Activated Hepatic Stellate Cells Stimulate Differentiation Of Stem Cells Into Hepatocytes And Improve Liver Recovery From Injury

【Background and Objective】Bone marrow mesenchymal stem cells(MSC) are nonhematopoietic cells that reside within the bone marrow stroma.These cells are multipotent and serve as precursors for various cells including osteoblast,smooth muscle cells,chondrocytes,adipocytes and hematopoietic supportive cells.The generation of hepatocytes from bone marrow derived stem cells is a compelling concept that has provoked much interest in the liver transplant field.Petersen et al.reported that some liver oval cells were derived from transplanted bone marrow.Hepatic oval cells are hepatic progenitor cells which are characterized by an ovoid nucleus,small size,and scant basophilic cytoplasm.Oval cells express phenotypical markers of both the biliary epithelium and hepatocyte lineages.After that,there are both positive and negative reports concerning the potency of bone marrow derived stem cells to differentiate into hepatocytes in vivo.The conflict results may attribute to the different stem cell microenvironment(or "niche") which is composed of non-parenchymal cells and secreted factors.The concept of the microenvironment was first developed in hematopoiesis,where other cells secrete factors to support proliferation,differentiation,and survival of distinct progenitor populations.The effect of niche cells on stem/progenitor cells in proliferation and differentiation was also demonstrated in other organs/tissues such as epidermal,heart, brain,etc.To date,the microenvironment which modulate differentiation of hepatic stem cells and control transdifferentiation of bone marrow derived stem cells migrated into liver are still unknown.Hepatic stellate cell(HSC) is one of major hepatic nonparenchymal cells which are located in the Disse space between the endothelium and the hepatocytes.Under conditions of stress or liver injury,quiescent HSCs are activated and acquire a myofibroblastic phenotype,contributing to excessive extracellular matrix deposition which gives rise to development of liver fibrosis and cirrhosis.On the other hand,activated HSCs were recently demonstrated to secrete factors to stimulate hepatocyte proliferation.Anatomically, bone marrow derived stem cells must circumvent the Disse space during translocation form liver sinusoids to the parenchyma and may be regulated by HSCs.Therefore,we assume that HSCs may present as a modulator for the differentiation of MSCs into hepatic cells and different states of HSCs may play variant roles.To prove this assumption,bone marrow MSCs were indirectly cocultured with quiescent HSCs or activated HSCs.Primary isolated quiescent HSCs were activated by being in vitro cultured alone or cocultured with Kupffer cells.Expression of hepatic specific markers and hepatocyte function of glycogen deposition were detected on differentiated MSCs.The variant effects of HSCs in different states on the differentiation of MSCs were compared.【Methods】1.MSCs Preparation and DeliveryRat bone marrow MSCs were harvested from mononuclear cells which were separated by Percoll density gradient.MSCs were plastic-adherent mononuclear bone marrow cells after expansion in culture.The resulting MSCs(passage 6-10) were used for our studies. To confirm the identity of bone marrow MSCs,the cells were grown in medium that is conducive to differentiation into osteoclasts and adipocytes.Adipocytes containing lipid droplets were observed 2 weeks later by Oil Red O(Sigma-Aldrich) staining.The deposition of bone mineral by osteoclasts differentitated from MSCs was observed 2 weeks later by Alizarin red(pH 4.1;Sigma-Aldrich) staining.2.Preparation of HSCs and Kupffer CellsPrimary HSCs and Kupffer cells were freshly isolated from rat liver nonparenchymal cells.The liver was perfused with collagenase and pronase solution.The supernatant was centrifuged at 50×g for 2 min for several rounds to collect and nonparenchymal cells containing the HSCs and Kupffer cells.HSCs were isolated by density gradient centrifugation.Kupffer cells were separated by density gradient and adhesion culture.3.Establishment of coculture system and investigation of the effects of HSCs in different activation state on the MSCs 3.1 MSCs were coculture with HSCs at different states:HSC-T6 cells,the immortalized activated HSCs cells;quiescent HSCs:freshly isolated HSCs within 5 days;culture activated HSCs:primarily isolated HSCs cultured for 7 to 20 days;Kupffer cell activated HSCs:primarily isolated HSCs cocultured with Kupffer cells for 7 to 20 days.MSCs were also cocultured with Kupffer cells as control group for Kupffer cells activated HSCs group. Culture medium was half changed every 3 days.3.2 Morphological changes of MSCs cocultured with HSCs were observed under microscopy.3.3 The hepatic specific mRNA expression of MSCs cocultured with HSCs was analyzed by reverse transcription-polymerase chain reaction(RT-PCR),including unmature hepatocyte marker alpha-fetoprotein(AFP),albumin,cytokeratin 18(CK18), glutamine synthetase(GS) and mature hepatocyte marker phosphoenolpyruvate carboxykinase(PEPCK),glucose 6 phosphate dehydrogenase(G6P).3.4 The MSCs were detected for expression of specific proteins after coculture with HSCs for 2 weeks with immunofluorescence,including biliary epithelial cells marker CK-19,hepatocyte marker CK-18,albumin and AFP,MSCs marker alpha-smooth muscle actin(α-SMA).3.5 Glycogen deposition was detected by Periodic Acid-Schiff Stain.4.Detection of HSCs activation state compared with hepatic oval cells proliferation.4.1 Establish 2-AAF/PH model to stimulate hepatic stem cells differentiation into hepatic oval cells(hepatic progenitor cells).4.2 Activated HSCs were detected by desmin staining.5.Activated HSCs were eliminated by gliotoxin administration.The number of hepatic oval cells and expression of hepatic stem/progenitor cells specific genes was evaluated.5.1 Gliotoxin were administrated 2 day after partial hepatectomy,the effects of eliminating activated HSCs was proved by desmin staining. 5.2 Effect of eliminating activated HSCs on liver regeneration and hepatic oval cells proliferation was evaluated.5.3 After gliotoxin administration,conditioned HSCs culture medium was supplied by intraperitoneal injection.Liver regeneration and hepatic oval cells proliferation of mouse were compared among experimental groups.【Results】1.Characteristics of MSCs Derived from Bone MarrowMSC cultures were initiated using mononuclear cells isolated from normal bone marrow samples and maintained for up to 10 passages.It took around 12 and 20 days to obtain a homogeneous adherent monolayer and establish primary culture of MSCs.Under adipogenic induction conditions for 3 weeks,the formation of intracellular microdroplets was noted,and stained positive for Oil Red O.While under osteogenic induction conditions for 3 weeks,bone marrow derived MSCs could differentiate into osteoblasts as proved by staining with Alizarin-Red-S for calciumphosphate precipitates.2.HSC Isolation and activation2.1 The purity of our HSC preparation was estimated to be greater than 95%according to desmin positive staining.Quiescent HSCs were negative forα-SMA staining.Cells cultured alone for 7 days acquired strongα-SMA staining with a typical filamentous distribution,indicating that HSCs were activated.HSCs cocultured with Kupffer cells were fully activated within 5 days,with strong expression ofα-SMA.2.2 The purity of our Kupffer cells preparation was estimated to be greater than 95% according to phagocytize assay.2.3 HGF mRNA expression in HSCs of different states were detected with RT-PCR. HGF was found to be either minimal or negligible in the quiescent HSCs and culture activated HSCs,whereas expressed by HSC-T6 cells and Kupffer cell activated HSCs.3.Hepatic Differentiation of Bone Marrow-Derived MSC In Vitro3.1 For HSC-T6 group and Kupffer cell activated HSCs group,the fibroblastic morphology of MSCs was lost and the cells developed a broadened and flattened morphology 1 week post-induction.A retraction of elongated ends was observed 2 weeks post-induction,and the cuboidal morphology of hepatocytes developed with increasing incubation which was retained up to 8 weeks.MSCs remained fibroblastic morphology in the groups of MSC cultured alone and MSCs co-cultured with Kupffer cells,quiescent HSCs or culture activated HSCs up to 8 weeks.3.2 Hepatocyte-specific genes,such as albumin,AFP,CK-18,GS,tyrosine aminotransferase(TAT),G6P were found to be either minimal or negligible in the undifferentiated MSCs,whereas they could be significantly up-regulated during differentiation into hepatocyte-like cells.In the HSC-T6 and Kupffer cell activated HSCs group,albumin,CK-18,AFP and GS appeared to be expressed at the first or second week of the initiation,whereas the mRNAs of PEPCK and G6P genes was significantly induced at 4 weeks3.3 On day 14,the majority of the differentiated MSCs in HSC-T6 group and Kupffer cell activated HSCs group expressed the hepatocyte markers,including CK-18,AFP,and albumin.Double-staining revealed that 12.4±3.7%(n=3) of the cells were positive for both albumin and biliary cell marker CK-19 two weeks post-induction,which decreased gradually and then disappeared 4 weeks post-induction.Meanwhile,the gain of hepatic function was also indicated by the gradual loss ofα-SMA,which was positive in nearly all cells up to day 7 and then gradually declined with ongoing culture to barely detectable levels at day 14.3.4 No glycogen was detected in the primarily isolated MSCs,neither in MSCs cocultured with Kupffer cells,quiescent HSCs or culture activated HSCs.In the differentiated MSCs cultured with HSC-T6 and Kupffer cell activated HSCs group, glycogen was negative at 2 weeks post-induction,but was visualized since 4 weeks post-induction.By 6 weeks,55.4±8.7%(n=4) differentiated cells revealed stored glycogen.4.Hepatic oval cells proliferation are companied with hepatic stellate cells activation.4.1 After 2-AAF/PH,hepatic oval cells were detected in liver tissue sections. 4.2 Desmin positive activated HSCs were detected companied with hepatic oval cells proliferation.5.Activated HSCs elimination defects liver regeneration and hepatic oval cells proliferation.5.1 Gliotoxin administration eliminated activated HSCs effectively.5.2 After eliminating activated HSCs,liver index and expression level of hepatic stem/progenitor cells specific genes were lower than that of control group,and were rebound after conditioned HSCs culture medium administration.【Conclusion】1.We had successfully isolated the MSCs with multipotency by density gradient and adhesion selection which is quick,simple and practical isolation method.2.The fully activated HSCs can induce the differentiation of bone marrow derived MSCs into hepatocyte-like cells,which was demonstrated by morphological changes and expression of hepatic specific mRNA and protein.3.Hepatic differentiation induced by activated HSCs was properly progressed from early to late stages of hepatogenesis.4.Hepatic oval cells proliferation were companied with hepatic stellate cells activation.5.Hepatic stellate cells activation is crucial for liver regeneration and hepatic oval cells proliferation.