Thy1-positive bone marrow stem cells express liver-specific genes in vitro and can mature into hepatocytes in vivo

The bone marrow contains stem cells that have the potential to differentiate into a variety of organ-specific mature cells, including the liver and the pancreas. Recently, the origin of hepatic progenitors and hepatocytes was identified to be the bone marrow. However, evidence that describes which cells, among all bone marrow cells, differentiate into hepatocytes, has not yet been presented. Based on recent reports, hematopoietic and hepatic stem cells share characteristic markers such as CD34, c-kit, and Thy1. In particular, both hematopoietic and hepatic stem cells express the Thy1 antigen. We investigated whether rat Thy1-positive bone marrow cells express liver-specific genes in vitro, and whether transplanted Thy1 BM cells differentiate into mature hepatocytes in vivo. For collection of Thy1 cells from bone marrow, FITC-conjugated anti-Thy1.1 monoclonal antibody was used with a Fluorescence-Activated Cell Sorter system. A coculture system of 2 separate layers was used for culture of Thy bone marrow cells. Cultured Thy1 cells expressed albumin protein, which was analyzed by immunofluorescent staining. Thy1 bone marrow cells obtained from wild-type dipeptidyl peptidase IV (DPPIV(+)) male rat were directly transplanted into the injured liver of DPPIV mutant (DPPIV(−)) Fisher 344 female rats and differentiated into mature hepatocytes in recipient liver on 60 days. Donor-derived hepatocytes were confirmed by DPPIV staining and Y-chromsome in situ hybridization. Our results suggest that Thy1-positive bone marrow cells have the potential to generate liver-specific genes in vitro and can differentiate into mature hepatocytes in adult liver in vivo. Thy1-positive bone marrow stem cells may represent preexisting hepatocyte-specific stem cells.     

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.     

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.     

骨髓干细胞在大鼠肝再生环境中的分化

研究大鼠骨髓干细胞在部分肝切除后肝再生环境中的分化。从部分肝切除模型大鼠的胫骨中提取骨髓细胞,应用流式细胞仪富集骨髓干细胞,以PKH26-GL体外标记后通过门静脉进行自体移植,2周后行白蛋白和角蛋白8免疫组化检查。结果肝板肝细胞间PKH26-GL标记骨髓干细胞表达白蛋白、角蛋白8。提示骨髓干细胞在部分肝切除后肝再生环境中能分化为肝细胞,骨髓干细胞可能参与部分肝切除后的肝再生过程。     

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.     

Mesenchymal stem cells: isolation, characterisation and in vivo fluorescent dye tracking

Cell therapies have been used to regenerate the heart by direct myocardial delivery, by coronary infusion and by surface attached scaffolds. Multipotent mesenchymal stem cells (MSC) with capacity to differentiate into cardiomyocytes and other cell lines have been predominantly trialled in rodents. However, large animal models are increasingly needed to translate basic research into new, safe regenerative therapies. Understanding the mode of action of cell therapies in the mammalian heart has been limited by cell tracking capability. This study examined the ability to track the fate of allogeneic MSC in sheep using various fluorescent dyes. MSC isolated from sheep bone marrow were grown in culture following extraction and flow cytometric characterisation. After labelling with fluorescent tracking dyes (e.g. CFSE and DiI) cells were tested for in vitro and in vivo signal up to six weeks. Labelling effect on cell division and differentiation was studied. Several dyes lost fluorescence and slowed cell division. However, the thiol reactive dye CM-DiI showed detectable in vivo fluorescence in labelled MSC six weeks after injection into sheep skeletal muscle and two weeks after implantation of an MSC coated biomaterial scaffold. CM-DiI labelled MSC differentiated in vitro showed label retention over four weeks. The fluorescent membrane dye CM-DiI tracks implanted sheep MSC and provides an alternative to traditional cell markers such as gene modified GFP.     

Differentiation of rat bone marrow stem cells in liver after partial hepatectomy

AIM: To investigate the differentiation of rat bone marrow stem cells in liver after partial hepatectomy. METHODS: Bone marrow cells were collected from the tibia of rat with partial hepatectomy, the medial and left hepatic lobes were excised. The bone marrow stem cells (Thy CD3-CD45RA- cells) were enriched from the bone marrow cells by depleting red cells and fluorescence-activated cell sorting. The sorted bone marrow stem cells were labeled by PKH26-GL in vitro and autotransplanted by portal vein injection. After 2 wk, the transplanted bone marrow stem cells in liver were examined by the immunohistochemistry of albumin (hepatocyte-specific marker). RESULTS: The bone marrow stem cells (Thy CD3-CD45RA- cells) accounted for 2.8% of bone marrow cells without red cells. The labeling rate of 10μM PKH26-GL on sorted bone marrow stem cells was about 95%. There were sporadic PKH26-GL-labeled cells among he-patocytes in liver tissue section, and some of the cells expressed albumin. CONCLUSION: Rat bone marrow stem cells can differentiate into hepatocytes in regenerative environment and may participate in liver regeneration after partial hepatectomy.     

Mesenchymal stem cells remain of host origin even a long time after allogeneic peripheral blood stem cell or bone marrow transplantation

OBJECTIVE: Plasticity of hematopoietic stem cells (HSC) has gained major interest in stem cell research. In order to investigate whether HSC may differentiate into mesenchymal stem cells (MSC), we assessed chimerism in peripheral blood (PB), mononuclear cell fractions (MNC) of bone marrow, and MSC derived from bone marrow (BM) from 27 up to 4225 days after allogeneic transplantation. PATIENTS AND METHODS: We applied fluorescence in situ hybridization using X/Y gene probes in sex-mismatched and STR-PCR in sex-matched patients. MSC could have been generated in 27 of 55 bone marrow samples derived from 20 patients. Fifteen patients received peripheral blood stem cell transplants (PBSCT), including CD34-selected PBSCT in two. Five patients received bone marrow. RESULTS: While all patients had chimerism in PB and MNC of the BM, in all but one patient BM-derived MSC were of recipient origin. This single patient showed reproducibly MSC of donor origin in a frequency of 1% after having received a CD34-selected PBSCT. Looking at graft collections, MSCs were easily generated from BM specimens, while no MSC could be derived from PBSC samples. CONCLUSION: Even though HSC have been found to differentiate into a variety of nonhematological cell types, they usually do not differentiate into MSC after allogeneic transplantation.     

Mesenchymal stem cells obtained after bone marrow transplantation or peripheral blood stem cell transplantation originate from host tissue

Mesenchymal stem cells (MSC) obtained from human bone marrow have been described as adult stem cells with the ability of extensive self-renewal and clonal expansion, as well as the capacity to differentiate into various tissue types and to modulate the immune system. Some data indicate that leukapheresis products may also contain non-hematopoietic stem cells, as they occur in whole bone marrow transplantation (BMT). However, there is still controversy whether MSC expand in the host after transplantation like blood progenitor cells do. Therefore, we were interested in finding out if graft MSC can be detected in leukapheresis products and in bone marrow after BMT and peripheral blood stem cell transplantation (PBSCT). Every sample from total bone marrow transplants exhibited growth of MSC after in vitro culture, but not one of nine leukapheresis products did. In addition, bone marrow aspirates of 9 patients receiving BMT and of 18 patients after PBSCT were examined for origin of MSC. Almost all MSC samples exhibited a complete host profile, whereas peripheral blood cells were of donor origin. We conclude that even if trace amounts of MSC are co-transplanted during PBSCT or BMT, they do not expand significantly in the host bone marrow.     

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells

AIM: To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell (MSC) in vitro, in order to find suitable resource of hepatocytes for bioartificial liver or liver transplantation. METHODS: The rat myeloid MSC was isolated and divided into three groups which were cultured by Frieden-steion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor (HGF), respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS: After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured with cholestatic serum. Furthermore, on the 35th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF. CONCLUSION: Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.     

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells

AIM: To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell (MSC)in vitro, in order to find suitable resource of hepatocytes for bioartificial liver or liver transplantation. METHODS: The rat myeloid MSC was isolated and divided into three groups which were cultured by Frieden steion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor (HGF), respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS: After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured withcholestatic serum. Furthermore, on the 35th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF. CONCLUSION: Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.     

Cell-to-cell contact induces mesenchymal stem cell to differentiate into cardiomyocyte and smooth muscle cell

BACKGROUND: Recent evidences have suggested that stem cell can differentiate into cardiomyocyte and smooth muscle cell (SMC) in vivo or in vitro. But the mechanism on how stem cell differentiates is still unknown. We investigated whether intercellular interaction or soluble chemical factors would induce mesenchymal stem cells (MSCs) to acquire the phenotypical characteristics of cardiomyocytes or SMC. METHODS: MSCs were isolated from rat bone marrow with density gradient centrifugation and amplified in vitro. Flow cytometry was used to monitor the expression of surface antigen profile. After labeled by GFP (green fluorescent protein) transfection, rat MSCs were used to culture with adult rat cardiomyocytes and rat aortic SMCs in direct co-culture, indirect co-culture and conditioned culture, respectively. One week later, immunofluorescence staining against alpha-actin, desmin, and cardiac troponin T (cTnT) for cardiomyocyte, smooth muscle calponin and SM-alpha-actin for SMC were performed. RESULTS: Immunofluorescence staining was positive against alpha-actin, desmin, and cTnT on MSCs in co-culture group with adult cardiomyocytes, positive against smooth muscle calponin and SM-alpha-actin on MSCs in co-culture group with SMCs. In contrast, no alpha-actin, desmin, and cTnT expression was observed in the indirect co-culture group and conditioned culture group; no smooth muscle calponin and SM-alpha-actin in the indirect co-culture group and conditioned culture group. CONCLUSIONS: Direct cell-to-cell contact between MSC and adult cardiomyocyte or SMC, but not the soluble signaling molecules is obligatory in the differentiation of MSC into cardiomyocytes or SMC.     

骨髓间充质干细胞向心肌分化的实验研究

目的:研究骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)分化为心肌样细胞的能力,用于心肌补片治疗心肌梗死的研究。方法:分离C57/BSL小鼠BMSCs,全培养差速贴壁法,经过贴壁培养至第3代,流式细胞仪鉴定细胞表面标志(CD34、CD45、CD73、CD90),经10μmol/L的5-氮杂胞苷诱导细胞,24 h后更换完全培养基培养,2 w后进行免疫荧光染色,荧光显微镜观察心肌钙蛋白T(cTnT)和连接素蛋白43(CX43)的表达。结果:流式鉴定结果显示CD34、CD45阴性,CD73强阳性,CD90弱阳性。免疫荧光染色显示,诱导后细胞高表达心肌细胞特异性蛋白cTnT,连接素蛋白CX43表达水平明显增加。结论:5-aza可以诱导BMSCs大量表达心肌特异性蛋白cTnT和细胞连接素蛋白(CX43),干细胞分化为心肌样细胞,为干细胞移植治疗小鼠心梗提供种子细胞。     

骨髓间(充)质干细胞体外定向诱导心肌样细胞超微结构特征研究

目的：研究兔骨髓间（充）质干细胞（mesenchymal stem cell，MSC）经5～氮杂胞苷（5-azacytidine，5-aza）诱导在体外定向分化的心肌样细胞超微结构特征。方法：取兔髂骨骨髓，分离并培养骨髓MSC，用5～aza定向诱导向心肌样细胞分化。以相差显微镜、透射电镜观察心肌样钏胞形态学变化及超微结构特征。结果：5-aza诱导后，部分细胞体积增大，呈“捧状”或“珠状”结构，有肌管样结构形成，透射电镜下见有肌丝、心房颗粒及线粒体等心肌样细胞超微等结构。结论：经5-氮杂胞苷诱导分化的骨髓间（充）质干细胞具有心肌样细胞超微结构特征．     

Characterization of the differentiation capacity of rat-derived hepatic stem cells

The liver in an adult rat maintains a balance between cell gain and cell loss. Although normally proliferatively quiescent, hepatocyte loss such as that caused by partial hepatectomy (PH) invokes a rapid regenerative response to restore liver mass. This restoration of moderate cell loss and "wear and tear" renewal is largely achieved by hepatocyte self-replication. Furthermore, hepatocyte transplants in rats, in which a selective pressure for the transplanted cells can be applied, have shown that a certain proportion of hepatocytes can undergo significant clonal expansion, suggesting that hepatocytes themselves are the functional stem cells of the liver. Fetal liver may also harbor bipotential stem cells capable of sustained clonal expansion. More severe liver injury activates a potential stem cell compartment located within the canals of Hering, giving rise to cords of bipotential oval cells that can differentiate into hepatocytes and biliary epithelial cells. Other cell populations with hepatic potential reside in the bone marrow; whether these hematopoietic cells can function as stem cells for the rat liver remains to be confirmed. Pancreatic cells have also been found to have hepatocytic potential.     

Human hepatic stem-like cells isolated using c-kit or CD34 can differentiate into biliary epithelium

BACKGROUND & AIMS: Recent reports suggest that after bone marrow transplantation into rodents and humans, hematopoietic stem cells migrate into the liver and give rise to oval cells, hepatocytes, and biliary epithelial cells. We investigated this hypothesis further in the human liver using the hematopoietic markers c-kit and CD34. METHODS: Immunofluorescence confocal microscopy was performed using cytokeratin 19 (CK-19; biliary cell marker) with either c-kit or CD34. Immunomagnetic separation was then used to select c-kit- or CD34-positive cells. After attachment, cells were cultured for up to 7 days, and their growth and phenotypic characteristics were examined. RESULTS: In cirrhotic tissue, c-kit- or CD34-positive cells were located in the portal tracts surrounding bile ducts. Occasionally c-kit- (but not CD34-) positive cells that coexpressed CK-19 were observed integrated into bile ducts. In vitro, immunoisolated c-kit or CD34 cells gave rise to colonies of at least 2 morphologies expressing CK-19 or CD31 (endothelial cell marker). CD34- or c-kit-positive cells with similar properties were also isolated from normal liver. CONCLUSIONS: These findings indicate that cells present in human liver that express the markers c-kit or CD34 have the capacity to differentiate into biliary epithelial cell lineage and may therefore represent human biliary epithelial progenitor cells.     

Isolation and characterization of multipotential mesenchymal stem cells from feline bone marrow

OBJECTIVE: Although several types of stem cells have been isolated from rodent and human tissues, very few data exist on stem cell isolation from nonrodent animals, which seriously limits the advancement of stem cell biology and its ultimate translation to human clinical applications. Domestic cats are used frequently in biomedical research and are the preferred species for studies of normal physiology and disease, particularly in neuroscience. Therefore, the objective of this study was to characterize mesenchymal stem cells (MSC) from feline bone marrow for use in research on the application of stem cells to human health problems for which cats are the preferred model. METHODS: Mesenchymal stem cells from feline bone marrow were isolated by standard methodology developed for other species and characterized according to morphology, growth traits, cell-surface antigen profile, and differentiation repertoire in vitro. RESULTS: Feline mesenchymal stem cells exhibit a fibroblast-like morphology with bipolar or polygonal cell bodies and possess a cell-surface antigen profile similar to their rodent and human counterparts. Feline MSC exist at a frequency of 1 in 3.8 x 10(5) bone marrow mononuclear cells and are capable of differentiation to adipocytic, osteocytic, and neuronal phenotypes when exposed to appropriate induction media. CONCLUSIONS: Mesenchymal stem cells isolated from feline bone marrow possess several traits typical of MSC from other species. Characterization of feline mesenchymal stem cells will facilitate future studies of stem cell biology and therapeutics for which the domestic cat is an indispensable model.