Objective To investigate whether pancreatic progenitors and islets differentiated from human embryonic stem cells(hESCs) could correct hyperglycemia in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice.Methods We obtained pancreatic progenitors and islets derived from hES cells line YT1 according to the optimized four-stage differentiation protocol.Stage 1:definitive endoderm formation; Stage 2:pan creatic specialization; Stage 3:amplification of pancreatic progenitors,Stage 4:maturation of pancreatic islets.To observe the morphological changes of each stage and immunofluorescent expression of pancreatic and duodenal homeobox gene(PDX-1),glucagon,insulin,C-peptide,glucose transporter-2(Glut-2).The differentiated cells from stage 3 and stage 4 were then transplanted into one of the epididymal fat pads(EFP) of NOD/SCID mice.The survival and function of the graft were measured by immunohistochemistry and blood glucose monitor.Results The stage 4-differentiated pancreatic islets expressed mature β cell-specific markers such as glucagon,insulin and Glut 2,and even PDX-1 and C-peptide-double-positive.The stage 4-pancreatic islets had nearly 17.1％ insulin-positive cells as assayed by flow cytometry analysis.Differentiated pancreatic islets released insulin/C-peptide in response to glucose stimulation.After implantation into EFP of NOD/SCID mice,hES cell-derived human pancreatic progenitors and islets corrected hyperglycemia for at least 12 weeks.Conclusions Pancreatic progenitors and islets differentiated from hESCs can correct hyperglycemia in NOD/SCID mice.

BACKGROUND: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.METHODS: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.RESULTS: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).CONCLUSIONS: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.

Objective To investigate whether pancreatic progenitors differentiated from human embryonic stem(hES) cells could correct hyperglycemia in non-obese diabetic(NOD)/severe combined immunodeficient(SCID) mice or not.Methods Pancreatic islets derived from hES cells line YT1 according to the optimized four-stage differentiation protocol in a chemical-defined culture system were observed.In the first stage,activin A and wortmannin were utilized to induce definitive endoderm formation.In the second stage,the differentiated endoderm cells were treated with all-trans retinoic acid(RA),NOGGIN and basic fibroblast growth factor(bFGF) to induce pancreatic specialization.In the third stage,pancreatic progenitors were induced by epidermal growth factor(EGF)-regulated expansion.Finally,in the fourth stage,a cocktail of factors was utilized to induce mature insulin-producing cells.The differentiated human pancreatic islet cells of(3-5)×106 were then transplanted into one of the epididymal fat pads(EFP) of NOD/SCID mice.Graft survival and function were measured by blood glucose levels.Results The differentiated hES cells obtained by the four-stage approach comprised nearly 17.1% insulin-positive cells and 3.8% C-peptide positive as assayed by flow cytometry analysis,which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets.Most of these insulin-producing cells co-expressed mature β cell-specific markers such as PDX1,insulin,C-peptide and glucagon.After implantation into EFP of NOD/SCID mice,hES cell-derived pancreatic islets corrected hyperglycemia for an least eight weeks.Conclusion Human terminal differentiation pancreatic islet cells can correct hyperglycemia in NOD/SCID mice.

人脐带间充质干细胞是一种多潜能细胞,具有来源广泛,采集容易,无异体排斥等优点,并且具有自我更新和多向分化潜能,成为细胞治疗和组织工程新的种子细胞.

目的探讨体外诱导人胚胎干细胞(hESs)定向分化为胰岛细胞的可行性。方法体外分三阶段诱导hESCs定向分化为胰岛细胞。第一阶段:予以活化素-A、渥曼青霉素诱导分化形成定型内胚层;第二阶段:采用维甲酸(RA)、NOGGIN、碱性成纤维细胞生长因子(bFGF)诱导胰腺细胞定向分化;以EGF扩增胰腺祖细胞;第三阶段:采用尼克酰胺、唾液素4、bFGF及骨形成蛋白(BMP4)促进胰岛细胞成熟;观察诱导各阶段细胞形态变化,免疫荧光法鉴定胰十二指肠同源异型盒基因(PDX-1)、胰高糖素、胰岛素、C肽、葡萄糖转运子2(glut-2)表达。结果诱导14 d时hESs出现胰高糖素荧光表达;20 d时细胞出现PDX-1和C肽共表达;22 d形成的成熟胰岛细胞出现glut-2和胰岛素的阳性表达;胰岛素阳性细胞占17.1%,C肽阳性细胞占3.8%。结论体外可简单高效的诱导hESs定向分化为成熟胰岛细胞。

精原干细胞是精子形成的前体细胞,具有自我增殖和多向分化潜能。本文对精原干细胞的微环境、体外培养和冻存的研究现状进行综述。

根据尿白蛋白排泄率(UAER),将60例2型糖尿病(T2DM)患者分为三组,即正常白蛋白尿组(A组)、微量白蛋白尿组(B组)、临床白蛋白尿组(C组)。应用流式细胞免疫学方法测定三组患者和健康对照者(对照组)外周血中CD44阳性细胞数,并将CD44表达与其空腹血糖、糖化血红蛋白、胆固醇、甘油三酯、高密度脂蛋白、UAER、尿N-乙酰-β-D-氨基葡萄糖苷酶(NAG)水平进行相关性分析。结果显示,T2DM患者的外周血CD44阳性细胞率均显著高于对照组(P<0.01),B、C组高于A组(P<0.01)。T2DM患者尿NAG均高于对照组,但A、B、C组间无统计学差异(P>0.05)。CD44表达与尿NAG呈正相关(P<0.01),与其他指标无相关性。提示外周血CD44可作为监测糖尿病肾病(DN)病情变化的指标之一,NAG与CD44在DN病变过程中可能有协同性。