León-Quinto T, Jones J, Skoudy A, Burcin M, Soria B
Institute of Bioengineering, School of Medicine, Miguel Hernández University, San Juan Campus, Alicante, Spain.
Diabetologia. 2004 Aug;47(8):1442-51. doi: 10.1007/s00125-004-1458-8. Epub 2004 Jul 29.
AIMS/HYPOTHESIS: We recently demonstrated that insulin-producing cells derived from embryonic stem cells normalise hyperglycaemia in transplanted diabetic mice. The differentiation and selection procedure, however, was successful in less than 5% of the assays performed. Thus, to improve its effectiveness, new strategies have been developed, which increase the number of islet cells or islet progenitors.
Mouse embryonic stem cells transfected with a plasmid containing the Nkx6.1 promoter gene followed by a neomycin-resistance gene, were cultured with factors known to participate in endocrine pancreatic development and factors that modulate signalling pathways involved in these processes. Neomycin was used to select the Nkx6.1-positive cells, which also express insulin. The transfected cells were differentiated using several exogenous agents, followed by selection of Nkx6.1-positive cells. The resulting cells were analysed for pancreatic gene and protein expression by immunocytochemistry, RT-PCR and radioimmunoassay. Also, proliferation assays were performed, as well as transplantation to streptozotocin-induced diabetic mice.
The protocols yielded cell cultures with approximately 20% of cells co-expressing insulin and Pdx-1. Cell trapping selection yielded an almost pure population of insulin-positive cells, which expressed the beta cell genes/proteins Pdx-1, Nkx6.1, insulin, glucokinase, GLUT-2 and Sur-1. Subsequent transplantation to streptozotocin-induced diabetic mice normalised their glycaemia during the time period of experimentation, proving the efficiency of the protocols.
CONCLUSIONS/INTERPRETATION: These methods were both highly efficient and very reproducible, resulting in a new strategy to obtain insulin-containing cells from stem cells with a near 100% success rate, while actively promoting the maturation of the exocytotic machinery.
目的/假设：我们最近证明，源自胚胎干细胞的胰岛素生成细胞可使移植的糖尿病小鼠的高血糖正常化。然而，分化和筛选程序在不到5%的实验中取得成功。因此，为提高其有效性，已开发出新策略，可增加胰岛细胞或胰岛祖细胞的数量。
用含有Nkx6.1启动子基因和新霉素抗性基因的质粒转染小鼠胚胎干细胞，将其与已知参与内分泌胰腺发育的因子以及调节这些过程中信号通路的因子一起培养。使用新霉素筛选同时表达胰岛素的Nkx6.1阳性细胞。用几种外源性试剂使转染细胞分化，随后筛选Nkx6.1阳性细胞。通过免疫细胞化学、逆转录-聚合酶链反应（RT-PCR）和放射免疫测定法分析所得细胞的胰腺基因和蛋白质表达。此外，进行增殖测定，并将细胞移植到链脲佐菌素诱导的糖尿病小鼠体内。
这些方案产生的细胞培养物中约20%的细胞同时表达胰岛素和Pdx-1。细胞捕获筛选产生了几乎纯的胰岛素阳性细胞群体，这些细胞表达β细胞基因/蛋白质Pdx-1、Nkx6.1、胰岛素、葡萄糖激酶、葡萄糖转运蛋白2（GLUT-2）和磺脲类受体1（Sur-1）。随后将其移植到链脲佐菌素诱导的糖尿病小鼠体内，在实验期间使它们的血糖正常化，证明了这些方案的有效性。
结论/解读：这些方法高效且可重复性强，产生了一种从干细胞中获得含胰岛素细胞的新策略，成功率接近100%，同时积极促进胞吐机制的成熟。
