In vitro cultivation of islet-like cell clusters from human umbilical cord blood-derived mesenchymal stem cells

A major obstacle to successful islet transplantation for both type 1 and 2 diabetes is an inadequate supply of insulin-producing tissue. In vitro transdifferentiation of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) into insulin-producing cells could provide an abundant source of cells for this procedure. For this study, we isolated and characterized human UCB-MSCs and induced them in vitro to differentiate into islet-like cell clusters using a 15-day protocol based on a combination of high-glucose, retinoic acid, nicotinamide, epidermal growth factor, and exendin-4. These clusters appeared about 9 days after pancreatic differentiation; expressed pancreatic beta-cell markers, including insulin, glucagon, Glut-2, PDX1, Pax4, and Ngn3; and could synthesize and secrete functional islet proteins at the end of the inducing protocol. The insulin-positive cells accounted for (25.2-3.36)% of whole induced cells. Although insulin secretion of those insulin-producing cells did not respond to glucose challenge very well, human UCB-MSCs have the ability to differentiate into islet-like cells in vitro and may be a potential new source for islet transplantation.     

脐带源间充质干细胞与大鼠胰腺细胞共培养向胰岛样细胞分化及移植治疗1型糖尿病

背景：脐带Wharton’s Jelly中间充质干细胞可以向胰岛样细胞诱导分化。 目的：验证脐带源间充质干细胞与大鼠胰腺细胞共培养向胰岛样细胞诱导分化的可能性,并观察移植后对糖尿病大鼠血糖的影响。 方法：分离、诱导、传代脐带Wharton’s Jelly中间充质干细胞,再与大鼠胰腺细胞共培养,诱导成胰岛细胞团样组织。将大鼠分为3组,正常对照组不进行移植及造模;模型组仅制备糖尿病大鼠模型;实验组造模后将胰岛样细胞移植入糖尿病大鼠肾脏包膜。 结果与结论：脐带Wharton’s Jelly细胞培养中有细胞从组织块中爬出,第7天形态发生变化,贴壁细胞部分变成梭形。分离培养的细胞表达具有间充质干细胞表面特有标志CD44、CD29、CD105,不表达CD34、CD45、CD14。诱导第7,10天PDX-1及人胰岛素强染色;胰岛素及C-肽浓度较单纯培养组明显升高;PDX-1及人胰岛素mRNA诱导第7、10天较高表达。移植第1周大鼠尾尖血糖链脲佐菌素实验组明显低于模型组(P < 0.01),但明显高于正常照组(P < 0.01)。8周链脲佐菌素实验组肾脏被膜下发现胞核染棕色染色的Brdu阳性、胞浆棕色染色的胰岛素阳性细胞。结果表明,脐带Wharton’s Jelly中存在脐带源间充质干细胞,与大鼠胰腺细胞共培养可促进间充质干细胞向胰岛样细胞诱导分化,移植入糖尿病大鼠肾脏被膜下,可显著降低糖尿病大鼠血糖。     

The differentiation of embryonic stem cells seeded on electrospun nanofibers into neural lineages

Due to advances in stem cell biology, embryonic stem (ES) cells can be induced to differentiate into a particular mature cell lineage when cultured as embryoid bodies. Although transplantation of ES cells-derived neural progenitor cells has been demonstrated with some success for either spinal cord injury repair in small animal model, control of ES cell differentiation into complex, viable, higher ordered tissues is still challenging. Mouse ES cells have been induced to become neural progenitors by adding retinoic acid to embryoid body cultures for 4 days. In this study, we examine the use of electrospun biodegradable polymers as scaffolds not only for enhancing the differentiation of mouse ES cells into neural lineages but also for promoting and guiding the neurite outgrowth. A combination of electrospun fiber scaffolds and ES cells-derived neural progenitor cells could lead to the development of a better strategy for nerve injury repair.     

Human marrow-derived mesodermal progenitor cells generate insulin-secreting islet-like clusters in vivo

Transplantation of pancreatic islet cells is the only known potential cure for diabetes mellitus. However, the difficulty in obtaining sufficient numbers of purified islets for transplantation severely limits its use. A renewable and clinically accessible source of stem cells capable of differentiating into insulin-secreting beta-cells might circumvent this limitation. Here, we report that human fetal bone marrow (BM)-derived mesodermal progenitor cells (MPCs) possess the potential to generate insulinsecreting islet-like clusters (ISILCs) when injected into human fetal pancreatic tissues implanted in severe combined immunodeficiency (SCID) mice. Seven essential genes involved in pancreatic endocrine development, including insulin, glucagon, somatostatin, pdx-1, glut-2, nkx 2.2, and nkx 6.1, are expressed in these BM-MPC-derived ISILCs, suggesting that ISILCs are generated through neogenesis of BM-MPCs. Our data further suggest that differentiation of BM-MPCs into ISILCs is not mediated by cell fusion. Insulin secretion from these ISILCs is regulated by glucose concentration in vitro, and transplantation of purified ISILCs normalizes hyperglycemia in streptozocin (STZ)- induced nonobese diabetic (NOD)/SCID mice.     

胎鼠胰腺干细胞原位移植治疗糖尿病的实验研究

目的：原位移植胎鼠胰腺干细胞，探讨其在糖尿病鼠胰腺微环境中转分化为胰岛样细胞团的可行性。方法：分离纯化SD大鼠胎鼠胰腺干细胞；荧光原位杂交（fluorescent in situ hybridization, FISH）检测SRY上的性别决定区（sex determining region on Y, SRY）以鉴别雄雌；免疫细胞化学检测巢蛋白（Nestin）、胰十二指肠同源异型盒基因（PDX-1）的表达及流式细胞术测定Nestin细胞含量以鉴定胰腺干细胞；分胰腺实质内移植组、实验对照组及空白对照组，10只/组；监测各组大鼠血糖及血浆胰岛素含量，8周后取大鼠胰腺组织切片观察，FISH检测SRY，逆转录-聚合酶链反应（RT-PCR）观察各组大鼠胰腺内Nestin、PDX-1及胰岛素等mRNA的表达情况，免疫印迹（Western blotting）检测PDX-1及胰岛素的蛋白表达水平。结果：有5只胎鼠经FISH检测为雄性。免疫组化示存在Nestin 和PDX-1阳性细胞，流式细胞术测定Nestin阳性细胞含量占74.1%。胰腺实质内移植组大鼠于移植后第3周血糖开始下降，血浆胰岛素水平逐渐升高；第5周血糖及血浆胰岛素均达到正常水平并维持。取第8周大鼠胰腺组织切片HE染色可见外源性细胞团，FISH检测SRY阳性。RT-PCR示胰腺实质内移植组胰腺组织内胰岛素的mRNA表达明显高于实验对照组（P<0.05），而Nestin及PDX-1的mRNA表达量高于实验对照组及正常组（P<0.05）。Western blotting示干细胞移植组胰腺组织内胰岛素含量接近正常大鼠（P>0.05），而PDX-1的含量高于正常大鼠(P<0.05)。结论：胎鼠胰腺干细胞原位移植后可在体内转分化为胰岛样细胞团且具有良好的功能，移植后可使血糖降至正常，胰腺干细胞原位移植可能为1型糖尿病的治疗提供了一个新的策略。     

Differentiation and transplantation of functional pancreatic Beta cells generated from induced pluripotent stem cells derived from a type 1 diabetes mouse model

The nonobese diabetic (NOD) mouse is a classical animal model for autoimmune type 1 diabetes (T1D), closely mimicking features of human T1D. Thus, the NOD mouse presents an opportunity to test the effectiveness of induced pluripotent stem cells (iPSCs) as a therapeutic modality for T1D. Here, we demonstrate a proof of concept for cellular therapy using NOD mouse-derived iPSCs (NOD-iPSCs). We generated iPSCs from NOD mouse embryonic fibroblasts or NOD mouse pancreas-derived epithelial cells (NPEs), and applied directed differentiation protocols to differentiate the NOD-iPSCs toward functional pancreatic beta cells. Finally, we investigated whether the NPE-iPSC-derived insulin-producing cells could normalize hyperglycemia in transplanted diabetic mice. The NOD-iPSCs showed typical embryonic stem cell-like characteristics such as expression of markers for pluripotency, in vitro differentiation, teratoma formation, and generation of chimeric mice. We developed a method for stepwise differentiation of NOD-iPSCs into insulin-producing cells, and found that NPE-iPSCs differentiate more readily into insulin-producing cells. The differentiated NPE-iPSCs expressed diverse pancreatic beta cell markers and released insulin in response to glucose and KCl stimulation. Transplantation of the differentiated NPE-iPSCs into diabetic mice resulted in kidney engraftment. The engrafted cells responded to glucose by secreting insulin, thereby normalizing blood glucose levels. We propose that NOD-iPSCs will provide a useful tool for investigating genetic susceptibility to autoimmune diseases and generating a cellular interaction model of T1D, paving the way for the potential application of patient-derived iPSCs in autologous beta cell transplantation for treating diabetes.     

骨髓间充质干细胞体外诱导分化为胰岛样细胞的降血糖作用

背景：移植胰岛及胰岛细胞治疗糖尿病已初见成效,但由于胰岛来源匮乏和免疫排斥反应而研究受阻。 目的：移植将大鼠骨髓间充质干细胞在体外诱导分化为胰岛样细胞,观察其对糖尿病大鼠的治疗作用。 方法：将大鼠骨髓间充质干细胞用碱性成纤维细胞生长因子、肝细胞生长因子等诱导,免疫细胞染色等检测诱导情况。SD大鼠腹腔注射链脲佐菌素建立糖尿病模型,建模成功后,随机分为对照组移植诱导胰岛样细胞的实验组,实验组经肾包囊移植诱导后的胰岛样细胞,对照组移植相同体积生理盐水,观察移植后糖尿病大鼠血糖和体质量变化。 结果与结论：大鼠骨髓间充质干细胞体外经肝细胞生长因子、碱性成纤维细胞生长因子等因子诱导后可以向胰岛样细胞转化。细胞移植后,对照组大鼠血糖无明显变化(P > 0.05),实验组大鼠血糖与对照组和移植前相比较,明显降低(P < 0.05)。大鼠骨髓间充质干细胞经含肝细胞生长因子、碱性成纤维细胞生长因子等的诱导体系可诱导成胰岛样细胞,经诱导的细胞有一定胰岛素分泌能力,将诱导后细胞通过肾包囊途径移植入糖尿病大鼠体内,可降低大鼠血糖水平。     

Induced differentiation of human umbilical cord mesenchymal stem modified by cells Pdx1gene into islet beta-like cells in vitro

This study was to explore the induced differentiation of human mesenchymal stem cells (MSCs) modified by pancreatic and duodenal homeobox factor 1 (Pdx1) gene into insulin-producing cells in vitro. After recombined adenovirus vector with Pdx1 gene infected MSCs for 7 d, cells were induced by induction factors. The genes' expressions related to islet beta cells such as Pdx1, insulin, glucose transporter-2 (Glut2), were detected with RT-PCR, immunocytochemistry and Western blot. The levels of insulin and C peptide secretion were examined with chemiluminescence immunoassay. Insulin(+) cell rate was detected by flow cytometry. After infected by recombined adenovirus with Pdx1 and combined with induction factors, MSCs were aggregated and islet-like cell clusters formed. Dithizone staining of these cells was positive. The genes' expression related to islet beta cells, such as Pdx1, insulin, Glut2, could be detected. After induction, the islet-like cell clusters secreted insulin and C peptide. The levels of insulin and C peptide secretion increased with glucose stimulation. Insulin(+) cell rate was (11.61 +/- 4.83)%. It could be concluded that Pdx1 gene modified MSCs from human umbilical cord could be induced to differentiate into islet beta-like cells.     

Chromatin-remodeling factors allow differentiation of bone marrow cells into insulin-producing cells

Type 1 diabetes is caused by the destruction of pancreatic beta-cells by T cells of the immune system. Islet transplantation is a promising therapy for diabetes mellitus. Bone marrow stem cells (BMSC) have the capacity to differentiate into various cell lineages including endocrine cells of the pancreas. To investigate the conditions that allow BMSC to differentiate into insulin-producing cells, a novel in vitro method was developed by using the histone deacetylase inhibitor, trichostatin A (TSA). BMSC, cultured in presence of TSA, differentiated into islet-like clusters under appropriate culture conditions. These islet-like clusters were similar to the cells of the islets of the pancreas. The islet-like clusters showed endocrine gene expression typical for pancreatic beta-cell development and function, such as insulin (I and II), glucagon, somatostatin, GLUT-2, pancreatic duodenal homeobox-1 (PDX-1), and Pax 4. Immunocytochemistry confirmed islet-like clusters contained pancreatic hormones. The colocalization of insulin and C-peptide was also observed. Enzyme-linked immunosorbent assay analysis demonstrated that insulin secretion was regulated by glucose. Western blot analysis demonstrated the presence of stored insulin. Electron microscopy of the islet-like cells revealed an ultrastructure similar to that of pancreatic beta-cells, which contain insulin granules within secretory vesicles. These findings suggest that histone-deacetylating agents could allow the differentiation of BMSC into insulin-producing beta-cells.     

Conophylline与尼克酰胺联合诱导脂肪源干细胞为功能性类胰岛细胞

背景：1型糖尿病的胰岛移植治疗一直面临供体来源不足与免疫排斥两大关键问题,寻找一种自体来源的种子细胞通过组织工程方法制备类胰岛组织可以提供充足新型供体、降低异基因供体移植的不良反应。 目的：分析成人脂肪干细胞体外分化为对葡萄糖敏感、可分泌胰岛素的功能性胰岛样细胞团的能力,探索体外制备类胰岛组织的技术路线。 方法：首先分离纯化人体脂肪干细胞,采用新型植物诱导剂Conophylline与其他诱导因子的不同组合将脂肪干细胞诱导分化为胰岛样细胞团,观察不同组合的诱导分化效率,并利用特异性染色、RT-PCR,免疫细胞化学等方法对诱导分化后的细胞团在基因水平与蛋白水平上进行鉴定,最后用ELISA法检测细胞团在不同浓度葡萄糖刺激下胰岛素的分泌情况。 结果与结论：脂肪干细胞具有多能干细胞特性,可诱导分化为具有胰岛素分泌和葡萄糖浓度反应性类胰岛细胞团;Conophylline与尼克酰胺联合诱导可大幅度提高诱导分化效率。     

胚胎干细胞定向分化为胰岛素分泌细胞的研究进展

胰岛移植是治疗糖尿病的有效方法之一。胚体干细胞在体外定向分化为胰岛素分泌细胞为胰岛移植提供了足够的细胞来源。概述了近年来胚胎干细胞向胰腺细胞系的诱导分化所取得的进展。     

PDX1联合NKX6.1促进骨髓间充质干细胞分化为胰岛β样细胞

目的： 为提高骨髓间充质干细胞(BMSCs)向胰腺β样细胞的分化效率,以产生足够用于移植的胰岛样细胞。方法: 构建含PDX1与NKX6.1双基因的重组腺病毒载体,用重组腺病毒感染并联合多种细胞因子分步诱导BMSCs。用RT-PCR、Western blotting等多种方法分别检测PDX1、NKX6.1、胰岛素及C-肽表达情况;观测植入鼠肾包膜下的细胞形态与胰岛素、C-肽等相应分子表达情况以及检测植入细胞对糖尿病模型大鼠的血糖水平的调节能力。结果: BMSCs经重组腺病毒pAdxsi-CMV-PDX1/CMV-NKX6.1联合相应细胞因子分步诱导,双硫腙染色细胞质呈亮红色,RT-PCR显示诱导后的细胞持续稳定表达胰岛素、葡萄糖转运蛋白2(GLUT2)等β细胞相关分子;Western blotting、免疫细胞化学与间接荧光结果亦相似。所诱导的实验组细胞经5.5和25mmol/L葡萄糖刺激后胰岛素分泌水平分别为(1 240.4±109.3) mU/L和(3 539.8±245.1) mU/L, 并显著高于对照组的分泌量。移植实验组细胞可恢复STZ糖尿病小鼠血糖正常水平。结论: PDX1与NKX6.1联合细胞因子在体外能有效地诱导BMSCs分化为胰岛β样细胞;这种胰岛β样细胞移植能有效恢复STZ糖尿病小鼠的血糖正常水平,维持小鼠良好的生存状态,这将为治疗糖尿病带来新的希望。     

Effect of monolayer cells on sphere cells--two types of cells that emerge during the neural differentiation of mouse embryonic stem cells

Embryonic stem (ES) cells represent a valuable resource for transplantation and tissue engineering applications. For derivation of neural cells, a five-stage differentiation protocol has been widely applied, which involves the propagation of ES cells, formation of embryoid bodies (EBs), selection of neural stem cells (NSCs), expansion of NSCs, and further maturation of NSCs to neurons. During the expansion stage (the fourth stage), two types of cells with distinct morphologies normally emerge, with one type being monolayer cells and the other sphere-like aggregates growing on top of the monolayer cells. In this study, we focus on how the monolayer cells may affect different aspects of aggregate cells, which may have important implications for regenerative medicine. We find that monolayer cells can support the proliferation and decrease the apoptosis rate of sphere cells, as well as facilitate the production of Tuj1-positive cells from sphere cells. In addition, transplantation of monolayer cells into nude mice does not result in tumor formation nor affects the tumorigenicity of sphere cells, when grafted together with monolayer cells.