| In the1960s, Leonard Hayflick proposed “Hayflick limit” theory to explain thelimited ability of mammalian cells to divide when cultured in vitro. Mammalian aging isa complex physiological phenomenon, which is a process accompanied by gradual lossof tissue homeostasis and organ function.Adult stem cells with proliferation andmulti-differentiation potential exist throughout the adult body,maintaining self-stablestate and supporting a continuous renewal and repair of tissues.With the aging of anindividual, the function of stem cells degenerate to the degree that they could notgenerate new cells to replace the damaged cells,leading to the destruction of the stabilityin the body,which in turn leads to aging.It has been reported that Human mesenchymalstem cells (MSCs) exhibit little differentiation potential during prolonged culture in vitro.Furthermore, the roles MSCs have received significant attention in the fields of cell andgene therapy due to the ease of acquisition, low immunomodulatory activity andmultiple differentiation potential. MSCs as a very rare population require initialexpansion in vitro prior to clinical use. However, senescence often occurs in MSCs whenthey are cultured in vitro. Serial passages of MSCs in culture lead to growth arrest andloss of stem cell differentiation potential, which will limit the success of cell-basedclinical applications. Thus, analysis of in vitro senescence in MSCs is crucial for basicresearch as well as therapeutic applications.With this in mind, we designed a study to explore the molecular mechanismsunderlying MSCs senescence and find a new way to extend MSCs lifespan in vitro. Therole of the NAD-dependent protein deacetylase silent information regulator2(Sir2) inmediating gene silencing, stress resistance and longevity in lower organisms hasattracted a large amount of interest in the last decade. In mammals, Sirt1NAD+-dependent protein deacetylase is the most closely related homologue of yeast Sir2and it regulates cell cycle, metabolism, apoptosis and cellular senescence byinteractions with a number of biological molecules such as p53, Ku70, FOXO, E2F1,NF-κB, and PGC-1α. However, the evidence that SIRT1delays the aging of normaldiploid cells is inconsistent. To investigate a role of Sirt1in MSCs senescence, weexamined the effects of Sirt1down-expression and up-regulation on the process ofMSCs senescence.In this study, we found that the main characteristic features of the MSCs wereacquired senescence-like growth arrest, a enlarged and flattened shape and highsenescence-associated β-galactosidase (SA-β-gal) activity as the passage numberincreases. NAD-dependent protein deacetylase SIRT1is markedly reduced in bothhuman bone marrow derived MSCs (B-MSCs) and adipose tissue-derived MSCs afterincreasing passages of cell culture, indicating that SIRT1may play an important role inthe process of senescence.To make it clear whether or not active SIRT1modulates senescence-like phenotype inMSCs, we first examined the effect of SIRT1inhibition.In this study, we specificallyknocked down SIRT1expression by using two different sets of lentiviral shRNA vectoragainst SIRT1in both B-MSCs and A-MSCs at an early passage in culture. In both typesof MSCs, SIRT1down-expression significantly decreases the percentage of cells in Sphase by use of BrdU incorporation assay and propidine iodine staining, and markedlyslowed down the growth rate. During this prolonged culture period, SA-β-gal activitywas assayed over time to investigate whether down-expression of SIRT1has any effecton the senescence of MSCs. In both kinds of MSCs, no difference could be observedbetween the effect of mock shRNA transfection and SIRT1knockdown in cells fromearly passages. However, in cells from later passages, more SA-β-gal+cells wereobserved in those cultures where SIRT1had been knocked down. These results suggestindicated that specific knockdown of SIRT1accelerated cellular senecence in both typesof MSCs.Next, we examined overexpression of SIRT1could offer any growth advantage toMSCs and protect these cells from senescence. We successfully overexpressed SIRT1 and the dominant negative mutant H363Y in B-MSCs by lentivirus-based strategy. Wefound that cells overexpressing the H363Y mutant like the SIRT1-knockdown cells hada significantly lower BrdU incorporation rate, higher SA-β-gal activity, and shorter lifespan. For the early cell passages following transduction, no difference in BrdUincorporation or SA-β-gal activity was observed between control cells and cellsoverexpressing SIRT1. However, after serial passages in vitro, compared to control theB-MSCs overexpressing SIRT1showed a significantly higher rate of BrdU incorporation,lower SA-β-gal activity, and could be cultured for a much longer time, althougheventually even these cells also reached a state of senescence. Thus, we concluded thatenforced SIRT1expression led to cell proliferation and delayed senescence of MSCs,but had no effection on reversion of senescent MSCs or ending the process ofsenescence in MSCs.Though overexpression of SIRT1in MSCs is a promising strategy for cell-basedtherapy, the SIRT1-modified MSCs must be able to maintain their potential todifferentiate. Thus, we tested whether MSCs overexpressing SIRT1at early passageretain the multidifferentiation potential of the parent cells. B-MSCs modified withwild-type or H363Y mutant SIRT1could be readily coaxed to differentiate into adiposecells or osteoblasts in differentiation conditions. We detected the expression of fourMSCs surface markers CD31, CD34, CD90, and CD105, by FACS. Our data indicatethat there is no significant change for the expression of these markers could be observedin SIRT1-overexpressing MSCs and SIRT1-knockdown MSCs. Taken together, thesefindings suggest that MSCs modified with SIRT1maintain the ability to differentiate andthe changes observed in cell proliferation after SIRT1knockdown or overexpression arenot likely due to cell differentiation.To further elucidate the mechanisms of the phenotype described above, we assayedthe levels of two key mediators-P21CIP1and P16INK4ainvolved in senescence signalingpathways. We found that expression of either protein does not change significantly in theearly passages following knockdown or overexpression of SIRT1. However, in lateculture passages, P16INK4abut not P21CIP1is accumulated faster in SIRT1-knockdown MSCs. Conversely, overexpression of SIRT1efficiently delayed the accumulation ofP16INK4ain the prolonged culture. These results were consistent with a previous reportshowing that expression of P16INK4a, but not P21Cip1, is closely associated withsenescence of human B-MSCs. Interestingly, in human A-MSCs, knockdown of SIRT1induces accumulation of both P16INK4aand P21Cip1, indicating that varying mechanismsare involved in different types of MSCs.Small molecules targeted for SIRT1is very promising to clinic medicine. Resveratrol,a natural agent found in grape skin, is considered to be an activator of SIRT1in manystudies. Resveratrol activates SIRT1and other targets. To test whether resveratrol canpotentially be used to prevent senescence of MSCs in vitro without the need for themodification of the SIRT1gene, we introduced various concentrations of resveratrol intocultures of B-MSCs. After being treated with5μM or20μM resveratrol for2days,B-MSCs exhibited a much faster rate of growth with more cells in the S phase and lesscells in the G0/G1phase. However, with continuous addition of20μM resveratrol, MSCsstarted to apoptosis. Resveratrol may be toxic at high doses. Our data demonstrated thatunlike overexpression of SIRT1, resveratrol only transiently promoted proliferation ofMSCs.In summary, our findings will help us understand the role of SIRT1in the aging ofnormal diploid cells, and offer a new way to prevent of human MSCs senescence. The end-stage liver diseases caused by drugs, alcohol and other factors affect ourhealth seriously. Hepatocyte transplantation, bioartificial liver and liver tissueengineering are considered as promising approches to the treatment of these patients.However, the number and vitality of hepatocyte are the biggest bottleneck of theseapproaches. On the other hand, embryonic stem cells with unlimited proliferation andmulti-differentiation potential are expected to become a new source of hepatocyte.Researchers simulated the environment of liver development and employed step by stepstrategy to differentiate ESCs into hepatocyte. Many teams continuously improve theinduction strategy and have made some progress. However, there is a big difference,such as incomplete function and short-term maintenance，between hepatocyte-derivedESCs and these isolated from liver. Therefore, we shifted our attention to the laterprocess of ESCs differentiation into hepatocyte, that is, the process of hepatic stem cellor hepatobalst differentiation into mature and functional hepatocyte.The liver is an extreme heterogenous and complicated organ. The cells in liver arearranged in order to maintain the communication among cells. In structure, the stableand ordered arrangement which is dependent on cell junctions is important forperformance of liver functionality. This gets us thinking if we could promote orderlyarrangement between the cells by enhancing the connection between the cells in order topromote the hepatic precursor cells to develop into mature hepatocytes. In particular, celljunctions between hepatocytes including gap junctions, adherens junctions, desmosomes,and tight junctions are known to play key roles in the performance of liver-specificfunction. Gap junctions as pathway for small and hydrophilic molecules transport areactive and contain binding sites for several proteins to play a bigger role. Gap junctional channel are grouped at the membrane surface and are composed of12connexin proteins.Connexin32/connexin43, as the two most important gap junctions in liver, have asynchronous change in the opposite direction during differentiation from hepaticprogenitor/stem cells into hepatocytes. We found in the stage of hepatic progenitor,Cx43is expressed at a high level and Cx32hardly detected, but in the stage ofhepatocytes, the opposite expression is noticed, the level of cells Cx43decreases whilethat of CX32increases. This phenomenon suggests connexin32/connexin43play crucialroles during hepatic/progenitor stem cells differentiation into hepatocytes. We’re alsolooking at what key molecules can control CX32/CX43expression simultaneously. Withthis in mind, we designed a study to explore the molecular mechanisms underlyingCX32/CX43involved in differentiation from hepatic stem cells into hepatocytes. Wehope that we could find the new mechanism of hepatic progenitor/stem cellsdifferentiation and hepatocytes maturation from the angle of the interaction betweencells, exploring possible molecular mechanism, to achieve the ultimate goal ofintroducing new elements to improve the differentiation of embryonic stem cells intohepatocytes.In the experiment, we use hepatic progenitor cells (WB-F344cells) that has beenestablished and employed to study differentiation of these cells into hepatocytes orcholangiocytes in vitro. WB-F344cell morphology and its differentiation potential aresimilar hepatic progenitor cells derived ECSs. We use specifici inducing conditions tostimulate WB-F344cells to differentiate into cholangiocytes and hepatocyte. Throughthe analysis of relative gene and protein expression by realtime PCR and western blot,we not only verify the effectiveness of the induction system, but also observe changes ofconnexins. These changes reflect the CX43and CX32distribution in liver tissue, that is,CX32only has high expression in the mature hepatocyte and CX43in the bile ductwhich is stationed by stem cells.To make it clear whether or not CX32/CX43modulates differentiation of hepaticprogenitor cells into hepatocytes, in this study, we specifically knocked down CX43expression by using lentiviral shRNA vector, and successfully overexpressed CX43and CX32in WB-F344cells by lentivirus-based strategy. We found that cells knocked downCX43and overexpressed CX32, compared to control, showed a higher expression ofhepatocytes-specific maker by realtime PCR and western blot analysis.To further elucidate the mechanisms of the phenotype described above, we assayedthe phosphorylation of p38MAPK involved in signaling pathways. We found that thephosphorylated p38MAPK and CX43do not coexpressed with ALB distribute in portaltriad, while expressed little in hepatocyte. To the contrary, CX32is expressed in maturehepatocyte. To further confirm the regulation relationship between p-p38MAPK andCX43/CX32, we examined changes in expression of connexin in rat livers treated withp38MAPK inhibitor after70%partial hepatectomy (PH). We can see through treatmentof the inhibitor, the expression of phosphorylated p38MAPK and CX43markedlydeceases and the expression of CX32increases at1day after PH.WB-F344treated withinhibitor expressed higher ALB and CYP1b1with the down-regulation of CX43and upregulation of CX32. To address whether expression of CX43is responsible for thefunction of p38MAPK, WB-F344overexpressed CX43was treated every two days withp38MAPK inhibitor. CX43overexpresion weakens the inhibitors role. All our datasupport p38Mapk as a key signaling molecule can promote the differentiation of hepaticprogenitor cells through connexin32/43.In our previous work, p38MAPK can promote the differentiation of hepatic stem/progenitor cells into hepatocytes through the regulation of connexin32/43expression,which is confirmed in WB-F344cells and hepatic stem/progenitor cells isolated fromrat liver. Therefore, we added p38MAPK inhibitors at the stage of hepatic stem/progenitor cells-derived from ESCs differentiation into mature hepatocytes. Thepreliminary experiment shows that the p38MAPK inhibitor declines CX43expressionobviously, and promotes the expression of CYP1B1.In summary, our findings tell us that p38MAPK inhibitor dephosphorylation ofp38MAPK promotes hepatocytes-specific maker ALB and CYP1b1expression withupregulation of CX32and downregulation of CX43. These results provide a solidfoundation for our future study to develop p38MAPK inhibitors to differentiation of human embryonic stem cells into hepatocytes. |
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