Transplanted bone marrow stromal cells are not cellular origin of hepatocellular carcinomas in a mouse model of carcinogenesis

AIM：To investigate the malignant potential of hepatic stem cells derived from the bone marrow stromal cells （BMSCs） in a mouse model of chemical hepatocarcinogenesis.
METHODS：BMSCs from male BALB/c mice were harvested and cultured, then transplanted into female syngenic BALB/ c mice via portal vein. Hepato-carcinogenesis was induced by 6 mo of treatment with diethylnitrosamine （DEN）. Six months later, the liver was removed from each treated mouse and evaluated by immunohistochemistry and fluorescence in situ hybridization （FISH）.
RESULTS：Twenty-six percent of recipient mice survived and developed multiple hepatocellular carcinomas （HCCs）. Immunohistochemically, HCC expressed placental form of glutathione-S-transferase （GST-P） and α-fetoprotein, but did not express cytokeratin 19. Y chromosome positive hepatocytes were detected by fluorescent in situ hybridization （FISH） in the liver of mice treated with DEN after BMSCs transplantation while no such hepatocytes were identified in the liver of mice not treated with DEN. No HCC was positive for the Y chromosome by FISH.
CONCLUSION：Hepatic stem cells derived from the bone marrow stromal cells have a low malignant potential in our mouse model of chemical hepatocarcingenesis.     

The origin and liver repopulating capacity of murine oval cells

The appearance of bipotential oval cells in chronic liver injury suggests the existence of hepatocyte progenitor/stem cells. To study the origin and properties of this cell population, oval cell proliferation was induced in adult mouse liver by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) and a method for their isolation was developed. Transplantation into fumarylacetoacetate hydrolase (Fah) deficient mice was used to determine their capacity for liver repopulation. In competitive repopulation experiments, hepatic oval cells were at least as efficient as mature hepatocytes in repopulating the liver. In mice with chimeric livers, the oval cells were not derived from hepatocytes but from liver nonparenchymal cells. This finding supports a model in which intrahepatic progenitors differentiate into hepatocytes irreversibly. To determine whether oval cells originated from stem cells residing in the bone marrow, bone marrow transplanted wild-type mice were treated with DDC for 8 months and oval cells were then serially transferred into Fah mutants. The liver repopulating cells in these secondary transplant recipients lacked the genetic markers of the original bone marrow donor. We conclude that hepatic oval cells do not originate in bone marrow but in the liver itself, and that they have valuable properties for therapeutic liver repopulation.     

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.     

Origin of hepatocellular carcinoma: role of stem cells

The question of whether hepatocellular carcinoma (HCC) arises from the differentiation block of stem cells or dedifferentiation of mature cells remains controversial. Recently, researchers suggested that HCC may originate from the transdifferentiation of bone marrow cells. Interestingly, there are four levels of cells in the hepatic stem cell lineage: bone marrow cells, hepato-pancreas stem cells, oval cells and hepatocytes. Hematopoietic stem cells and the liver are known to have a close relationship in early development. Bone marrow stem cells could differentiate into oval cells, which could differentiate into hepatocytes and duct cells. The development of pancreatic and liver buds in embryogenesis suggests the existence of a common progenitor cell to both the pancreas and liver. Cellular events during hepatocarcinogenesis illustrate that HCC may arise from cells at various stages of differentiation in the hepatic stem cell lineage.     

Use of recombinant adeno-associated viral vectors as a tool for labeling bone marrow cells

We have tested the feasibility of using recombinant adeno-associated virus (rAAV) vectors as a tool for labeling bone marrow (BM) cells in vivo. We infected BM cells of donor FVB mice with rAAV vectors containing the lacZ gene for 2 h. We then injected the rAAV-infected cells to lethally irradiated-recipient FVB mice. Peripheral blood (PB), BM and spleen harvested at 4 weeks after BM transplant (BMT) demonstrated stable engraftment in beta-galactosidase (beta-gal) expression. In contrast, Dil-labeling displayed only a faint signal 4 weeks after BMT. To analyze the kinetics of BM cells, we injected vascular endothelial growth factor (VEGF), which promotes mobilization of BM cells. Administration of VEGF protein significantly increased the rAAV-mediated beta-gal expression in PB and BM of recipient mice. Moreover, when myocardial infarction was induced in BMT mice, the ischemic area exhibited significant beta-gal staining in rAAV-labeled BMT group. rAAV vectors programmed stable transduction in BM cells in vivo through rapid infection. rAAV appears to represent a useful vector for labeling BM cells ex vivo prior to BMT for analysis of cardiovascular therapeutic purposes.     

Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells

The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT. CONCLUSION: The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure.     

大鼠移植骨髓细胞向肝细胞转化的实验研究

目的 探讨体内骨髓细胞向肝细胞转化的可行性。方法 将雌性SD大鼠随机分为3组，每组15只。①R BMT(全身照射 骨髓移植)；②2—AAF R BMT；③2—AAF PH(部分肝切) BMT。进行交叉性别骨髓细胞移植，雄性骨髓植入雄性受体，分别于第5、10、20天处死雌鼠。以雄性性别决定基因sry作为细胞标记，用原位杂交和FISH作为检测方法对骨髓细胞的肝细胞转化进行分析。结果 PCR移植效果初步分析可见，R BMT组11例中有10例PCR阳性；2AAF PH BMT组11例中有7例阳性，2AAF B BMT组10例中有6例阳性。sry原位杂交染色发现，第5天各组雌性受体肝索中均未见sry阳性的肝细胞。第10天R BMT组可见1例sry阳性的细胞位于肝细胞索,FISH染色可见这一细胞白蛋白mRNA阳性。第20天各组PCR阳性各例均可在肝索中检测到sry阳性的细胞。FISH染色可见白蛋白mRNA阳性。经统计学分析第20天各组sry阳性细胞数无明显差异。结论 在B BMT、2—AAF PH BMT和2—AAF R BMT模型中移植的骨髓细胞均可以植入肝脏，并存在于肝细胞索。植入肝索的骨髓细胞最早可见于移植后第10天，并发生转分化，表达白蛋白mRNA。不经过全身照射的2—AAF PH BMT组，移植的骨髓细胞也可以进入肝脏发生转分化，因此全身照射并不一定是移植骨髓细胞活化、植入和转化的必须条件。     

A preliminary study of liver cells derived from bone marrow

OBJECTIVE: To investigate whether hepatocytes can be derived from bone marrow cells in vivo. METHODS: A cohort of lethally irradiated BALB/C female mice received whole bone marrow transplants from age‐matched male donors and were killed at 1, 2, 4 and 8 weeks post transplantation. Fluorescence in situ hybridization (FISH) for the Y‐chromosome was performed using liver tissue. RESULTS: A few Y‐chromosome positive hepatocytes were found in the liver tissues at 2 months post transplantation. CONCLUSION: Hepatocytes can be derived from bone marrow cells after transplantation in lethally irradiated mice without severe acute hepatic injury.     

骨髓间充质干细胞在小鼠肝脏局部分化为肝细胞的实验研究

目的 探讨小鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）在肝内分化为肝细胞的可行性。方法腹腔注射丙烯醇致雌性C57BL／6小鼠全肝损伤后局部接受雄性同品系绿色荧光蛋白（green fluorescence protein，GFP）转基因小鼠骨髓间充质干细胞移植。移植后3周取骨髓嵌合小鼠肝脏做冰冻切片，荧光显微镜下观察GFP阳性细胞在肝内分布，石蜡切片行GFP抗体和白蛋白（albumin，Alb）抗体免疫组化双标法检测GFP阳性细胞在受体小鼠肝内的分化情况。结果移植后3周小鼠肝内可见GFP阳性细胞，并可见GFP和Alb双阳性细胞。结论异体骨髓间充质干细胞可在小鼠肝内分化为表达Alb的肝细胞，为肝组织的再生和修复提供了新思路，显示了其在肝病治疗中的应用潜力。     

细胞移植疗法在肝病中的研究与应用

人们对使用细胞移植疗法恢复肝脏功能的前景寄予厚望,期待其最终能够代替肝脏移植手术.众多学者对成人成熟肝细胞,不同来源干细胞的再生能力方面进行了大量研究,特别是间充质干细胞来源的肝细胞移植疗法在恢复肝脏再生方面的效果在动物模型实验中取得令人鼓舞的结果.本文将对近年来细胞移植治疗各种肝病的机制研究、动物实验以及临床试验等方...     

骨髓来源肝细胞的初步研究

目的 验证骨髓细胞是否可以转化为肝细胞。方法 通过放射致死剂量照射的BALB/C雌性小鼠接受周龄相配的同系雄性小鼠的全骨髓移植后，分别在移植后1，2，4周和2个月分批处死移植存活的小鼠。分别在游离的肝细胞和肝脏的石蜡切片上用荧光原位杂交法检测Y染色体阳性肝细胞。结果 在移植后2个月的小鼠肝脏游离细胞和石蜡切片上均发现有Y染色体阳性肝细胞，数量极少，但确实存在。结论 在接受放射致死剂量照射但没有导致严重的急性肝脏损伤情况下，骨髓细胞可以转化为成熟的肝细胞。     

Hepatic gene induction in murine bone marrow after hepatectomy

BACKGROUND/AIMS: Bone marrow cells are highly plastic and differentiate into various cell types, including hepatocytes. To explore the mechanisms underlying these processes, we focused on the initial responses of bone marrow to hepatectomy, using a mouse model. METHODS: To evaluate hepatic differentiation in bone marrow cells we measured hepatocyte-related gene expression in mice undergoing partial hepatectomy with or without pretreatment for 1 week with 2-acetyl aminofluorene (AAF). RESULTS: Hepatectomy induced several genes related to early hepatic differentiation in bone marrow. Expression of these genes was enhanced by the administration of AAF, whereas genes specific for mature hepatocytes were not detected. We characterised the bone marrow cell population expressing hepatocyte differentiation genes. alpha-fetoprotein mRNA was induced in Lin- and either CD34+, c-kit+, Sca-1+, CD49f+ or CD45+ cells. The genes upregulated in the liver after AAF treatment and hepatectomy were identified using oligonucleotide microarrays. These included genes associated with the acute phase response. Dexamethasone inhibited the expression of early hepatic differentiation genes in the bone marrow of AAF/PHx mice. CONCLUSIONS: Early hepatic differentiation genes were induced in bone marrow in response to hepatectomy, especially when regeneration of the remnant liver was suppressed. Circulating signals generated in the AAF/PHx liver might activate this differentiation.     

Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation

Following a report of skeletal muscle regeneration from bone marrow cells, we investigated whether hepatocytes could also derive in vivo from bone marrow cells. A cohort of lethally irradiated B6D2F1 female mice received whole bone marrow transplants from age-matched male donors and were sacrificed at days 1, 3, 5, and 7 and months 2, 4, and 6 posttransplantation (n = 3 for each time point). Additionally, 2 archival female mice of the same strain who had previously been recipients of 200 male fluorescence-activated cell sorter (FACS)-sorted CD34(+)lin(-) cells were sacrificed 8 months posttransplantation under the same protocol. Fluorescence in situ hybridization (FISH) for the Y-chromosome was performed on liver tissue. Y-positive hepatocytes, up to 2.2% of total hepatocytes, were identified in 1 animal at 7 days posttransplantation and in all animals sacrificed 2 months or longer posttransplantation. Simultaneous FISH for the Y-chromosome and albumin messenger RNA (mRNA) confirmed male-derived cells were mature hepatocytes. These animals had received lethal doses of irradiation at the time of bone marrow transplantation, but this induced no overt, histologically demonstrable, acute hepatic injury, including inflammation, necrosis, oval cell proliferation, or scarring. We conclude that hepatocytes can derive from bone marrow cells after irradiation in the absence of severe acute injury. Also, the small subpopulation of CD34(+)lin(-) bone marrow cells is capable of such hepatic engraftment.     

Hematopoietic stem cell transplantation prevents diabetes in NOD mice but does not contribute to significant islet cell regeneration once disease is established

The treatment of type I diabetes by islet cell transplantation, while promising, remains restricted due to the incomplete efficacy and toxicity associated with current immunosuppression, and by limited organ availability. Given reports suggesting bone marrow derived stem cell plasticity, we sought to determine whether such cells could give rise to pancreatic islet cells in vivo. In the context of autoimmune diabetes, we transplanted unfractionated bone marrow from beta-gal trangenic donor mice into NOD mice prior to, at, and two weeks beyond the onset of disease. Successful bone marrow engraftment before diabetes onset prevented disease in all mice and for 1 year after transplant. However, despite obtaining full hematopoietic engraftment in over 50 transplanted mice, only one mouse became insulin independent, and no beta-Gal positive islets were detected in any of the mice. To test whether tolerance to islets was achieved, we injected islets obtained from the same allogeneic donor strain as the hematopoietic cells into 4 transplant recipients, and 2 had a reversion of their diabetes. Thus allogeneic bone marrow transplantation prevents autoimmune diabetes and tolerizes the recipient to donor islet grants, even in diabetic animals, yet the capacity of bone marrow derived cells to differentiate into functional islet cells, at least without additional manipulation, is limited in our model.     

Development,expansion and maturation of hematopoietic stem cells in mouse embryo

During embryonic development, hematopoiesis occurs in different tissues. Primitive hematopoiesis first occurs in the yolk sac and definitive hematopoietic stem cells (HSCs) arise in the aorta/gonad/mesonephros (AGM) region. Fetal liver then functions as the major hematopoietic organ from the mid to late gestation, before hematopoiesis starts in the bone marrow. We have developed a primary culture system of mouse AGM cells which produces both hematopoietic cells and endothelial cells and found that hemangioblasts, the common precursor of hematopotieic and endothelial cells, are enriched in the cell population which express podocalyxin like protein 1, a CD34-related molecule, but lacks CD45 in the AGM region. We also developed a culture system of E14.5 fetal hepatic cells which support hematopoiesis. AGM-derived HSCs proliferated most vigorously in the presence of such fetal hepatic cells, however HSCs derived from later stages of development proliferated less efficiently in the same co-culture system, suggesting that the proliferation potential of HSCs declines along with development. While AGM-derived HSCs poorly engrafted in the bone marrow of irradiated adult mice, co-culture of AGM-derived HSCs with fetal hepatic cells dramatically increased the bone marrow engraftment. These results indicate that fetal hepatic cells alter the characteristics of HSCs. Interestingly, Oncostatin M (OSM) strongly induced differentiation of immature hepatocytes to metabolically active mature hepatocytes, but it simultaneously reduced their ability to support hematopoiesis. As OSM is produced by hematopoietic cells in the fetal liver, it is likely that OSM plays a role for coordinating the development of hematopoietic cells and the liver. In conclusion, our in vitro system provides a means to study the molecular basis of development, expansion and maturation of HSCs.     

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.     

骨髓干细胞在大鼠肝纤维化形成环境中的分化

目的 研究骨髓干细胞在肝纤维化形成环境中向肝细胞定向分化。 方法 采用四氯化碳皮下注射法诱导大鼠肝纤维化,应用流式细胞仪分选富集Thy+CD3-CD45RA-的骨髓干细胞,采用红色荧光染料PKH26-GL对其标记后进行自体移植,6周后通过免疫组织化学方法检测大鼠肝组织白蛋白、ck 8、α-平滑肌肌动蛋白表达。 结果 PKH 26-GL标记的细胞在纤维化肝脏中表达白蛋白和ck8,占肝细胞总数的(0.17±0.02)％;未见表达α-平滑肌肌动蛋白。 结论 骨髓干细胞在肝纤维化形成环境中可以向肝细胞定向分化,不向肌成纤维样细胞分化。     

Liver from bone marrow in humans

It has been shown in animal models that hepatocytes and cholangiocytes can derive from bone marrow cells. We have investigated whether such a process occurs in humans. Archival autopsy and biopsy liver specimens were obtained from 2 female recipients of therapeutic bone marrow transplantations with male donors and from 4 male recipients of orthotopic liver transplantations from female donors. Immunohistochemical staining with monoclonal antibody CAM5.2, specific for cytokeratins 8, 18, and 19, gave typical strong staining of hepatocytes, cholangiocytes, and ductular reactions in all tissues, to the exclusion of all nonepithelial cells. Slides were systematically photographed and then restained by fluorescence in situ hybridization (FISH) for X and Y chromosomes. Using morphologic criteria, field-by-field comparison of the fluorescent images with the prior photomicrographs, and persistence of the diaminiobenzidene (DAB) stain through the FISH protease digestion, Y-positive hepatocytes and cholangiocytes could be identified in male control liver tissue and in all study specimens. Cell counts were adjusted based on the number of Y-positive cells in the male control liver to correct for partial sampling of nuclei in the 3-micron thin tissue sections. Adjusted Y-positive hepatocyte and cholangiocyte engraftment ranged from 4% to 43% and from 4% to 38%, respectively, in study specimens, with the peak values being found in a case of fibrosing cholestatic recurrent hepatitis C in one of the liver transplant recipients. We therefore show that in humans, hepatocytes and cholangiocytes can be derived from extrahepatic circulating stem cells, probably of bone marrow origin, and such "transdifferentiation can replenish large numbers of hepatic parenchymal cells.     

Bone marrow transplantation in mice leads to a minor population of hepatocytes that can be selectively amplified in vivo

Cell-based therapy may some day be a therapeutic alternative to liver transplantation. Recent observations indicating that hematopoietic stem cells can differentiate into hepatocytes have opened new therapeutic prospects. However, the clinical relevance of this phenomenon is unknown. We have previously developed a strategy based on the protective effect of Bcl-2 against Fas-mediated apoptosis to selectively amplify a small number of hepatocytes in vivo. We now show that this approach can be used to amplify a minor population of bone marrow-derived hepatocytes. Normal mice were transplanted with unfractionated bone marrow cells from transgenic animals expressing Bcl-2 under the control of a liver-specific promoter. Recipients were then submitted to weekly injections of the anti-Fas antibody, Jo2. Upon sacrifice, the liver of the recipients showed bone marrow-derived clusters of mature hepatocytes expressing Bcl-2, which showed that the hepatocyte progeny of a genetically modified bone marrow can be selectively expanded in vivo. In contrast, no Bcl-2 expression could be detected without the selective pressure of Jo2, suggesting that differentiation of bone marrow cells into mature hepatocytes is very inefficient under physiologic conditions. We conclude that a selection strategy will be required to achieve a therapeutic level of liver repopulation with bone marrow-derived hepatocytes.