Center for Engineering in Medicine, Harvard Medical School, Massachusetts General Hospital, Boston, MA 02114, USA.
J Tissue Eng Regen Med. 2011 Apr;5(4):313-23. doi: 10.1002/term.317. Epub 2010 Aug 17.
Promise of cellular therapy for type 1 diabetes has inspired the search for transplantable cell sources, and embryonic stem cells (ESCs) have emerged as strong candidates. We have developed a directed differentiation protocol to obtain insulin-producing cells from ESCs. The ESCs are first induced towards a homogeneous monolayer of definitive endoderm-like cells by co-culture with primary hepatocytes. Pancreatic commitment is induced by plating the ESC-derived endoderms on Matrigel, along with Sonic hedgehog inhibition and retinoid induction. More than 70% of differentiated cells positively upregulated Pdx-1, along with pro-endocrine transcription factors Ngn3, β2/neroD1, Nkx2.2 and Nkx6.1. Final maturation to islet-specific cells is achieved by co-culturing the ESC-derived pancreatic endocrine cells with endothelial cells, which resulted in Insulin 1 upregulation in 60% of the cell population, along with high levels of IAPP and Glut2. The differentiated cell population also secreted high levels of insulin. Our findings illustrate the significant effect of co-culture in different stages of differentiation and maturation of ESCs in vitro. Such a high yield of pancreatic islet cells has not yet been reported. Our findings establish a robust protocol for islet differentiation.
细胞疗法治疗 1 型糖尿病的前景激发了人们对可移植细胞来源的探索，胚胎干细胞（ESCs）成为强有力的候选者。我们开发了一种定向分化方案，从 ESCs 中获得产生胰岛素的细胞。首先，通过与原代肝细胞共培养，将 ESCs 诱导为同质的单层确定内胚层样细胞。通过在 Matrigel 上接种 ESC 衍生的内胚层，并抑制 Sonic hedgehog 和诱导类视黄醇，诱导胰腺分化。超过 70%的分化细胞上调 Pdx-1，同时上调前内分泌转录因子 Ngn3、β2/neroD1、Nkx2.2 和 Nkx6.1。通过将 ESC 衍生的胰腺内分泌细胞与内皮细胞共培养，实现向胰岛特异性细胞的最终成熟，这导致 60%的细胞群体中胰岛素 1 的上调，同时高水平的 IAPP 和 Glut2。分化细胞群体也分泌高水平的胰岛素。我们的研究结果表明，共培养在 ESCs 体外分化和成熟的不同阶段具有重要作用。尚未有报道显示如此高产量的胰岛细胞。我们的研究结果建立了一种用于胰岛分化的稳健方案。
