Shim J H, Kim S E, Woo D H, Kim S K, Oh C H, McKay R, Kim J H
Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, West Building/Room 304, Science Campus, 1 Anam-dong 5-ga, Sungbuk-goo, Seoul 136-713, Republic of Korea.
Diabetologia. 2007 Jun;50(6):1228-38. doi: 10.1007/s00125-007-0634-z. Epub 2007 Apr 18.
AIMS/HYPOTHESIS: The relative lack of successful pancreatic differentiation of human embryonic stem cells (hESCs) may suggest that directed differentiation of hESCs into definitive endoderm and subsequent commitment towards a pancreatic fate are not readily achieved. The aim of this study was to investigate whether sequential exposure of hESCs to epigenetic signals that mimic in vivo pancreatic development can efficiently generate pancreatic endodermal cells, and whether these cells can be further matured and reverse hyperglycaemia upon transplantation.
The hESCs were sequentially treated with serum, activin and retinoic acid (RA) during embryoid body formation. The patterns of gene expression and protein production associated with embryonic germ layers and pancreatic endoderm were analysed by RT-PCR and immunostaining. The developmental competence and function of hESC-derived PDX1-positive cells were evaluated after in vivo transplantation.
Sequential treatment with serum, activin and RA highly upregulated the expression of the genes encoding forkhead box protein A2 (FOXA2), SRY-box containing gene 17 (SOX17), pancreatic and duodenal homeobox 1 (PDX1) and homeobox HB9 (HLXB9). The population of pancreatic endodermal cells that produced PDX1 was significantly increased at the expense of ectodermal differentiation, and a subset of the PDX1-positive cells also produced FOXA2, caudal-type homeobox transcription factor 2 (CDX2), and nestin (NES). After transplantation, the PDX1-positive cells further differentiated into mature cell types producing insulin and glucagon, resulting in amelioration of hyperglycaemia and weight loss in streptozotocin-treated diabetic mice.
CONCLUSIONS/INTERPRETATION: Our strategy allows the progressive differentiation of hESCs into pancreatic endoderm capable of generating mature pancreatic cell types that function in vivo. These findings may establish the basis of further investigations for the purification of transplantable islet progenitors derived from hESCs.
目的/假设：人类胚胎干细胞（hESC）相对缺乏成功的胰腺分化，这可能表明将hESC定向分化为确定的内胚层并随后定向分化为胰腺命运并不容易实现。本研究的目的是调查hESC依次暴露于模拟体内胰腺发育的表观遗传信号是否能有效产生胰腺内胚层细胞，以及这些细胞在移植后是否能进一步成熟并逆转高血糖症。
在胚胎体形成过程中，hESC依次用血清、激活素和视黄酸（RA）处理。通过逆转录-聚合酶链反应（RT-PCR）和免疫染色分析与胚胎胚层和胰腺内胚层相关的基因表达和蛋白质产生模式。在体内移植后评估hESC来源的胰腺十二指肠同源盒1（PDX1）阳性细胞的发育能力和功能。
依次用血清、激活素和RA处理可高度上调编码叉头框蛋白A2（FOXA2）、含SRY框基因17（SOX17）、胰腺和十二指肠同源盒1（PDX1）和同源盒HB9（HLXB9）的基因表达。产生PDX1的胰腺内胚层细胞群体显著增加，以外胚层分化为代价，并且一部分PDX1阳性细胞还产生FOXA2、尾型同源盒转录因子2（CDX2）和巢蛋白（NES）。移植后，PDX1阳性细胞进一步分化为产生胰岛素和胰高血糖素的成熟细胞类型，从而改善链脲佐菌素诱导的糖尿病小鼠的高血糖症和体重减轻。
结论/解读：我们的策略允许hESC逐步分化为能够产生在体内发挥功能的成熟胰腺细胞类型的胰腺内胚层。这些发现可能为进一步研究从hESC中纯化可移植胰岛祖细胞奠定基础。
