脂肪间充质干细胞向肝细胞横向分化的研究

目的探讨脂肪源性间充质干细胞（AMSC）向肝细胞横向分化的可能性。方法胶原酶消化脂肪组织,贴壁培养,体外扩增后以流式细胞仪鉴定其表面标志。取扩增3代的AMSC分为2组,诱导分化组在含有2％FBS的DMEM-F12培养基中加入肝细胞生长因子20 ng/ml和成纤维细胞生长因子4 10 ng/ml、1×ITS和地塞米松0．1μmol/L,培养14 d；空白对照组则不加任何细胞因子。RT-PCR检测诱导分化过程中肝细胞核因子1、GATA4等基因转录水平的变化。2周后,采用流式细胞术检测AFP和Alb阳性细胞在两组细胞中的比例,检测肝细胞特异性细胞角蛋白（CK） 18、CK19的表达。结果分离、培养的AMSC呈成纤维细胞样生长,可以稳定传代。流式细胞术检测结果显示第3代脂肪间充质干细胞高表达表面CD29、CD44；不表达CD34、CD45。RT-PCR检测诱导5、8、11、14 d的细胞,显示有肝细胞特异性转录因子GATA4和肝细胞核因子1A基因的表达,并随时间延长而逐渐增多。流式细胞术检测诱导14 d的细胞,发现30．0％的细胞表达Alb,17．8％细胞表达AFP,双阳性的细胞为6．9％；免疫荧光检测发现诱导细胞表达CK18、CK19。空白对照组脂肪间充质干细胞则未见上述各项变化。结论在低血清培养体系中,采用细胞因子联合诱导,显示脂肪间充质细胞在体外能定向分化为肝细胞样细胞。     

胚胎干细胞用于肝组织工程研究的初步评价

目的在生物材料上进行胚胎干细胞（embryonic stem cell,ESc）的三维培养和肝细胞定向诱导分化,评价其用于构建肝组织工程的可行性。方法胰酶消化生物反应器悬浮旋转培养5天的拟胚体（embryoid bodies,EBs）,将细胞悬液与细胞外基质Matrigel 1∶1混合接种于聚乳酸（poly-L-lactic acid,PLLA）和聚乙醇酸（polyglycolic acid,PGA）共聚合三维支架内,用系列诱导剂定向诱导肝细胞分化。镜下动态观察ES细胞的培育和生长情况,并用HE染色、糖原染色、免疫荧光染色检测组织结构形态、肝细胞功能及其肝细胞标志物白蛋白（ALB）的表达。结果显微镜和扫描电镜见ESc可在聚合支架材料上三维立体状态生长,随培养时间不断增殖,交织成网状。HE染色提示形成了致密的类肝组织样结构。糖原染色和免疫荧光染色结果提示形成的类肝组织表达Alb,并有大量糖原存在。结论可在PLLA/PGA三维支架材料及Matrigel上培养ESc和定向肝细胞诱导分化,长时间培养后可形成类肝样组织。     

共同培养诱导骨髓基质干细胞向肝细胞分化的研究

目的 探索大鼠骨髓皋质干细胞（MsCS）向肝细胞分化的能力，及肝细胞生长的微环境埘其诱导分化的作用。方法 采用梯度离心法，获取大鼠骨髓基质干细胞；改良的两步法获取大鼠肝细胞。将鉴定的MsCS和肝细胞以半透膜相隔共同培养，以单独培养的MSCs作对照。在第1、3、7．14．21、28天，分别以逆转录聚合酶链反应（RT-PCR）和免疫细胞化学分析检测甲胎蛋白（AFP）、白蛋白、细胞角蛋白l8（CK-l8）的基因和蛋白表达。结果在MSCs与肝细胞共同培养过程中，MSCs出现明显的细胞形态、体积和数量变化，可见双核或多核细胞，细胞轮廓较清晰。RT-PCR检测：共同培养的MSCs第7天即出现AFP基因表达，第14天表达增强，第21天表达减弱；第14天开始出现白蛋白、CK-18基因表达，并持续表达。单独培养的MSCs均无表达。共同培养的MSCs，于第7天进行免疫细胞化学检测，AFP即呈阳性；第14天白蛋白和CK-18也呈阳性；单独培养的MSCs未见AFP、白蛋白及CK-18表达。结论 大鼠骨髓基质干细胞与肝细胞共同培养，可被诱导分化为肝细胞。     

肝细胞条件培养液对人脂肪问充质干细胞向肝细胞分化和增殖的作用

目的 探讨人脂肪间充质干细胞在改良的诱导体系下向肝细胞的分化和增殖情况,为肝组织工程提供新的种子细胞来源.方法 从人脂肪组织分离出脂肪间充质干细胞,用含有碱性成纤维细胞生长因子、肝细胞生长因子、成纤维细胞生长因子-4的肝细胞诱导液进行诱导,并于诱导7 d后加入抑瘤素M.用细胞计数试剂盒-8法检测整个诱导过程细胞的增殖情况;通过光学显微镜观察诱导细胞的形态变化;用RT-PCR法和免疫荧光法分别检测肝细胞特异性基因和蛋白的表达;并对多种肝细胞特异性功能进行检测.组间比较采用t-test检验.结果 用改良肝细胞诱导液培养的人脂肪间充质干细胞在培养第5、7、14、21天时,细胞数均明显多于用对照培养液培养的细胞(f值分别为6.59、8.69、15.94和24.64,P值均＜0.05).诱导细胞表现出上皮样肝细胞形态,表达肝细胞特异性基因和蛋白;具有多种肝细胞特异性功能,如靛青绿摄取/排泌、糖原合成以及白蛋白分泌功能.结论 人脂肪间充质干细胞在含碱性成纤维细胞生长因子、肝细胞生长因子、成纤维细胞生长因子-4和抑瘤索M的诱导体系中能够分化为更加成熟的具有多种肝细胞特异性功能的细胞,且此诱导体系同时具有促进细胞增殖的作用.     

人骨髓间充质干细胞体外分化为肝细胞样细胞

目的 探讨人骨髓间充质干细胞(MSCs)的体外培养及特异性诱导为肝细胞样细胞的能力。方法 骨髓标本来源于健康志愿者的胸骨,年龄2～35岁,采用淋巴细胞分离液(密度1.077)分离人MSCs,并分别采用HGF、FGF4、HGF FGF4以及无生长因子四种处理因素体外诱导第三代人MSC向肝细胞样细胞分化。通过流式细胞术分析鉴定.MSCs的纯度,于诱导培养的0、7、14、21、28天时留取细胞检测CK18、AFP和白蛋白的表达情况,同时进行糖原染色验证细胞功能。结果 用淋巴细胞分离液分离出的人MSCs纯度可达90％,采用HGF、FGF4及HGF FGF4三种处理因素均可在体外诱导人MSCs特异分化为具有肝细胞样细胞表型和功能的细胞。结论 人MSCs能在体外扩增并定向诱导为肝细胞样细胞。     

生物反应器内EBs来源细胞的肝细胞分化与成熟

目的 观察微重力生物反应器内拟胚体(EBs)来源细胞的肝细胞分化与成熟.方法 将未分化鼠胚胎干细胞(ES细胞)以1×106/mL移入微重力反应器内,在DMSO、地塞米松、FGF4、HGF等刺激作用下,进行15 d的旋转培养.采用ELISA法动态检测培养液中鼠白蛋白的产生量.培养结束时转移EBs于载玻片和培养板,分别检测EBs来源细胞的糖原储存和对靛青绿(ICG)、荧光素标记低密度脂蛋白(DiI-Ac-LDL)的摄取能力.结果 在生物反应器旋转培养的第5、10 d,未从培养液中检出特异的鼠白蛋白,而在第15 d的培养液中检出一定量的鼠白蛋白.与原代培养的成熟肝细胞一样,转种的EBs来源细胞periodic acid-Schiff(PAS)糖原染色阳性,ICG摄取试验阳性,DiI-Ac-LDL摄取试验阳性.未分化的ES细胞均呈阴性.结论 微重力生物反应器不仅能加快EBs的形成与肝细胞分化,而且能促进分化肝细胞的成熟.     

人肝细胞L02诱导MSCs分化为肝样细胞的观察

目的 探讨骨髓间充质干细胞(MSCs)在与肝细胞L02在体外共培养条件下诱导分化为肝细胞的可行性.方法 密度梯度离心联合贴壁筛选法获取MSCs.将接种于盖玻片上的MSCs和人肝细胞L02共置于培养皿中.在2、4、6、8 d时分别用免疫细胞化学检测AFP、细胞角蛋白(CK18),同时检测染色体核型.结果 成功分离培养出MSCs,其表型为CD29阳性,CD34阴性.共培养的MSCs可在2～8 d时,形态变为三角形、多角型或类圆形.第2天时,共培养细胞AFP表现为强阳性表达,以后减弱,第8天AFP的阳性表达很少.第2、4天检测出CK18均为阴性表达,第6、8天CK18为阳性表达.阳性对照组细胞CK18有较强的阳性表达,AFP弱阳性表达.阴性对照组均为阴性表达.共培养细胞染色体核型无融合现象.结论 人肝细胞L02能够诱导MSCs分化为肝样细胞.     

人胎肝非实质间充质干细胞体外诱导分化为类肝细胞

目的体外诱导人胎肝非实质间充质干细胞(NPMSCs)分化为类肝细胞,对类肝细胞进行分子生物学及功能鉴定。方法采用体外细胞培养技术,分离培养人胎肝NPMSCs,在1%基质胶作基质,2.5 mmol/L氮胞苷预处理10～12 h,肝细胞生长因子10μg/L加成纤维细胞生长因子4 10μg/L加肝细胞生长培养基中诱导。用显微摄像和四甲基偶氮唑盐研究细胞增殖及生长特征,用流式细胞仪、免疫组织化学和逆转录聚合酶链反应鉴定细胞表型。采用酶联免疫吸附法检测培养上清液中人Alb水平,过碘酸希夫试验进行糖原染色。结果从人胎肝获得贴壁细胞种植后生长分裂良好,连续传10代后,每份人胎肝NPMSCs可扩增达109个细胞。NPMSCs表型为CD166阳性,CD34阴性。在添加成纤维细胞生长因子4和肝细胞生长因子的基质胶上诱导培养的NPMSCs在21～28 d时,形态由长梭形变为三角形、多角形或类圆形。细胞转圆率为40%,双核细胞比率5%。免疫组织化学和逆转录聚合酶链反应检测显示未诱导培养的NPMSCs中,有较少的细胞表达甲胎蛋白及其mRNA,未见其他肝脏特有的转录因子或者细胞质蛋白标志。诱导早期可见较多细胞表达GATA4、甲胎蛋白和CK18及其mRNA,至诱导后期表达下降,而Alb、CK18、谷胱甘肽S转移酶-π和肝细胞核因子1α表达逐渐上升。Alb、CK18阳性细胞比例达60%。未诱导分化的NPMSCs不分泌Alb,诱导分化的NPMSCs以时间依赖方式产生Alb。NPMSCs诱导14d时首先见到部分细胞出现红紫色染色的糖原积聚物,28 d后阳性染色细胞数量增多。结论在本实验诱导条件下可获得在复制及翻译各环节肝细胞标志阳性的类肝细胞。诱导后NPMSCs已具备肝细胞特有的功能性特征。     

胚胎干细胞向胰岛素分泌细胞的定向分化

在适当的条件下，胚胎干细胞可在体外被诱导分化为胰岛素分泌细胞。这一过程包括胚胎干细胞的诱导分化以及分化细胞的筛选和成熟。通过检测胰岛细胞的细胞标志和应用组织化学方法可鉴定胚胎干细胞是否已分化为胰岛素分泌细胞。研究显示，来源于小鼠胚胎干细胞的胰岛素分泌细胞可使糖尿病模型动物的血糖恢复到正常水平，但在胚胎干细胞进入临床应用之前，仍有很多问题需进一步研究加以解决。     

Embryoid-body cells derived from a mouse embryonic stem cell line show differentiation into functional hepatocytes

Embryonic stem (ES) cells have a potential to differentiate into various progenitor cells. Here we investigated the differentiation capacity of mouse ES cells into hepatocytes both in vitro and in vivo. During the culture of embryoid bodies (EBs) derived from ES cells, albumin (ALB) messenger RNA (mRNA) was expressed within 12 days after removal of leukemia inhibitory factor, and alpha-fetoprotein (AFP) mRNA was observed within 9 days without additional exogenous growth factors. In ES cells and early EBs, by contrast, neither ALB mRNA nor AFP mRNA was observed. ALB protein was first detected at day 15 and the level increased with the culture period. The differentiation of EBs facilitated the synthesis of urea with the culture period, whereas early EBs and ES cells produced no urea. These results suggest that cultured EBs contain hepatocytes capable of producing ALB and urea. ES cells and the isolated cells from EBs were transplanted through portal vein to the liver after 30% partial hepatectomy of female mice pretreated with 2-acetylaminofluorene. Four weeks after transplantation with isolated cells from day-9 EBs, ES-derived cells containing Y-chromosome in the liver were positive for ALB (0.2% of total liver cells), whereas teratoma was found in mice transplanted with ES cells or EBs up to day 6. The incidence of teratoma was decreased with the culture duration and no teratoma was observed in the liver transplanted with isolated cells from day-9 EBs. In conclusion, our in vitro and in vivo experiments revealed that cultured EBs contain functional hepatocytes or hepatocyte-like cells.     

Directed differentiation of human embryonic stem cells into functional hepatic cells

The differentiation capacity of human embryonic stem cells (hESCs) holds great promise for therapeutic applications. We report a novel three-stage method to efficiently direct the differentiation of human embryonic stem cells into hepatic cells in serum-free medium. Human ESCs were first differentiated into definitive endoderm cells by 3 days of Activin A treatment. Next, the presence of fibroblast growth factor-4 and bone morphogenetic protein-2 in the culture medium for 5 days induced efficient hepatic differentiation from definitive endoderm cells. Approximately 70% of the cells expressed the hepatic marker albumin. After 10 days of further in vitro maturation, these cells expressed the adult liver cell markers tyrosine aminotransferase, tryptophan oxygenase 2, phosphoenolpyruvate carboxykinase (PEPCK), Cyp7A1, Cyp3A4 and Cyp2B6. Furthermore, these cells exhibited functions associated with mature hepatocytes including albumin secretion, glycogen storage, indocyanine green, and low-density lipoprotein uptake, and inducible cytochrome P450 activity. When transplanted into CCl4 injured severe combined immunodeficiency mice, these cells integrated into the mouse liver and expressed human alpha-1 antitrypsin for at least 2 months. In addition, we found that the hESC-derived hepatic cells were readily infected by human immunodeficiency virus-hepatitis C virus pseudotype viruses. CONCLUSION: We have developed an efficient way to direct the differentiation of human embryonic stem cells into cells that exhibit characteristics of mature hepatocytes. Our studies should facilitate searching the molecular mechanisms underlying human liver development, and form the basis for hepatocyte transplantation and drug tests.     

小鼠胚胎干细胞体外定向心肌细胞分化的研究

目的：小鼠胚胎干细胞（embryonic stem cell,ES细胞）体外定向心肌细胞分化模型的建立。方法：通过悬滴培养技术定向诱导ES细胞分化为拟胚体,进而分化为心肌细胞,并用RT－PCR对四种肌肉肌动蛋白在EBs中的表达进行了鉴定。结果：在拟胚体和进一步分化的ES细胞中观察到自发节律性收缩的心肌细胞,α－平滑肌肌动蛋白（血管和胃肠道平滑肌）以及纹状肌肉肌动蛋白（心肌和骨骼肌）在这些心肌细胞样的细胞中均能表达。结论：小鼠胚胎干细胞体外定向心肌细胞分化模型的建立,可用于研究相关基因在心肌发育过程中的直接或间接作用,有助于胚胎干细胞衍生的心肌细胞用于临床治疗作用的研究。     

Differentiation of mouse embryonic stem cells into hepatocytes in vitro and in vivo

OBJECTIVE: Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages. Cell‐to‐cell contact is important for cell differentiation. Mouse ES cells were cocultured with mouse fetal liver cells and the green fluorescent protein (GFP) positive ES cells were transplanted into rats liver through the portal vein in order to investigate their potential to differentiate into hepatocytes. METHODS: Mouse ES cells were cocultured with the mouse fetal liver cell line, BNL.CL₂. They did not make direct contact; instead the culture media was exchanged freely. After coculture for 48 h, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4 and SEK1 mRNA were assayed by RT‐PCR, and alpha‐fetoprotein by immunohistochemistry. The morphology was investigated by microscopy. After transplantion of the GFP‐positive ES cells, the whole liver was removed from a rat every four days. The liver slices were examined under a fluorescent microscope to detect the GFP‐positive cells. Albumin was detected on the same slices by immunohistochemistry. RESULTS: After coculture with BNL.CL₂ cells, the differentiated ES cells had the same morphology as the BNL.CL₂ cells, and albumin, transthyretin, glucose 6 phosphates and SEK‐1 mRNA were found by RT‐PCR, and alpha‐fetoprotein was detected immuno­histochemically. The transplanted GFP‐positive ES cells were found in the rats’ liver slices by GFP fluorescence, and development of teratomas was not observed. The immunohistochemistry results indicated that the transplanted GFP‐positive ES cells retained an albumin‐producing ability. CONCLUSIONS: Cell‐to‐cell contact is important for the differentiation of ES cells. Mouse embryonic stem cells can differentiate into hepatocytes directly either in vitro or in vivo.     

Differentiation of embryonic stem cells into hepatocytes: biological functions and therapeutic application

Embryonic stem (ES) cells provide a unique source for tissue regeneration. We examined whether mouse ES cells can efficiently differentiate into transplantable hepatocytes. ES cells were implanted into mouse livers 24 hours after carbon tetrachloride intoxication; ES-derived cells with several hepatocyte-cell-markers were generated. They were able to grow in vitro and showed morphology consistent with typical mature hepatocytes and expressed hepatocyte-specific genes. After transplantation into the carbon tetrachloride-injured mouse liver, ES-derived green fluorescent protein-positive cells were incorporated into liver tissue and rescued mice from hepatic injury. No teratoma formation was observed in the transplant recipients. In conclusion, ES cells can provide a valuable tool for studying the molecular basis for differentiation of hepatocytes and form the basis for cell therapies.     

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