Hua Xiu-feng, Wang Yan-wei, Tang Yu-xiao, Yu Sheng-qiang, Jin Shao-hua, Meng Xiao-mei, Li Hua-feng, Liu Fu-jun, Sun Qiang, Wang Hai-yan, Li Jian-yuan
Department of Endocrinology, Yuhuangding Hospital, Yantai, Shandong Province, China.
Research Center of Stem Cell Engineering of Shandong, Central Laboratory of Yuhuangding Hospital, Yantai, Shandong Province, China.
PLoS One. 2014 Jul 10;9(7):e102198. doi: 10.1371/journal.pone.0102198. eCollection 2014.
Human pancreatic islet transplantation is a prospective curative treatment for diabetes. However, the lack of donor pancreases greatly limits this approach. One approach to overcome the limited supply of donor pancreases is to generate functional islets from human embryonic stem cells (hESCs), a cell line with unlimited proliferative capacity, through rapid directed differentiation. This study investigated whether pancreatic insulin-producing cells (IPCs) differentiated from hESCs could correct hyperglycemia in severe combined immunodeficient (SCID)/non-obese diabetic (NOD) mice, an animal model of diabetes.
We generated pancreatic IPCs from two hESC lines, YT1 and YT2, using an optimized four-stage differentiation protocol in a chemically defined culture system. Then, about 5-7 × 10(6) differentiated cells were transplanted into the epididymal fat pad of SCID/NOD mice (n = 20). The control group were transplanted with undifferentiated hESCs (n = 6). Graft survival and function were assessed using immunohistochemistry, and measuring serum human C-peptide and blood glucose levels.
The pancreatic IPCs were generated by the four-stage differentiation protocol using hESCs. About 17.1% of differentiated cells expressed insulin, as determined by flow cytometry. These cells secreted insulin/C-peptide following glucose stimulation, similarly to adult human islets. Most of these IPCs co-expressed mature β cell-specific markers, including human C-peptide, GLUT2, PDX1, insulin, and glucagon. After implantation into the epididymal fat pad of SCID/NOD mice, the hESC-derived pancreatic IPCs corrected hyperglycemia for ≥ 8 weeks. None of the animals transplanted with pancreatic IPCs developed tumors during the time. The mean survival of recipients was increased by implanted IPCs as compared to implanted undifferentiated hESCs (P<0.0001).
The results of this study confirmed that human terminally differentiated pancreatic IPCs derived from hESCs can correct hyperglycemia in SCID/NOD mice for ≥8 weeks.
人类胰岛移植是一种有前景的糖尿病治疗方法。然而，供体胰腺的短缺极大地限制了这种方法。克服供体胰腺供应有限的一种方法是通过快速定向分化，从具有无限增殖能力的人类胚胎干细胞（hESCs）中生成功能性胰岛。本研究调查了从hESCs分化而来的胰腺胰岛素分泌细胞（IPCs）是否能纠正重症联合免疫缺陷（SCID）/非肥胖糖尿病（NOD）小鼠（一种糖尿病动物模型）的高血糖症。
我们在化学限定培养系统中，使用优化的四阶段分化方案，从两个hESC系YT1和YT2中生成胰腺IPCs。然后，将约5 - 7×10⁶个分化细胞移植到SCID/NOD小鼠（n = 20）的附睾脂肪垫中。对照组移植未分化的hESCs（n = 6）。使用免疫组织化学以及测量血清人C肽和血糖水平来评估移植物的存活和功能。
通过使用hESCs的四阶段分化方案生成了胰腺IPCs。通过流式细胞术测定，约17.1%的分化细胞表达胰岛素。这些细胞在葡萄糖刺激后分泌胰岛素/C肽，类似于成人胰岛。这些IPCs中的大多数共表达成熟的β细胞特异性标志物，包括人C肽、GLUT2、PDX1、胰岛素和胰高血糖素。植入SCID/NOD小鼠的附睾脂肪垫后，hESC来源的胰腺IPCs纠正高血糖症达≥8周。在此期间，移植胰腺IPCs的动物均未发生肿瘤。与移植未分化hESCs相比，植入IPCs可提高受体的平均生存期（P<0.0001）。
本研究结果证实，源自hESCs的人类终末分化胰腺IPCs可在SCID/NOD小鼠中纠正高血糖症达≥8周。
