Examining the therapeutic potential of various stem cell sources for differentiation into insulin-producing cells to treat diabetes

Novel strategies are being developed to generate stem-cell-derived insulin-producing cells (IPCs), which could reverse the growing incidence of diabetes worldwide. We reviewed studies of stem-cell-based therapies for pancreatic β-cell regeneration published between 1997 and 2017. Differentiation into IPCs can be achieved using various stem-cell sources: embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and several types of adult stem cells such as pancreatic, hepatic and mesenchymal stem cells. However, reliable cell replacement therapy for diabetes is still in its early stages, and safety and ethical concerns are pressing issues. It will be necessary to find means of identifying optimal stem-cell sources and of inducing β-cell differentiation without using genetic mutations. The present article examines the potential of various stem-cell candidates for IPC generation, and the current obstacles preventing emergence of a candidate source.     

In vitro directed differentiation of mouse embryonic stem cells into insulin-producing cells

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. Methods 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.Results 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.Abbreviations Anti-Shh antibody against sonic hedgehog - D3 undifferentiated D3 stem cell line - EB embryoid bodies - ES embryonic stem - FBS fetal bovine serum - LIF leukaemia inhibitory factor - mES mouse embryonic stem - Ngn3 neurogenin 3 - P gelatine-coated plates - Pdx-1 pancreatic duodenum homeobox 1     

高糖在联合转录因子诱导大鼠骨髓间充质干细胞分化为胰岛样细胞中的作用

目的 探讨高糖在胰-十二指肠同源盒因子-1(Pdx-1)、神经元素3(Ngn 3)诱导大鼠骨髓间充质干细胞(BMSC)分化为胰岛样细胞(IPC)中的作用.方法 构建真核表达载体pcDNA3.1(+)/(zeo)-Ngn 3、pcDNA3.1(+)-Pdx-1,贴壁培养法分离Sprauge-Dawley雄性大鼠BMSC,流式细胞术鉴定BMSC,脂质体介导转染BMSC,并筛选稳定转染细胞培养于高糖(4.5 g/L) DMEM培养液,RT-PCR检测Ngn 3、巢蛋白、Pdx-1 mRNA的表达,ELISA法检测胰岛素浓度.结果 分离的细胞表面高表达CD44、CD105,而低表达CD34,符合BMSC特征.诱导过程中BMSC形态上趋向IPC分化,高糖培养组较低糖培养组细胞上清胰岛素浓度明显提高(F=25.77,P＜0.05),高糖质粒培养组Pdx-1、Ngn 3及巢蛋白mRNA表达水平明显高于高糖培养组及质粒转染组(F=35.12、12.76、8.34,P均＜0.05).结论 高糖对Pdx-1联合Ngn 3诱导BMSC向IPC分化具有促进作用.     

肝细胞生长因子及葡萄糖对人胰腺干细胞向胰岛素分泌细胞分化的影响

目的 探讨人胰腺干细胞体外扩增及其向胰岛素分泌细胞分化的影响因素.方法 应用剪碎消化法从孕4～6月龄引产的人胎儿胰腺中获得胰岛组织,选用差速贴壁法从胰岛中分离胰腺干细胞,经扩增后定向诱导成具有胰岛素分泌功能和胰岛样结构的胰岛样细胞团,观察肝细胞生长因子和葡萄糖对胰腺干细胞向胰岛素分泌细胞诱导分化以及胰岛样细胞团胰岛素分泌功能的影响.采用流式细胞仪和免疫组织化学法检测扩增前后nestin阳性细胞和ABCG2阳性细胞.行葡萄糖刺激的胰岛素释放试验,以酶联免疫吸附法（ELISA）检测每1×105个扩增细胞经定向诱导后形成的胰岛样细胞团释放的胰岛素量,比较肝细胞生长因子和葡萄糖对定向分化的影响.采用单因素方差分析或多样本均数两两比较进行统计学分析.结果 经过15代连续扩增,nestin阳性细胞和ABCG2阳性细胞分别扩增了27.9倍和37.8倍.扩增后的细胞经15 d定向诱导后形成具有胰岛素分泌功能和胰岛样结构的细胞团.葡萄糖刺激的胰岛素分泌试验显示,当诱导培养基中肝细胞生长因子浓度为0、5、10、15μg/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为5.00、12.93、14.91和11.23μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为19.91、35.53、47.43和23.33 μU.诱导培养基中的葡萄糖浓度为0、2.8、5.6和11.2 mmol/L时,5.6 mmol/L葡萄糖刺激后,胰岛素分泌量分别为9.07、14.21、6.91和3.53 μU;25.0 mmol/L葡萄糖刺激后,胰岛素分泌量分别为26.64、48.97、37.63和34.20 μU.结论 人胰腺中nestin和ABCG2阳性胰腺干细胞具有很强的体外扩增能力.扩增后的胰腺干细胞仍然具有形成胰岛素分泌细胞的功能,经体外诱导后能形成胰岛样细胞团.在胰腺干细胞向胰岛素分泌细胞分化诱导过程中,适当浓度的肝细胞生长因子和葡萄糖有利于胰岛样细胞团的形成和胰岛素分泌.     

细胞因子对骨髓间充质干细胞增殖及其向心肌细胞分化的影响

目的探讨干细胞因子（SCF）和粒细胞集落刺激因子（G-CSF）预处理对骨髓间充质干细胞（MSCs）增殖分化的作用。方法将sD大鼠随机分成对照组、SCF组、G—CSF组和SCF＋G—CSF联合处理组。流式细胞仪检测第4代MsCs细胞周期。用DAPI（25mg／m1）标记的P4MSCs与心肌细胞共培养。在共培养的第1天至第5天用免疫荧光技术分别检测心肌特异性肌节肌球蛋白重链（MHC）、肌钙蛋白T（TnT）的表达，统计向心肌样细胞分化的MSCs百分率。结果（1）SCF和G—CSF联合应用能明显诱导MSCs进入S期，单因子SCF组和G-CSF组与对照组相比，也明显的促进MSCs由G0／G1期进入S期（P〈0．01）。（2）MSCs与心肌细胞共培养后，SCF和G—CSF联合组MSCs表达心肌特异性蛋白MHC及TnT的阳性百分率均显著增高，单细胞因子组MSCs的心肌特异性蛋白MHC及TnT的阳性表达亦较对照组高。结论SCF和G—CSF对MSCs具有促增殖、促分化的作用。     

体外胰岛细胞共培养及化学因子联合诱导NOD鼠源性诱导性多能干细胞分化为胰岛素分泌细胞的研究

目的 建立更为高效的NOD鼠体外胰岛素分泌细胞诱导体系. 方法 化学因子分步加入培养基,将大鼠胰岛细胞以半透膜相隔与NOD鼠源性诱导性多能干细胞（NOD-iPSCs）共培养约20 d,检测胰岛素分泌细胞的相关功能,并与纯化学因子诱导方法进行对比. 结果 诱导后成团生长,双硫腙染色呈棕红色,RT-PCR显示胰十二脂肠同源异型盒基因1（PDX-1）从第8天起开始表达,并逐渐增强.免疫荧光染色显示细胞表达胰岛素及C-P,联合组及纯化学因子组诱导培养每隔4d检测至20 d,高糖刺激后两组胰岛素分泌均呈逐渐升高趋势. 结论 NOD-iPSCs能够在体外诱导生成胰岛素分泌细胞.联合培养体系提供大鼠胰岛细胞功能因子可加速分化,提高效率,可能为一种更高效的诱导方法.