Insulin-secreting cells from induced pluripotent stem cells regulate blood glucose levels in vitro

doi:10.3969/j.issn.2095-4344.2016.28.016

Abstract

Abstract:

BACKGROUND: Mouse pluripotent stem cells are induced to differentiate into insulin-secreting cells that can effectively improve blood glucose levels in diabetic mice.
OBJECTIVE: To detect mRNA and protein levels of insulin-like cell clusters from induced pluripotent stem cells and to investigate the function of insulin-secreting cells in vitro and in vivo.
METHODS: Mouse induced pluripotent stem cells cultured in vitro were induced to differentiate into insulin-secreting cells using combined inducers through three stages. The morphology of endodermal cells, islet-derived progenitor cells and mature islet cells in each stage was observed and relative gene expression levels were detected by PCR. Mature insulin-like cell clusters underwent dithizone staining and functions of insulin released in vitro were observed by ELISA assay. Finally, the insulin-secreting cells were transplanted into the subrenal capsule of diabetic mice, and then blood glucose levels were observed.
RESULTS AND CONCLUSION: The mature spherical insulin-like cell clusters were successfully obtained in vitro, which were in iron red by dithizone staining, and expression of insulin mRNA was determined by PCR. The insulin-like cell clusters could secrete insulin in response to various blood glucose levels by ELISA assay. In addition, after the cells clusters were transplanted into the subrenal capsule of mice with type 1 diabetes, the blood glucose levels were marbedly improved.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Pluripotent Stem Cells, Diabetes Mellitus, Type 1, Insulin-Secreting Cells, Cell Differentiation, Blood Glucose, Tissue Engineering

CLC Number:

R394.2

Lei Lei, Liang Ying-zi, Su Ying-jun, Ma Xian-jie, Cui Xin, Guo Shu-zhong . Insulin-secreting cells from induced pluripotent stem cells regulate blood glucose levels in vitro[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(28): 4210-4217.

Figures/Tables 2

2.1 诱导型多能干细胞的鉴定具有多能性的诱导型多能干细胞贴壁后呈克隆样生长，碱性磷酸酶染色呈红紫色，而分化的细胞和饲养层细胞不能着色(图2)。结果表明实验中所使用的诱导型多能干细胞具有多能性。 2.2 定向诱导分化为胰岛分泌细胞的形态诱导型多能干细胞在生长培养基中能够维持其多能性和自我更新的能力，在加入特定的生长因子后，诱导型多能干细胞启动向相应方向分化的过程。在第一阶段诱导分化后，呈集落样生长的细胞形态开始发生改变，细胞克隆的表面开始由光滑变得毛糙，紧凑的细胞间距变得疏松，多数细胞开始向周围分散(图3A)。随后，加入第二阶段诱导分化的培养基，未分化为内胚层的细胞脱落，分化为内胚层的细胞成簇，此阶段的细胞生长减缓(图3B)。最后，在分化为胰岛前体细胞中加入第三阶段的诱导分化培养基，使其进一步分化为成熟的胰岛细胞，细胞呈球形聚集，聚集的细胞排列紧密，细胞体积较小(图3C)。 2.3 诱导分化的胰岛素分泌细胞功能基因的检测结果分析诱导后的细胞逐渐表达内胚层标志基因Sox 17和Fox2A的mRNA、胰岛前体细胞标志基因NeuroD1、NGN3和Sox9的mRNA以及成熟胰岛细胞标志基因Insulin、ISL、Glu2和GCG的mRNA(图4)。对诱导21 d的细胞团进行双硫腙染色，细胞团呈猩红色，表明诱导分化的胰岛样细胞团具有与正常胰岛相似的特性。 2.4 体外胰岛素分泌的检测结果为了验证诱导型多能干细胞来源的胰岛素分泌细胞是否具有分泌胰岛素的功能。在体外分别用低浓度和高浓度的葡萄糖刺激诱导的胰岛素分泌细胞1 h，用ELISA法测定胰岛素分泌量(图5)。 2.5 糖尿病小鼠肾包膜移植诱导的胰岛素分泌细胞后随机血糖检测结果根据随机血糖检测结果，糖尿病小鼠肾包膜下移植诱导的胰岛素分泌细胞，小鼠的高血糖症状得到明显改善，并且维持接近正常的血糖水平达3周。未进行移植的糖尿病小鼠血糖在整个实验期间一直维持在高血糖水平(图6)。 2.6 糖尿病小鼠肾包膜下移植物组织学观察见图7。诱导的胰岛素分泌细胞移植3周后取材，对移植物样本进行胰岛素免疫荧光染色，镜下观察显示类似胰岛样细胞分布于肾包膜下，并表达绿色荧光(胰岛素)，表明诱导的胰岛素分泌细胞具有胰岛β细胞分泌胰岛素的功能(图7C)。另外对移植诱导后的胰岛素分泌细胞的糖尿病小鼠胰腺进行苏木精-伊红染色，观察显示糖尿病小鼠在移植实验期间并无胰岛细胞再生现象(图7B)，这表明移植组糖尿病小鼠血糖水平降低是由于诱导分化的胰岛素分泌细胞植入所致，而并非自身胰岛细胞的恢复。"

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