人脐带间充质干细胞生物学特性及向类肝细胞的分化

目的: 研究脐带间充质干细胞(umbilical cord-mesenchymal stem cells, UC-MSCs)生物学的特性及向肝细胞分化的可能性.方法:从脐带中分离间充质干细胞, 体外行传代培养, 检测脐带间充质干细胞表面免疫标志、细胞周期和生长活性等, 利用肝细胞生长因子、成纤维生长因子4和抑瘤素等细胞因子诱导脐带间充质干细胞向肝细胞分化, 用免疫细胞方法对诱导和未诱导的细胞进行免疫学检测, 糖原染色进行功能鉴定.结果: 从人脐带中可分离到贴壁生长的间充质干细胞, 细胞形态类似成纤维细胞,可在体外进行长期稳定培养; CD29、CD105和Vimentin表达阳性, 基本不表达CD34、CD31, 经加入细胞因子可成功将间充质干细胞向肝细胞诱导分化, 分化的细胞表达肝细胞表面标志物ALB、AFP、CK18和CK19, 糖原染色呈现阳性.结论:人脐带中可成功分离到间充质干细胞, 细胞可实现体外长期培养, 表达脐带间充质干细胞的表面标志, 在体外脐带间充质干细胞诱导分化为肝细胞, 有望成为细胞替代治疗的理想来源之一.     

人脐血间充质干细胞诱导分化成心肌细胞的研究

目的探讨体外脐血间充质干细胞诱导分化成心肌细胞的可行性和最佳方法。方法收集获知情同意的健康产妇脐血细胞,分离单个核细胞,从中进一步分离间充质干细胞,传代培养至第3代,应用免疫荧光流式细胞仪标记间充质干细胞特异性抗原CD34、CD44和CD90。5-氮胞苷诱导分化4周后,免疫组织化学染色和RT-PCR法分别检测心肌细胞标记物肌钙蛋白Ⅰ、转录因子GATA 4和β-肌球蛋白重链。结果在第3代细胞中,可检测到CD44、CD90的表达,未检测到CD34的表达。脐血间充质干细胞经5-氮胞苷诱导分化后,呈现成纤维细胞样形态和克隆增殖特点。免疫组织化学染色和RT-PCR可检测到肌钙蛋白Ⅰ,GATA 4和β-肌球蛋白重链的表达。结论脐血间充质干细胞能够被诱导分化成心肌样细胞,可成为干细胞移植的细胞来源。     

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。     

人脂肪间充质干细胞体外分离、培养的方法研究

目的：探讨人脂肪间充质干细胞体外分离及培养方法。方法用消化离心的方法获得人脂肪间充质干细胞,以1×104/cm2接种于体积分数为0．1％胎牛血清的LG-DMEM培养基中,并进行细胞形态学观察,绘制细胞生长曲线,流式细胞仪检测细胞表面抗原,行成脂及成骨诱导检测其多向分化潜能。结果获取的脂肪间充质干细胞大小较为均匀,呈梭形的成纤维细胞样,细胞增殖良好;细胞生长曲线测定表明接种后第4天细胞进入指数增长期,第8天后生长进入平台期,体外能长期培养存活,并保持不分化状态;流式细胞仪检测表明细胞高表达 CD13、CD73,低表达 CD34、CD45及 HLA-DR;能诱导成脂及成骨细胞。结论利用消化离心法在体外能分离得到脂肪间充质干细胞,细胞生长良好,可作为组织工程的种子细胞。     

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.     

体外培养成人脂肪间充质干细胞及向平滑肌细胞诱导分化

目的 体外培养成人脂肪间充质干细胞,并应用转化生长因子β将脂肪间充质干细胞诱导分化为平滑肌细胞.方法 采用酶消化法和贴壁培养法分离培养脂肪间充质干细胞,流式细胞仪对第3代和第5代细胞进行表面抗原检测,对第5代细胞进行转化生长因子β诱导,于诱导后第10天进行免疫化学鉴定.结果 体外培养的脂肪间充质干细胞呈扁平的长梭形,细胞形态均一,传代稳定.干细胞相关标志CD29和CD44表达阳性,造血干细胞相关标志CD34随传代次数的增加由弱阳性逐渐转为阴性,内皮细胞相关标志CD31表达阴性.流式细胞仪检测结果 发现脂肪间充质干细胞中G0/G1、S和G2/M期的细胞分别占90.14%、3.77%和6.09%.转化生长因子β定向诱导后倒置显微镜下观察细胞呈"峰"、"谷"样形态,免疫荧光化学检测发现诱导组细胞α平滑肌肌动蛋白表达阳性.结论 成人脂肪组织中含有间充质干细胞,且可在转化生长因子β诱导后分化为平滑肌细胞.     

体外诱导人脐血间充质干细胞向肝细胞样细胞分化的研究

目的 探讨人脐血间充质干细胞能否在体外诱导分化为肝细胞样细胞,并探索其定向诱导分化为肝细胞样细胞的分化机制。方法 无菌条件下采集正常产妇脐血,用相对密度为1.077的淋巴细胞分离液分离脐血MNC,进而采用贴壁培养法获得MSC,流式细胞仪检测其表面际志。分别用HGF、FGF4、俩者联合、无生长因子四种处理因素,及含2％FBS的DMEM,1×ITS讲行诱导培养,并于诱导前及诱导后的第7、14、21、28天留取细胞,RT～PCR法检测AFP、白蛋白及C-met FGFR2mRNA的表达,免疫细胞化学法检测AFP、白蛋白、抗肝细胞抗体和CK18的表达,PAS法进行糖原染色,分析是否诱导出肝细胞样细胞。结果 MSC强表达CD29、CD44,不表达CD34。诱导后7,21天分别检测出AFP,白蛋白mRNA及蛋白的表达,28天检测出CK18、抗肝细胞抗体的表达,21天、28天均可检测到糖原染色阳性细胞,随诱导时间的延长C-met,FGFR2 mRNA表达增高,HGF FGF4诱导组肝系细胞标志阳性率均高于单独生长因子诱导组(P<0.05);单独生长因子诱导组间无明显差异(P>0.05)。无生长因子诱导组在以上时间点均未检测到上述指标。结论HGF、FGF4均能诱导人脐血MSC分化为具有肝系细胞表型和功能的细胞,且二者在一定程度上有联合作用。生长因子与其相幢受体之间,可能存在正反馈调节机制。     

损伤肝脏条件培养液体外诱导小鼠骨髓间充质干细胞分化为肝细胞

目的探索损伤肝脏条件培养液体外诱导小鼠骨髓间充质干细胞（MSCs）分化为肝细胞的可行性及方法。方法培养注射CCl4所致的损伤小鼠肝脏组织块,收集条件培养液,进行MSCs诱导分化。通过形态学观察、RT-PCR、免疫荧光反应和过碘酸Schiff反应鉴定分化细胞。结果诱导组细胞呈肝细胞样变化。RT-PCR检测显示：AFP基因第5天开始表达,第10天表达增强,此后表达减弱；细胞角蛋白18和Alb基因第10天开始表达,持续到第20天；第20天检测到酪氨酸氨基转移酶基因表达。免疫荧光反应显示诱导20 d的细胞AFP、细胞角蛋白18、Alb表达阳性,诱导20 d细胞过碘酸Schiff反应呈阳性。结论MSCs在损伤肝脏条件培养液诱导下可分化为肝细胞。     

体外诱导人脂肪干细胞向内皮细胞分化的研究

目的摘要目的诱导人脂肪间充质干细胞（hADAS cells）成内皮细胞,探索最佳诱导条件,为血管组织工程种子细胞的来源及其临床应用提供基础。方法分为内皮细胞生长因子（VEGF）50ng／ml和20ng／ml、碱性成纤维细胞生长因子（b-FGF）10ng／ml两组诱导脂肪干细胞成内皮细胞,流式细胞仪检测其表面抗原CD34、CD31表达,荧光显微镜下观察其吞噬乙酰化低密度脂蛋白（DiI—ac—LDL）和结合植物凝集素（FITC—Lectin）的能力,细胞免疫荧光法检测Ⅷ因子的表达。结果流式证实间充质干细胞在诱导第4和8天CD31、CD34表达逐渐增加;可吞噬DiI—ac—LDL,与Letin结合;细胞免疫荧光法可检测到Ⅷ因子表达。结论VEGF、b-FGF可诱导脂肪干细胞成内皮样细胞,对VEGF有很大的依赖性,成为血管组织工程种子细胞的选择之一。     

肝细胞生长因子诱导华通胶干细胞向肝细胞样细胞分化的体外实验

目的观察外源性肝细胞生长因子对华通胶干细胞向肝细胞样细胞分化的诱导作用。方法取人脐带华通胶干细胞,进行细胞表型分析和端粒酶活性评价;通过检测华通胶干细胞中甲胎蛋白(AFP)和白蛋白(Alb)两种基因的相对含量,对两种浓度肝细胞生长因子诱导能力进行初步评估。分组:A组:华通胶干细胞+肝细胞生长因子(HGF)(20 ng/ml);B组:华通胶干细胞+HGF(40 ng/ml);C组:HL-7702人肝细胞株;D组:华通胶干细胞。结果华通胶干细胞表达间充质细胞表型:CD73、CD90、CD105与HLA-ABC阳性;不表达造血干细胞表型:CD45、HLA-DR阴性;华通胶干细胞端粒酶表达呈阳性。AFP基因相对含量在第7 d表达增高,仍未达到阳性对照组水平(P<0.05);高剂量组含量高于低剂量组(P<0.05);在21、42 d两组相对含量与阳性对照组差异无统计学意义;Alb基因相对含量在第7 d表达增高,高剂量与低剂量组差异无统计学意义,低于阳性对照组(P<0.05);在21 d两组相对含量与阳性对照组差异无统计学意义;在42 d,两组相对含量高于阳性对照组(P<0.05)。结论肝细胞生长因子可以诱导华通胶干细胞向肝细胞样细胞进行分化,该诱导作用与肝细胞生长因子剂量有关。     

Conditionally immortalized mouse hepatocytes for use in liver gene therapy

BACKGROUND AND AIMS: The use of hepatocytes for gene therapy is limited by the difficulty of maintaining and altering primary liver cells in culture. A conditionally immortalized mouse hepatocyte cell line has been developed which can be passaged indefinitely at the permissive temperature (33 degrees C), but fails to proliferate and dies at the non-permissive temperature (39 degrees C) in vitro. METHODS: Hepatocytes were harvested from a 6 week-old male transgenic mouse ('immortomouse') carrying a thermolabile SV40 Large T gene, using a modified two-step collagenase perfusion method, and serially passaged at 33 degrees C for more than 1 year. To assess the ability of immortohepatocytes to engraft and populate mouse liver, cells were infused into partially hepatectomized congenic mice via the portal vein (n = 10) or the spleen with (n = 2) and without (n = 2) partial hepatectomy. The ability to transfect immortohepatocytes was assessed using the reporter gene enhanced green fluorescent protein (EGFP). RESULTS: All immortohepatocytes in culture stained positive by immunohistochemistry for the hepatocyte markers albumin, AFP, CK8 and CK18. In early cultures a proportion of cells also stained strongly for the biliary epithelial markers CK7 and CK19. Late cell cultures were negative for M2PK and CK7 and stained variably with anti-CK19 antibodies. Cells transferred to the non-permissive temperature of 39 degrees C ceased proliferation and died within 1 week in vitro. Large T DNA was detected in the liver of all postoperative mice up to 2 weeks post-hepatocellular transplantation via PCR and Southern blot analysis. The immortohepatocytes were easily transfected with a reporter gene. CONCLUSIONS: Immortohepatocytes can survive in vivo after transfer to liver, and will be useful as a model for hepatic gene therapy.     

Characterization and enrichment of hepatic progenitor cells in adult rat liver

AIM: To detect the markers of oval cells in adult rat liver and to enrich them for further analysis of characterization in vitro. METHODS: Rat model for hepatic oval cell proliferation was established with 2-acetylaminofluorene and two third partial hepatectomy (2-AAF/PH). Paraffin embedded rat liver sections from model (11 d after hepatectomy) and control groups were stained with HE and OV6, cytokeratin19 (CK19), albumin, alpha fetoprotein (AFP), connexin43, and c-kit antibodies by immunohistochemistry. Oval cell proliferation was measured with BrdU incorporation test. C-kit positive oval cells were enriched by using magnetic activated cell sorting (MACS).The sorted oval cells were cultured in a low density to observe colony formation and to examine their characterization in vitro by immunocytochemistry and RT-PCR. RESULTS: A 2-AAF/PH model was successfully established to activate the oval cell compartment in rat liver. BrdU incorporation test of oval cell was positive. The hepatic oval cells coexpressed oval cell specific marker OV6, hepatocyte-marker albumin and cholangiocyte-marker CK19. They also expressed AFP and connexin 43. C-kit, one hematopoietic stem cell receptor, was expressed in hepatic oval cells at high levels. By using c-kit antibody in conjunction with MACS, we developed a rapid oval cell isolation protocol. The sorted cells formed colony when cultured in vitro. Cells in the colony expressed albumin or CK19 or coexpressed both and BrdU incorporation test was positive. RT-PCR on colony showed expression of albumin and CK19 gene. CONCLUSION: Hepatic oval cells in the 2-AAF/PH model had the properties of hepatic stem/progenitor cells. Using MACS, we established a method to isolate oval cells. The sorted hepatic oval cells can form colony in vitro which expresses different combinations of phenotypic markers and genes from both hepatocytes and cholangiocyte lineage.     

Fibroblast growth factor-4 and hepatocyte growth factor induce differentiation of human umbilical cord blood-derived mesenchymal stem cells into hepatocytes

AIM: To investigate the differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into hepatocytes by induction of fibroblast growth factor-4 (FGF-4) and hepatocyte growth factor (HGF), and to find a new source of cell types for therapies of hepatic diseases. METHODS: MSCs were isolated by combining gradient density centrifugation with plastic adherence. When HUCB-derived MSCs reached 70% confluence, they were cultured in Iscove modified Dulbecco medium (IMDM) supplemented with 10 mL/L FBS, 20 ng/mL HGF and 10 ng/mL FGF-4. The medium was changed every 4 d and stored for albumin, alpha-fetoprotein (AFP) and urea assay. Expression of CK-18 was detected by immunocytochemistry. Glycogen storage in hepatocytes was determined by PAS staining. RESULTS: By combining gradient density centrifugation with plastic adherence, we could isolate MSCs from 25.6% of human umbilical cord blood. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on d 28 by morphology. Compared with the control, the level of AFP increased significantly from d 12 to 18.20±1.16 μg/L (t = 2.884, P<0.05) in MSCs cultured with FGF-4 and HGF, and was higher (54.28±3.11 μg/L) on d 28 (t = 13.493, P<0.01). Albumin increased significantly on d 16 (t = 6.68, P<0.01) to 1.02±0.15 μg/mL, and to 3.63±0.30 μg/mL on d 28 (t = 11.748, P<0.01). Urea (4.72±1.03 μmol/L) was detected on d 20 (t = 4.272, P<0.01), and continued to increase to 10.28±1.06 μmol/L on d 28 (t = 9.276,P<0.01). Cells expressed CK-18 on d 16. Glycogen storage was observed on d 24. CONCLUSION: HUCB-derived MSCs can differentiate into hepatocytes by induction of FGF-4 and HGF. HUCB-derived MSCs are a new source of cell types for cell transplantation therapy of hepatic diseases.     

人骨髓来源多能成体祖细胞与人肝细胞系L02体外共培养向肝样细胞分化的实验研究

目的探索人骨髓来源多能成体祖细胞(hMAPCs)在与人肝细胞系L02在体外共培养条件下诱导分化为肝细胞的可行性,为其在组织工程学研究和临床医学中的广泛应用奠定基础。方法(1)取健康成人志愿者适量骨髓采用CD45、糖化蛋白A(GlyA)免疫微磁珠负分选,纯化培养CD45、GlyA hMAPCs。(2)间接共培养:将分别接种于盖玻片上的传代hMAPCs和人肝细胞系L02(密度均为1×105/ml)按照各50%比例共置于直径10 cm培养皿中,培养液共通,实现间接共培养。分别于第1、3、5、7天免疫细胞化学鉴定hMAPCs的Alb、AFP、细胞角蛋白18(CK18)、细胞角蛋白19(CK19)等肝细胞特征性表型表达变化情况,并设阳性对照(单独培养的L02细胞)和阴性对照(单独培养未经诱导的hMAPCs),计数阳性细胞比率。(3)直接共培养:将CFSE荧光(绿色)标记的hMAPCs与L02(密度均为1×104/ml)按照各50%比例混合后接种于激光共聚焦显微镜检查专用培养皿内,实现直接共培养。5 d后用SABC-Cy3免疫荧光(红色)双标后在激光共聚焦显微镜下分别观察hMAPCs表达Alb、AFP、CK18的情况。同样设阳性对照(单独培养的CFSE标记L02细胞)和阴性对照(单独培养的CFSE标记MAPCs)。镜下表达黄色荧光的细胞即是向肝细胞分化的MAPCs,为阳性细胞;单纯表达绿色荧光的细胞为未分化的MAPCs;单纯表达红色荧光的细胞为L02细胞。结果(1)间接共培养结果:AFP作为不成熟肝细胞的表型标志,在间接共培养第1天表现为强阳性表达,随后逐渐减弱,至第7天,AFP的阳性表达已经很少。而Alb在第1天有可疑阳性表达,第3天,Alb即出现较强的阳性表达,第5天,Alb的阳性表达就达到高峰,第7天,密集生长的细胞仍广泛的阳性表达Alb。第1、3天检测CK18均为阴性,至第5天CK18开始出现阳性表达,第7天CK18阳性表达较前增强。CK19在各时间点均为阴性着色。阳性对照细胞Alb及CK18有较强的阳性表达,AFP弱阳性表达,CK19阴性表达。阴性对照细胞均为阴性表达。(2)直接共培养结果:Alb和CK18检测时,较多细胞质内出现黄色荧光表达,而AFP阳性荧光表达则仅出现在极个别细胞质内,该结果与阳性对照细胞荧光表达类似,而阴性对照细胞未见有阳性荧光表达。结论(1)与人肝细胞系L02间接共培养能够诱导人骨髓来源的MAPCs向成熟肝样细胞定向分化。(2)与人肝细胞系L02直接共培养能够诱导人骨髓来源的MAPCs向成熟肝样细胞定向分化,且时间有提前趋势。     

Searching for common stem cells of the hepatic and hematopoietic systems in the human fetal liver: CD34+ cytokeratin 7/8+ cells express markers for stellate cells

BACKGROUND/AIMS: The hematopoietic and hepatic systems are intertwined in the liver during fetal life. Cells expressing the hematopoietic stem cell marker CD34 and cytokeratin 7/8 (CK7/8) are hypothesized to be common stem cells for the hematopoietic and hepatic systems. Our aim was to determine if human fetal liver cells expressing CD34 and CK7/8 represent a common stem cell for both the hematopoietic and hepatic systems. METHODS: CD34+CK7/8+ cells from midgestation livers were analyzed for the expression of various markers by flow cytometry and isolated based on their expression of CD34, nerve growth factor receptor (NGFR) and lack of CD45 expression. CD34+CD38- hematopoietic stem cells were also isolated and cultured in the presence of various hepatopoietins. RESULTS: CD34+CK7/8+ cells comprised 3.4-8.5% of the erythrocyte-depleted liver. CD34+CK7/8+ cells had unique light-scatter properties compared to hematopoietic precursors and did not express most markers associated with hematopoietic cells. They did stain with CD13, CD59, NGFR, desmin and alpha-smooth muscle actin. In culture, these cells had a stellate appearance. Cultured hematopoietic stem cells failed to generate hepatocytes. CONCLUSIONS: CD34+CK7/8+ cells are not common stem cells but rather appear to be hepatic stellate cells. A link between the hematopoietic and hepatic systems during fetal life requires further investigation.     

aFGF、HGF体外诱导小鼠ESC向肝细胞定向分化及标志物研究

目的观察酸性成纤维细胞生长因子(acid fibroblast growth factor,a FGF)、肝细胞生长因子(hepatocyte growth factor,HGF)对小鼠胚胎干细胞(embryonic stem cell,ESC)体外定向诱导分化作用及诱导后肝细胞标志物的表达水平。方法体外培养小鼠ESC使其发育成拟胚体,然后加入a FGF、HGF诱导ESC定向分化成肝细胞。收集培养上清液,RIA法测定甲胎蛋白(alpha fetoprotein,AFP)、白蛋白(albumin,ALB)浓度。PCR和免疫印迹法检测ALB、细胞角蛋白8(CK8)以及细胞角蛋白18(CK18)在细胞内mRNA和蛋白表达水平。结果 ESC培养5 d后发育成为拟胚体,加入不同浓度a FGF继续培养,5 d后AFP浓度降低,ALB浓度升高,与对照组相比有显著性差异。细胞内ALB、CK8及CK18表达水平明显升高。一次诱导后加入HGF,继续诱导5 d,上清液中AFP浓度降低,ALB浓度升高,具有浓度依赖性。ALB、CK8及CK18在细胞内表达升高。结论体外培养小鼠ESC,加入a FGF、HGF后可诱导其向肝细胞定向分化。肝细胞标志物AFP水平明显降低,ALB、CK8以及CK18表达水平明显升高。     

Expression of liver‐specific markers in naïve adipose‐derived mesenchymal stem cells

Background: Increasing evidence suggests that adipose tissue contains mesenchymal stem cells (MSC) that possess the ability to transdifferentiate into other cell types including hepatocytes, similar to bone marrow‐derived stem cells. The existence of precommitted cells in the MSC population may explain transdifferentiation. Aims: Our aim was to identify a population of putative hepatocyte‐like precursor cells in human adipose tissue. Methods: We analysed the ‘basal’ hepatic potential of undifferentiated, naïve human adipose‐derived mesenchymal stem cells (hADMSC). hADMSC were isolated from human adipose tissue and characterized for cell surface markers and for liver‐specific gene expression. Results: The isolated undifferentiated naïve hADMSCs expressed MSC surface markers. They also expressed α‐fetoprotein, CK18, CK19 and HNF4, which are known as early liver expressing genes. Interestingly, the undifferentiated naïve hADMSC were also positive for albumin, G‐6‐P and α‐1‐antitrypsin (AAT), which are all known to be predominantly expressed in adult liver cells. These cells acquired a hepatocyte‐specific phenotype and function upon treatment with a differentiation medium, resulting in the upregulation of albumin, G‐6‐P and AAT. Moreover, urea production, glycogen storage ability and cellular uptake of indocyanine green, which were absent in the basal state, were evident in the treated cells. Conclusions: Our findings suggest the presence of cells with hepatocyte‐like properties that are isolated from human adipose tissue and that can readily acquire hepatocyte‐like functions. Adipose tissue could thus be an exciting alternative means for repopulating the liver after various injuries, and might serve as a source for the transplantation of liver cells.