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.     

生长因子及细胞外基质对肝前体细胞体外增殖及分化的影响

目的 明确多种生长因子对胚胎肝脏前体细胞体外增殖分化的影响。方法 用胶原酶从胎龄14．5d SD大鼠胚胎肝脏分离单个细胞，采用^3H-TdR掺入法检测生长因子对胎肝细胞体外增殖的促进作用，并观察生长因子及细胞外基质成分对胎肝干细胞集落形成的影响。采用免疫细胞双标记及G-6-P酶活性测定以检测肝前体细胞表面标记的表达及向成熟肝细胞的分化能力。结果 肝前体细胞在体外培养时显示克隆样生长的特性。促肝细胞生长因子(HGF)、表皮生长因子(EGF)能促进肝前体细胞的增殖，使DNA合成加速，并促进干细胞集落的形成及角蛋白19、白蛋白、G-6-P的表达。转化生长因子α对肝前体细胞的促增殖作用较弱。转化生长因子β对肝前体细胞的增殖起到抑制作用。细胞基质成分Ⅰ型胶原、Ⅳ型胶原、层黏连蛋白能促进肝干细胞集落的形成，而纤维连接蛋白的作用较弱。肝干细胞单克隆增殖需要生长因子及细胞外基质的共同参与，加入新鲜分离胎肝细胞培养液上清液时单细胞增殖较快，于第5天即形成细胞集落。结论 HGF、EGF对肝前体细胞的增殖分化起重要作用，细胞外基质成分亦参与了其增殖分化过程。肝干细胞单克隆培养除需生长因子和细胞外基质外，可能亦有某些造血细胞、间质细胞分泌的因子参与其增殖分化的调控。     

小鼠胚胎与成体肝脏中干细胞变化的探讨

目的 依据干细胞生物学特性,探讨从胚胎到成体肝脏内具干细胞特性细胞的变化状况.方法 分离不同胎龄与日龄小鼠肝脏细胞进行体外培养,观察分离获得的细胞数、培养形成的集落数及生长状态,并对集落进行干细胞表面标记物(c-kit、c-Met、Nestin)、肝细胞表面标记物(AFP、ALB)及胆管上皮细胞表面标记物(CK19)的标记.结果 各组分离的细胞经体外培养均有集落形成,肝干细胞的表面标记提示形成的集落具有肝干细胞特性.其中以第15天胚胎肝脏分离所获的细胞数和培养形成的集落数最多、生长状态最好.15 d后观察的各项指标渐减,细胞生长状态渐差.结论 早期胚胎肝脏中大部分是具有干细胞特性的细胞,随胎龄增加,有干细胞特性的细胞逐渐减少,直至出生第5天仅有少数保持未分化状态的细胞存留在肝脏内.     

胎脑神经干细胞的培养及鉴定

检测3个月胎龄人胎脑各个解剖部分神经巢蛋白（Nestin）的表达,分离神经干细胞进行培养,诱导其分化,利用免疫荧光细胞化学技术对培养的神经干细胞及分化细胞进行鉴定.结果 在人胎脑室管膜下区、海马、纹状体、丘脑、嗅球、皮层部位发现数量不等的Nestin阳性表达细胞,从海马和皮层组织分离培养得到的大量悬浮生长具有连续增殖能力的神经干细胞球,能够传代培养,表达神经干细胞的标志物Nestin,经诱导分化为神经元和胶质细胞.     

胎肝来源间充质干细胞的分离、培养与多向分化

目的从胎肝中分离培养间充质下细胞，并研究其牛物学特性。方法用优化的方法从胎肝中分离获得间充质干细胞。利用流式细胞仪分析细胞表型和细胞周期分布，并体外诱导成骨、成脂肪和成肝组织细胞分化。并用染色方法鉴定成骨、成脂肪分化结合形态学方法和RT-PCR方法鉴定成肝组织分化结果。结果从胎肝中分离培养的细胞为成纤维样，贴壁生长．表型相对均一，表面标志为CD90，CD44，CD147，而CD34，CD45，HLA-DR，具有向肝组织分化的潜能，并可向成骨和成脂肪分化。结论从胎肝中可分离培养得到具有多向分化潜能的间充质干细胞。     

新生大鼠海马神经干细胞的分离、培养、分化和鉴定

目的:探讨从新生大鼠海马分离神经干细胞并进一步培养、诱导分化和鉴定的可行性。方法:分离出生1d大鼠海马,吸管吹打机械分离制成单细胞悬液,采用无血清培养和胎牛血清诱导分化,应用免疫荧光技术鉴定神经干细胞及其分化的子代细胞。结果:从新生大鼠海马分离的细胞呈巢蛋白免疫阳性并能在体外传代培养和连续形成克隆;血清诱导分化后的细胞可分别表达神经元和星形胶质细胞的特异性抗原β-微管蛋白Ⅲ和神经胶质原纤维酸性蛋白。结论:分离和培养的细胞能表达神经干细胞的特异性标志物巢蛋白,并且具有很强的增殖能力和多向分化潜能,说明新生大鼠海马存在神经干细胞,并可用于进一步的实验研究。     

肝病患者血清诱导间充质干细胞表达肝细胞特异性标志物的实验研究

目的：观察重型肝病患者血清对人骨髓间充质干细胞（MSCs）的诱导分化作用，探讨人MSCs分离培养、体外扩增的条件和方法。方法：从肋骨分离、培养人骨髓MSCs、体外扩增培养、鉴定，并采用重型肝病患者血清诱导培养，分别在诱导培养0、7、14、21、28天时留取细胞，并采用免疫细胞化学方法检测肝特异性标志物（AFP、Alb、CK-18）、用PAS进行糖原染色实验。结果：诱导后5天、MSCs表现为肝细胞样细胞，随着诱导培养时间的延长，肝特异性标志物逐渐出现和成熟。AFP在7天时表达水平最高，培养14、21、28天时表达逐渐减弱；Alb、CK- 18和糖原随着诱导时间的延长，表达逐渐增强。结论：密度梯度离心，贴壁培养和消化时间控制相结合，是一种较为有效的分离纯化方法。重型肝病患者血清能诱导骨髓MSCs表达肝细胞的特异性标志物。     

丁酸钠诱导体外培养的大鼠肝卵圆细胞分化为成熟肝细胞

目的探讨分化刺激剂丁酸钠对体外培养的大鼠肝卵圆细胞分化的影响。方法从喂养含0．1%乙硫氨酸的胆碱缺乏性饮食4～6周的人鼠肝脏中分离出盱卵圆细胞,用免疫细胞化学和逆转录聚合酶链反应(RT-PCR)等方法对其进行鉴定。用0．75mmol/L酸钠处理大鼠肝卵圆细咆后．姬姆萨染色观察细胞表型改变,western blot检测细胞白蛋白的表达水平。结果免疫细胞化学结果显示分离出的细胞既表达成熟肝细咆的标志物白蛋白,也表达胆管细胞的标志物细胞角蛋白19,RT-PCR结果显示这些细胞还表达干细胞的标志物c-kit,但不表达造血干细胞的标志物CD34,表明这些细胞是大鼠肝前体细胞——肝卵圆细胞。0．75mmol/L丁酸钠能诱导大鼠肝卵圆细胞出现明显的表型改变,细胞变大,变圆,核浆比减小,且双核细胞数增多,约占总细胞数的50%左右,同时western blot的结果显示0．75mmol/L丁酸钠能够提高大鼠肝卵圆细胞白蛋白的表达水平。。结论分化刺激剂丁酸钠能诱导体外培养的大鼠肝卵圆细胞向成熟肝细胞分化。     

三七皂甙诱导骨髓基质细胞分化为心肌样细胞的实验研究

目的探讨三七皂甙对体外定向诱导猪骨髓基质细胞(MSCs)分化为心肌样细胞的影响。方法用穿刺方法抽取猪骨髓,分离并培养骨髓基质细胞,中药三七皂甙定向诱导分化为心肌样细胞。倒置显微镜下观察细胞形态,免疫细胞化学法鉴定心肌细胞特异性抗原标志物肌钙蛋白T,用RT-PCR对心肌肌球蛋白重链(β-MHC)进行鉴定。取培养的猪心肌细胞作为阳性对照,未经三七皂甙诱导正常培养的骨髓基质细胞作为阴性对照。结果猪MSCs经三七皂甙诱导2h后,大部分MSCs可分化为心肌样细胞,免疫细胞化学检测细胞中的肌钙蛋白T呈阳性,RT-PCR显示能表达β-MHC。结论MSCs是骨髓来源的具有多向分化潜能的干细胞,在三七皂甙的定向诱导下,可以向心肌样细胞分化,有望成为心衰及心肌梗死等心肌损伤时干细胞移植治疗的理想细胞材料。     

大鼠骨髓间充质干细胞的分离、培养及鉴定

目的体外分离、培养及鉴定大鼠骨髓间充质干细胞（MSC）。方法全骨髓贴壁法体外分离、培养3月龄SD大鼠MSC,利用差速贴壁原理纯化MSC,并通过形态学观察、细胞表面抗原标志物、多细胞系诱导分化对其进行鉴定。结果P3代不表达抗原CD34和CD45,表达抗原CD29和CD44,符合MSC表面标志物特征。P3代MSC定向诱导后经ALP染色、矿化结节染色、油红O染色、Ⅱ型胶原荧光染色后鉴定具有骨向分化、脂向分化及软骨分化能力。结论全骨髓贴壁法能够建立稳定的MSC体外分离培养体系。     

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.     

Characterization of cell types during rat liver development

Hepatic stem cells have been identified in adult liver. Recently, the origin of hepatic progenitors and hepatocytes from bone marrow was demonstrated. Hematopoietic and hepatic stem cells share the markers CD 34, c-kit, and Thy1. Little is known about liver stem cells during liver development. In this study, we investigated the potential stem cell marker Thy1 and hepatocytic marker CK-18 during liver development to identify putative fetal liver stem cell candidates. Livers were harvested from embryonic and fetal day (ED) 16, ED 18, ED 20, and neonatal ED 22 stage rat fetuses from Sprague-Dawley rats. Fetal livers were digested by collagenase-DNAse solution and purified by percoll centrifugation. Magnetic cell sorting (MACS) depletion of fetal liver cells was performed using OX43 and OX44 antibodies. Cells were characterized by immunocytochemistry for Thy1, CK-18, and proliferating cell antigen Ki-67 and double labeling for Thy1 and CK-18. Thy1 expression was found at all stages of liver development before and after MACS in immunocytochemistry. Thy1 positive cells were enriched after MACS only in early developmental stages. An enrichment of CK-18 positive cells was found after MACS at all developmental stages. Cells coexpressing Thy1 and CK-18 were identified by double labeling of fetal liver cell isolates. In conclusion, hepatic progenitor cells (CK-18 positive) in fetal rat liver express Thy1. Other progenitors express only CK-18. This indicates the coexistence of different hepatic cell compartments. Isolation and further characterization of such cells is needed to demonstrate their biologic properties.     

Comparison of hepatic properties and transplantation of Thy-1(+) and Thy-1(-) cells isolated from embryonic day 14 rat fetal liver

Thy-1, a marker of hematopoietic progenitor cells, is also expressed in activated oval cells of rat liver. Thy-1(+) cells are also in rat fetal liver and exhibit properties of bipotent hepatic epithelial progenitor cells in culture. However, no information is available concerning liver repopulation by Thy-1(+) fetal liver cells. Therefore, we isolated Thy-1(+) and Thy-1(-) cells from embryonic day (ED) 14 fetal liver and compared their gene expression characteristics in vitro and proliferative and differentiation potential after transplantation into adult rat liver. Fetal liver cells selected for Thy-1 expression using immunomagnetic microbeads were enriched from 5.2%-87.2% Thy-1(+). The vast majority of alpha fetoprotein(+), albumin(+), cytokine-19(+), and E-cadherin(+) cells were found in cultured Thy-1(-) cells, whereas nearly all CD45(+) cells were in the Thy-1(+) fraction. In normal rat liver, transplanted Thy-1(+) cells produced only rare, small DPPIV(+) cell clusters, very few of which exhibited a hepatocytic phenotype. In retrorsine-treated liver, transplanted Thy-1(+) fetal liver cells achieved a 4.6%-23.5% repopulation. In contrast, Thy-1(-) fetal liver cells substantially repopulated normal adult liver and totally repopulated retrorsine-treated liver. Regarding the stromal cell-derived factor (SDF)-1/chemokine (C-X-C motif) receptor 4 (CXCR4) axis for stem cell homing, Thy-1(+) and Thy-1(-) fetal hepatic epithelial cells equally expressed CXCR4. However, SDF-1alpha expression was augmented in bile ducts and oval cells in retrorsine/partial hepatectomy-treated liver, and this correlated with liver repopulation by Thy-1(+) cells. CONCLUSION: Highly enriched Thy-1(+) ED14 fetal liver cells proliferate and repopulate the liver only after extensive liver injury and represent a fetal hepatic progenitor cell population distinct from Thy-1(-) stem/progenitor cells, which repopulate the normal adult liver.     

Hepatic stem cells and liver repopulation

Research on hepatic stem cells has entered a new era of controversy, excitement, and great expectations. Although adult liver stem cells have not yet been isolated, an enormous repopulating capacity of transplanted mature hepatocytes under conditions of continuous liver injury has been discovered. Stem/progenitor cells from fetal liver have been successfully isolated and transplanted, repopulating up to 10% of normal liver. However, progenitor cell lines from adult and embryonic liver have not shown significant repopulating activity. Intensive research on embryonic stem cells has revealed the first promising attempts to use these cells as a source of hepatic progenitors. Conditions for their differentiation in vitro, isolation of purified hepatic progenitor cells, and liver repopulation are currently being evaluated. Multilineage adult progenitor cells of mesenchymal origin from bone marrow, muscle, and brain may turn out to be the long-sought primitive potential stem cells remaining in adult tissues.     

Liver-specific gene expression in mesenchymal stem cells is induced by liver cells

AIM: The origin of putative liver cells from distinct bone marrow stem cells, e.g. hematopoietic stem cells or multipotent adult progenitor cells was found in recent in vitro studies. Cell culture experiments revealed a key role of growth factors for the induction of liver-specific genes in stem cell cultures. We investigated the potential of rat mesenchymal stem cells (MSC) from bone marrow to differentiate into hepatocytic cells in vitro. Furthermore, we assessed the influence of cocultured liver cells on induction of liver-specific gene expression. METHODS: Mesenchymal stem cells were marked with green fluorescent protein (GFP) by retroviral gene transduction. Clonal marked MSC were either cultured under liver stimulating conditions using fibronectin-coated culture dishes and medium supplemented with SCF, HGF, EGF, and FGF-4 alone, or in presence of freshly isolated rat liver cells. Cells in cocultures were harvested and GFP+ or GFP-cells were separated using fluorescence activated cell sorting. RT-PCR analysis for the stem cell marker Thy1 and the hepatocytic markers CK-18, albumin, CK-19, and AFP was performed in the different cell populations. RESULTS: Under the specified culture conditions, rat MSC cocultured with liver cells expressed albumin-, CK-18, CK-19, and AFP-RNA over 3 weeks, whereas MSC cultured alone did not show liver specific gene expression. CONCLUSION: The results indicate that (1) rat MSC from bone marrow can differentiate towards hepatocytic lineage in vitro, and (2) that the microenvironment plays a decisive role for the induction of hepatic differentiation of rMSC.     

Flow cytometric isolation and clonal identification of self-renewing bipotent hepatic progenitor cells in adult mouse liver

The adult liver progenitor cells appear in response to several types of pathological liver injury, especially when hepatocyte replication is blocked. These cells are histologically identified as cells that express cholangiocyte markers and proliferate in the portal area of the hepatic lobule. Although these cells play an important role in liver regeneration, the precise characterization that determines these cells as self-renewing bipotent primitive hepatic cells remains to be shown. Here we attempted to isolate cells that express a cholangiocyte marker from the adult mouse liver and perform single cell-based analysis to examine precisely bilineage differentiation potential and self-renewing capability of these cells. Based on the results of microarray analysis and immunohistochemistry, we used an antibody against CD133 and isolate CD133(+) cells via flow cytometry. We then cultured and propagated isolated cells in a single cell culture condition and examined their potential for proliferation and differentiation in vitro and in vivo. Isolated cells that could form large colonies (LCs) in culture gave rise to both hepatocytes and cholangiocytes as descendants, while maintaining undifferentiated cells by self-renewing cell divisions. The clonogenic progeny of an LC-forming cell is capable of reconstituting hepatic tissues in vivo by differentiating into fully functional hepatocytes. Moreover, the deletion of p53 in isolated LC-forming cells resulted in the formation of tumors with some characteristics of hepatocellular carcinoma and cholangiocarcinoma upon subcutaneous injection into immunodeficient mutant mice. These data provide evidence for the stem cell-like capacity of isolated and clonally cultured CD133(+) LC-forming cells. Conclusion: Our method for prospectively isolating hepatic progenitor cells from the adult mouse liver will facilitate study of their roles in liver regeneration and carcinogenesis.