Division of Endocrinology and Diabetology, Department of Internal Medicine II, University Hospital of Freiburg , Freiburg, Germany.
Cytotherapy. 2011 Aug;13(7):802-13. doi: 10.3109/14653249.2011.571248. Epub 2011 Apr 21.
Reprogramming of multipotent adult bone marrow (BM)-derived mesenchymal stromal/stem cells (MSC) (BM-MSC) represents one of several strategies for cell-based therapy of diabetes. However, reprogramming primary BM-MSC into pancreatic endocrine lineages has not yet been consistently demonstrated.
To unravel the role and interaction of key factors governing this process, we used well-characterized telomerase-immortalized human MSC (hMSC-TERT). Pancreatic endocrine differentiation in hMSC-TERT was induced by two major in vitro strategies: (i) endocrine-promoting culture conditions and (ii) ectopic expression of two master regulatory genes of the endocrine lineage, human neurogenin 3 (NGN3) and human pancreatic duodenal homeobox 1 (PDX1).
Both approaches triggered pancreatic endocrine gene expression, notably insulin, glucose-transporter 2 and somatostatin. Transgenic overexpression of NGN3 and/or PDX1 proteins not only induced direct target genes, such as NEUROD1 and insulin, and but also triggered parts of the gene expression cascade that is involved in pancreatic endocrine differentiation. Notably, ectopic NGN3 alone was sufficient to initiate the expression of specific beta-cell lineage-associated genes, most importantly PDX1 and insulin. This was demonstrated both transcriptionally by mRNA expression and reporter gene analyzes and at a protein level by Western blotting. Such reprogramming of hMSC-TERT cells induced glucose-insensitive insulin biosynthesis and secretion.
Our results indicate that establishment of glucose-dependent insulin secretion in partially reprogrammed human MSC may depend on additional maturation factors. Moreover, hMSC-TERT provides a suitable cell model for investigating further the molecular mechanisms of reprogramming and maturation of adult MSC towards pancreatic endocrine lineages.
多能成体骨髓（BM）来源间充质基质/干细胞（MSC）（BM-MSC）的重编程是糖尿病细胞治疗的几种策略之一。然而，将原代 BM-MSC 重编程为胰腺内分泌谱系尚未得到一致证明。
为了揭示调控这一过程的关键因素的作用和相互作用，我们使用了经过充分特征鉴定的端粒酶永生化人 MSC（hMSC-TERT）。通过两种主要的体外策略诱导 hMSC-TERT 的胰腺内分泌分化：（i）内分泌促进培养条件和（ii）内源性表达两个内分泌谱系的主调控基因，人神经基因 3（NGN3）和人胰腺十二指肠同源盒 1（PDX1）。
这两种方法都触发了胰腺内分泌基因的表达，特别是胰岛素、葡萄糖转运蛋白 2 和生长抑素。NGN3 和/或 PDX1 蛋白的转基因过表达不仅诱导了直接靶基因，如 NEUROD1 和胰岛素，而且还触发了参与胰腺内分泌分化的部分基因表达级联。值得注意的是，单独的异位 NGN3 足以启动特定的β细胞谱系相关基因的表达，最重要的是 PDX1 和胰岛素。这通过 mRNA 表达和报告基因分析的转录以及 Western blot 的蛋白质水平得到证实。这种 hMSC-TERT 细胞的重编程诱导了葡萄糖不敏感的胰岛素生物合成和分泌。
我们的结果表明，在部分重编程的人 MSC 中建立葡萄糖依赖性胰岛素分泌可能依赖于其他成熟因子。此外，hMSC-TERT 为进一步研究成人 MSC 向胰腺内分泌谱系重编程和成熟的分子机制提供了一个合适的细胞模型。
