion of one cell type to another without an intermediate pluripotent stage is also possible but, at present, requires the viral transfection of appropriate transcription factors, limiting its therapeutic potential. The aim of this study was to investigate whether it is possible to achieve the direct conversion of an adult cell by exposing it to a demethylating agent immediately followed by differentiating culture conditions. Adult human skin fibroblasts were exposed for 18 h to the DNA methyltransferase inhibitor 5-azacytidine, followed by a three-step protocol for the induction of endocrine pancreatic differentiation that lasted 36 d. At the end of this treatment, 35 ± 8.9% fibroblasts became pancreatic converted cells that acquired an epithelial morphology, produced insulin, and then released the hormone in response to a physiological glucose challenge in vitro. Furthermore, pancreatic converted cells were able to protect recipient mice against streptozotocin-induced diabetes, restoring a physiological response to glucose tolerance tests. This work shows that it is possible to convert adult fibroblasts into insulin-secreting cells, avoiding both a stable pluripotent stage and any transgenic modification.
成熟细胞的分化状态是通过基因表达的表观遗传调控来实现和维持的，其中包括几种机制，如 DNA 甲基化。诱导多能干细胞技术的出现使成年细胞能够通过稳定的多能性状态转化为任何其他细胞类型。然而，无限的多能性是不符合生理的，本质上是不稳定的，并且使细胞容易受到培养诱导的改变。一种细胞类型到另一种细胞类型的直接转化也是可能的，但目前需要适当的转录因子的病毒转染，限制了其治疗潜力。本研究旨在探讨是否可以通过使成年细胞暴露于去甲基化剂，然后立即进行分化培养条件，从而实现其直接转化。将成人皮肤成纤维细胞暴露于 DNA 甲基转移酶抑制剂 5-氮杂胞苷 18 小时，然后进行为期 36 天的三步内分泌胰腺分化诱导方案。在该治疗结束时，35±8.9%的成纤维细胞成为胰腺转化细胞，获得上皮形态，产生胰岛素，然后对体外生理葡萄糖挑战作出激素释放反应。此外，胰腺转化细胞能够保护受体小鼠免受链脲佐菌素诱导的糖尿病，恢复对葡萄糖耐量试验的生理反应。这项工作表明，有可能将成纤维细胞转化为分泌胰岛素的细胞，避免稳定的多能性阶段和任何转基因修饰。
