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.     

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。     

肝细胞生长因子联合成纤维细胞生长因子-4诱导人骨髓来源的多能成体祖细胞向肝样细胞分化

目的 探索肝细胞生长因子（HGF）-4成纤维生长因子4-（FGF-4）诱导人骨髓来源多能成体祖细胞（hMAPCs）分化为肝细胞的可行性，为肝组织工程提供新的种子细胞来源。方法 （1）取志愿者适量骨髓后采用梯度密度离心＋贴壁培养获取骨髓间充质干细胞（MSCs），将MSCs通过CD45、GlyA免疫微磁珠负分选得到hMAPCS。（2）将hMAPCs用HGF＋FGF-4进行诱导分化。实验分组：A组：HGF（20ng／m1）＋（FGF-4）10ng／m1诱导hMAPCS；B组（阳性对照组）：L-02人肝细胞株；C组（阴性对照组）：未加任何诱导因素的hMAPCs。（3）免疫细胞化学鉴定不同诱导分化阶段细胞的白蛋白（A1b）、甲胎蛋白（AFP），细胞角蛋白-18（CK-18）等肝细胞特征的表型变化评计数阳性细胞比率。（4）逆转录-聚合酶链反应检测不同诱导分化阶段细胞的A1b、AFP，CK-18的mRNA转录。（5）Western blot检测诱导分化第21，35天后细胞的A1b表达。结果（1）免疫细胞化学结果：A1b、CK18在诱导组中不同时间段基本为阳性着色；AFP在诱导分化第7天为阳性着色，在诱导第14，21天为阴性着色。（2）逆转录聚合酶链反应结果：作为不成熟肝细胞表型的AFP，在诱导分化的第7天有mRNA阳性表达；作为成熟肝细胞表型的A1b及CK-18，在不同时间段mRNA均为阳性表达。（3）Western blot检测诱导分化第21、35天后细胞的A1b表达。结论hMAPCS在一定诱导条件下具有向肝样细胞分化的潜能。     

脐带组织来源基质干细胞的肝细胞相关标志物分析

目的研究脐带组织来源基质干细胞（umbilical cord-matrix stem cells,UCMSCs）具备的部分肝细胞生物学特性。方法观察分离脐带基质干细胞细胞形态和免疫表型,利用免疫组化检测脐带基质干细胞的CK-18、AFP、ALB和肝细胞抗原（hepatocyte paraf-fin 1,HepPar1）的表达,通过RT-PCR方法评价脐带基质干细胞的多种肝细胞特异性基因表达的稳定性。结果从脐带组织内分离出类成纤维细胞样的脐带基质干细胞,CD105表达阳性,CD45表达阴性;第4代脐带基质干细胞表达CK-18、AFP、ALB等蛋白,而不表达HepPar1;RT-PCR结果表明10代内脐带基质干细胞持续稳定表达ALB、CK-19、CPS-1、CYP1A1,而不表达CYP3A4。结论脐带基质干细胞是一种本身表达一定肝细胞标志物的间充质干细胞,可能更有利于向肝细胞分化或在移植后发挥更好的治疗作用。     

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.     

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.     

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.     

胆汁化血清对骨髓间充质干细胞向肝细胞样细胞定向分化的诱导

目的:寻找生物人工肝和肝细胞移植需要合适的肝细胞来源,研究不同诱导方法对骨髓间充质干细胞(MSC)在体外分化为肝细胞样细胞的影响。方法:本研究应用贴壁法对MSC进行分离和培养后,应用胆汁化血清及肝细胞生长因子不同诱导方式对之进行诱导分化,研究MSC转化为肝细胞样细胞的可能性,并进行转化的鉴定。结果:经贴壁分离和培养的MSC在5%胆汁化血清诱导作用下,21 d后可分化为形态似肝细胞样的细胞,经免疫组织化学显示,该细胞可表达细胞角蛋白18(CK18)和甲胎蛋白(AFP),且原位杂交显示,在培养的第35天,部分细胞还可合成清蛋白,与肝细胞生长因子(HGF)有着相似的诱导效果。结论:在合适的条件下,MSC可向肝细胞方向转化,且本方法操作简捷,胆汁化血清易于获得,具有潜在的应用价值。     

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.     

间充质干细胞对狼疮肾炎T淋巴细胞的免疫调节作用

目的 探讨间充质干细胞(MSCs)在体外对狼疮肾炎(LN)外周血T淋巴细胞的免疫调节作用.方法 从人骨髓中分离培养MSCs,采用流式细胞仪(FCM)分析鉴定MSCs的纯度.在植物血凝素(PHA)刺激下,LN外周血T淋巴细胞与不同数量的MSCs共培养.分组:A组:T淋巴细胞;B组:MSCsI+T淋巴细胞(MSCsl:T=1:5);C组:MSCs2+T淋巴细胞(MSCs2:T=1:20);D组:MSCs3+T淋巴细胞(MSCs3:T=1:100).用四甲基偶氮唑蓝(MTT)比色法检测各组T淋巴细胞的增殖情况,FCM分析各组T淋巴细胞CD28和CD152的表达及T淋巴细胞调亡情况,实时定量反转录-聚合酶链反应(RT-PCR)检测各组T淋巴细胞的干扰素(IFN)-γ、白细胞介素(IL)-10、转化生长因子(TGF)-β1基因的水平.结果 在体外,MSCs对由PHA诱导的LNT淋巴细胞的增殖、凋亡均有抑制作用,且抑制作用与MSCs呈剂量依赖性.MSCs抑制T淋巴细胞CD28表达,对CD152的表达无明显影响.MSCs能促进LN患者T淋巴细胞TGF-β1基因的表达,抑制IL-10、IFN-γ基因的表达.结论 MSCs可能通过抑制T淋巴细胞增殖、减少T淋巴细胞凋亡、抑制T淋巴细胞CD28表达和促进T淋巴细胞TGF-β1,基因表达及抑制IL-10、IFN-γ基因表达来下调LN的免疫反应.     

Embryonic stem cells develop into hepatocytes after intrasplenic transplantation in CCl4-treated mice

AIM： To transplant undifferentiated embryonic stem （ES） cells into the spleens of carbon tetrachloride （CCl4）-treated mice to determine their ability to differentiate into hepatocytes in the liver.
METHODS： CCh, 0.5 mL/kg body weight, was injected into the peritoneum of C57BL/6 mice twice a week for 5 wk. In group 1 （n = 12）, 1 × 10^5 undifferentiated ES cells （0.1 mL of 1 × 10^6/mL solution）, genetically labeled with GFP, were transplanted into the spleens 1 d after the second injection. Group 2 mice （n = 12） were injected with 0.2 mL of saline twice a week, instead of CCh, and the same amount of ES cells was transplanted into the spleens. Group 3 mice （n = 6） were treated with CCh and injected with 0.1 mL of saline into the spleen, instead of ES cells. Histochemical analyses of the livers were performed on post-transplantation d （PD） 10, 20, and 30.
RESULTS： Considerable numbers of GFP-immunopositive cells were found in the periportal regions in group 1 mice （CCh-treated） on PD 10, however, not in those untreated with CCh （group 2）. The GFP-positive cells were also immunopositive for albumin （ALB）, alpha-1 antitrypsin, cytokeratin 18, and hepatocyte nuclear factor 4 alpha on PD 20. Interestingly, most of the GFP-positive cells were immunopositive for DLK, a hepatoblast marker, on PD 10. Although very few ES-derived cells were demonstrated immunohistologically in the livers of group 1 mice on PD 30, improvements in liver fibrosis were observed. Unexpectedly, liver tumor formation was not observed in any of the mice that received ES cell transplantation during the experimental period
CONCLUSION： Undifferentiated ES cells developed into hepatocyte-like cells with appropriate integration into tissue, without uncontrolled cell growth.     

In vitro transdifferentiation of adult bone marrow Sca-1+ cKit- cells cocultured with fetal liver cells into hepatic-like cells without fusion

Several research groups have recently reported that certain bone marrow cells (BMCs) differentiate into hepatocytes in vitro as well as in vivo in rodents. However, it has yet to be elucidated what factors effectively trigger and sustain transdifferentiation of BMCs. In the present study, we specifically asked whether the presence of murine fetal liver cells (FLCs) triggered and supported in vitro transdifferentiation of murine BMCs. Fractionated BMCs from green fluorescence protein (GFP)-expressing transgenic mice and FLCs from ROSA26 mice (X-gal(+) FLCs) were cocultured in the presence of hepatocyte growth factor in laminin-coated dishes. We found that Sca-1(+) BMCs gave rise to adherent hepatic-like cells, which expressed albumin as assessed with immunocytochemistry and RNA-polymerase chain reaction (PCR), and alpha-fetoprotein and cytokeratin 19 as examined with RNA-PCR. When GFP(+)Sca-1(+)cKit(-) cells were cocultured with X-gal(+) FLCs, all GFP(+) albumin-producing cells were negative for X-gal, showing that cell fusion was not associated in the observed BMCs' differentiation into hepatic-like cells. Titration analysis revealed that 1 of 5,943 Sca-1(+)cKit(-) cells had the ability to proliferate and differentiate into hepatic-like cells. These data strongly suggest that BMCs differentiate into hepatic-like cells in the presence of FLCs and that the present method may be useful for propagating BMC-derived hepatocytic progenitors and for investigating the nature of those cells.     

Targeted migration of mesenchymal stem cells modified with CXCR4 to acute failing liver improves liver regeneration

AIM: To improve the colonization rate of transplanted mesenchymal stem cells (MSCs) in the liver and effect of MSC transplantation for acute liver failure (ALF).METHODS: MSC was modified with the chemokine CXC receptor 4 (CXCR4) gene (CXCR4-MSC) or not (Null-MSC) through lentiviral transduction. The characteristics of CXCR4-MSCs and Null-MSCs were determined by real-time quantitative polymerase chain reaction, Western blotting and flow cytometry. CXCR4-MSCs and Null-MSCs were infused intravenously 24 h after administration of CCl₄ in nude mice. The distribution of the MSCs, survival rates, liver function, hepatocyte regeneration and growth factors of the recipient mice were analyzed.RESULTS: In vitro, CXCR4-MSCs showed better migration capability toward stromal cell-derived factor-1α and a protective effect against thioacetamide in hepatocytes. In vivo imaging showed that CXCR4-MSCs migrated to the liver in larger numbers than Null-MSCs 1 and 5 d after ALF. Higher colonization led to a longer lifetime and better liver function. Either CXCR4-MSCs or Null-MSCs exhibited a paracrine effect through secreting hepatocyte growth factor and vascular endothelial growth factor. Immunohistochemical analysis of Ki-67 showed increased cell proliferation in the damaged liver of CXCR4-MSC-treated animals.CONCLUSION: Genetically modified MSCs expressing CXCR4 showed greater colonization and conferred better functional recovery in damaged liver.     

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。     

Dynamic tracking of stem cells in an acute liver failure model

AIM:To investigate a dual labeling technique,which would enable real-time monitoring of transplanted embryonic stem cell(ESC) kinetics,as well as long-term tracking.METHODS:Liver damage was induced in C57/BL6 male mice(n = 40) by acetaminophen(APAP) 300 mg/kg administered intraperitoneally.Green fluorescence protein(GFP) positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide(DiR) immediately before transplantationinto the spleen.Each of the animals in the cell therapy group(n = 20) received 5 × 10 6 ESCs 4 h following treatment with APAP.The control group(n = 20) received the vehicle only.The distribution and dynamics of the cells were monitored in real-time with the IVIS Lumina-2 at 30 min post transplantation,then at 3,12,24,48 and 72 h,and after one and 2 wk.Immunohistochemical examination of liver tissue was used to identify expression of GFP and albumin.Plasma alanine aminotransferase(ALT) was measured as an indication of liver damage.RESULTS:DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence.The transplanted cells were confined inside the spleen at 30 min post-transplantation,gradually moved into the splenic vein,and were detectable in parts of the liver at the 3 h time-point.Within 24 h of transplantation,homing of almost 90% of cells was confirmed in the liver.On day three,however,the DiR signal started to fade out,and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imaging,and confirmed that the highest photon emission was in the liver(P 0.0001).At 2 wk,the DiRsignal was no longer detectable in vivo ;however,immunohistochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells.GFP +ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma,with the presence of a larger number of GFP +ve cells incorporated within the sinusoidal endothelial lining.Very faint albumin expression was detected in the transplanted GFP +ve cells at 72 h;however at 2 wk,few cells that were positive for GFP were also strongly positive for albumin.There was a significant improvement in serum levels of ALT,albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point.In the cell therapy group,serum ALT was significantly(P = 0.016) lower and albumin(P = 0.009) was significantly higher when compared with the control group at the 2 wk time-point;however there was no difference in mortality between the two groups.CONCLUSION:Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribution,survival and engraftment of transplanted cells,and could be used for cell therapy models.     

体外不同诱导条件对人脐血间充质干细胞分化为神经细胞的研究

目的观察体外不同诱导条件对人脐血间充质干细胞(MSCs)向神经细胞分化的作用,探讨最佳诱导方法。方法取第3代脐血MSCs进行诱导,分为化学诱导剂组、生长因子诱导组、丹参素联合生长因子诱导组。采用免疫细胞化学方法和免疫荧光方法检测诱导前后神经元特异性标志小鼠抗神经元核抗原(NeuN)、兔抗微管蛋白(β-TubulinⅢ)和星形胶质细胞特异性标志神经胶质纤维酸性蛋白(GFAP)表达的变化。结果 MSCs经3种方法诱导后出现类似神经元样细胞的形态改变,伸出长突起。免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能特异性表达NeuN和β-TubulinⅢ,而GFAP阳性细胞较少。丹参素联合生长因子诱导组β-TubulinⅢ及NeuN的阳性细胞率均明显高于生长因子诱导组和化学诱导剂组,而GFAP阳性细胞率明显低于生长因子诱导组,差异均有统计学意义。结论丹参素联合表皮生长因子和碱性成纤维细胞生长因子在体外可定向诱导人脐血MSCs分化为神经元,诱导效果最佳。     

Functional integration of hepatocytes derived from human mesenchymal stem cells into mouse livers

AIMS: At present, clinical success of hepatocyte transplantation as an alternative to whole liver transplantation is hampered by the limited availability of suitable donor organs for the isolation of transplantable hepatocytes. Hence, novel cell sources are required to deliver hepatocytes of adequate quality for clinical use. Mesenchymal stem cells (MSCs) from human bone marrow may have the potential to differentiate into hepatocytes in vitro and in vivo. METHODS: Isolated MSCs were selected by density gradient centrifugation and plastic adherence, differentiated in the presence of human hepatocyte growth medium and transplanted in immunodeficient Pfp/Rag2 mice. RESULTS: Here, we demonstrate that human MSCs gain in vitro the characteristic morphology and function of hepatocytes in response to specified growth factors. Specifically, preconditioned MSCs store glycogen, synthesise urea and feature the active hepatocyte-specific gene promoter of phosphoenolpyruvate carboxykinase (PCK1). After transplantation into livers of immunodeficient mice, preconditioned MSCs engraft predominantly in the periportal portion of the liver lobule. In situ, the cells continue to store glycogen and express PCK1, connexin32, albumin and the human hepatocyte-specific antigen HepPar1, indicating that the transplanted cells retain prominent qualities of hepatocytes after their regional integration. CONCLUSION: MSCs derived from human bone marrow may serve as a novel source for the propagation of hepatocyte-like cells suitable for cell therapy in liver diseases.