Differentiation Of Bone Marrow Mesenchymal Stem Cells Into Epithelial Cells Induced By Vaginal Epithelial Cells In Vitro

Part one：Isolation, culture and characterization of rat bone marrowmesenchymal stem cells in vitroObjective: To establish a simple, high-yield, and repeatable protocol forisolation and culture of mesenchymal stem cells in vitro, as the foundation ofthe vaginal tissue engineering.Methods: Bone marrow cells were isolated and cultured following thewhole bone marrow adherent protocol. Bone marrow from the bilateral femursand tibias were flushed out using DMEM/F12medium under asepticconditions. The DMEM/F12marrow suspension was then filtered through a70μm cell strainer, collected, and centrifuged for5minutes at200g. Afterremoval of the supernatant, the resulting pellet was resuspended inDMEM/F12supplemented with10%fetal bovine serum,2mM–glutamine,1%penicillin, and streptomycin and plated at a density of1.5×107cells/cm~2.It was then maintained in a humidified atmosphere of95%air and5%CO2at37°C. Nonadherent cells were discarded after48h. The cells were fed every3–4days and passaged when70-80%confluent with0.25%trypsin/0.02%EDTA. After discarded the culture medium, wash the cells with37℃preheating phosphate-buffered saline and lift cells by incubation in0.5ml of0.25%trypsin/0.02%EDTA for2min at room temperature. Neutralize thetrypsin by adding complete medium, and culture all lifted cells in a25cm~2flask at a density of1×104cells/cm~2. The proliferation of MSCs at passage1to passage5was evaluated by MTT protocol. After several subculturingpurification，the MSCs at passage three to five were tested for the markersCD45, CD29, and CD90by flow cytometry analysis. Osteoblast and adipocytedifferentiation of MSCs were conducted to determine whether the cells hadmulti-potential properties. After14d of osteoblast differentiation, the alkaline phosphatase (ALP) activity of the cells was assessed using an ALP Kitfollowing the manufacturer’s instructions. The resulting red, insoluble,granular dye deposit indicated sites of ALP activity. Following an additional7-14d culture, the wells were stained with Alizarin Red to evaluate themineralization capacity. After14days of adipogenic differentiation, Oil Red Ostaining was conducted to assess lipid accumulation.Results: After7–10days of initial culture, distinct colonies offibroblastic cells formed in various sizes. When cell confluence achieved70%-80%, the cells were subcultured to passage one. After the subcultures, allthe living cells could adherent within24h. Cells grew more rapidly afterpassage. The cells were confluent and had a spiral whorl-like outlook after3-5days, which indicating another passaging. After the subculture, MSCs were inadaptive phase at1-2days, and went into logarithmic phase at3-4days. After5days, the proliferation of MSCs slow down and the curve flattened gradually,which suggesting they were in plateau phase. Flow cytometry analysis showedthat MSCs expressed CD29and CD90at99.91%and99.95%separately, butnot CD45. For osteogenic differentiation, ALP activity increased after14d ofculture, and mineralized nodules formed after4weeks of induction. Foradipogenic differentiation, intracellular Oil-red-O-stained lipids appeared after2weeks of culture.Conclusions: Whole bone marrow adherent protocol is a simple, efficientmethod to yield MSCs with high purification and multi-potential properties.Part two：Improved protocol for primary culture of rat vaginal epithelialcells in vitroObjective: To explore factors impacting on primary culture of rat vaginalepithelial cells (VECs) in vitro, to improve the current protocol of VECsseparation and culture, and establish a reproducible protocol for VECsseparation and culture that could be applied in tissue engineering.Methods: VECs were harvested by enzyme digestion protocol. Thevaginal tissues were dissected out, cleaned of as much connective tissue aspossible, minced into small pieces (10×5mm~2), and rinsed three times with PBS supplemented with penicillin/streptomycin (100units/ml,100mg/mL) at4℃. Vaginal tissues were incubated in Dispase II overnight at4°C. VECs wereisolated from the epithelium and peeled away from vaginal pieces by threerounds of enzymatic digestion with0.25%trypsin/0.02EDTA at37°C. Withgentle pipette suction and neutralize the trypsin by adding complete medium,the cell-fluid suspension was centrifuged at200g for5min. The pellet wasresuspended in keratinocyte growth medium with growth supplement,distributed into culture dishes with a density of1.0×105cells/cm~2, andmaintained in KGM with medium changes every48h. Based on the protocolmentioned above, we compared influence of rats in different estrous cycles(preoestrus, oestrus, metaoestrus, and anestrus), different Dispase Ⅱpreparation (0.6U/ml DispaseⅡsolved in PBS and0.6U/ml DispaseⅡsolvedin KBM), different Dispase Ⅱ concentration (0.6U/ml DispaseⅡsolved inKBM and1.2U/ml DispaseⅡsolved in KBM), different trypsine digestion(30min×1and10min×3), different coating solution (FN/C and deionizedwater), and different culture medium (KBM and KBM supplemented with6%FBS) on VECs culture. Detach scores of vaginal tissue and available live cellswere evaluated. At the first feed, the attachments of VECs on48h after theinitial culture were also observed. Epithelial cell phenotypes were confirmedby morphology, immunohistochemical and immunoflurorescence staining withantibodies to pan cytokeratins (AE1/AE3). The ultrastructure was oberservedby scanning electron microscope.Results: Epithelial layer of rat in preoestrus was more abundant thanother phases (P=0.0346). More VECs could attach after digestion withDispase Ⅱ solving in KBM than in PBS. Detach scores of epithelium fromunderlying connective tissues with1.2U/ml Dispase and available live cellsamout were higher than that of0.6U/ml Dispase (P=0.025; P=0.034). MoreVECs could attach after digestion with trypsine by3steps than that by onestep, and more VECs could attach after seeding on Fibronectin/collagen (FN/C)coating plastic culture vessels. VECs grew fast in medium containing fetalbovine serum, but were vulnerable to fibroblast cells contamination. The expression of AE1/AE3staining of the cultured cells was positive. Microvilliridges were observed on cells’ surface with scanning electron microscope.Conclutions: More VECs demonstrating epithelial characterization canbe obtained by improved protocol.Part three: Differentiation of bone marrow mesenchymal stem cells intoepithelial cellsObjective: To explore the feasibility of differentiation of bone marrowmesenchymal stem cells (MSCs) into epithelial cells in vitro.Methods: MSCs were gathered following the whole bone marrowadherent culture protocol. Vaginal epithelial cells (VECs) were collected bydispase and trypsin digestion protocol. For differentiation, MSCs wereco-cultured with VECs using cell culture transwell inserts (0.4μm pore,4.5cm~2) for14days. Fibronectin/collagen type I (FN/C)-coated inserts weredehydrated, loaded with VECs in KGM and incubated for48h. Then, MSCswere dispensed into the culture well or coverglass and incubated for24h. Atthe end of the incubation period, VECs in the top chamber and MSCs in thebottom chamber were washed, and3ml of growth medium was added to theculture wells. VECs in the top chamber and MSCs in the bottom chamberwere co-cultured using cell culture transwell inserts. The culture system wasassembled and incubated with KGM with growth supplement and FBS at afinal concentration of3%for indirect co-culture. The cells were feed every2-3days. Then,7–14days after the initial plating, morphological analysis(microscopy) and protein expression of pan-cytokeratin AE1/AE3(Westernblotting and immunocytochemistry) were assessed. VECs and MSCs wereused as positive and negative controls, respectively. Western Blot：Cells werewashed twice with PBS and lysed in RIPA extraction buffer for30min on ice.After centrifugation at12,000rpm for30minutes, the supernatants werecollected and the protein concentration was estimated using NanodropND-1000Spectrophotometer. Proteins were separated on8%SDS-PAGE gels,and electroblotted onto polyvinylidene difluoride (PVDF) membrane at4°C.The blots were blocked with blocking buffer (5%non-fat dry milk in PBS) for 1hour. The blots were incubated with anti-pan-cytokeratins (AE1/AE3), orGAPDH antibodies overnight in TBS-T at4°C. After washing3times inTBS-T for10minutes each, blots were incubated with fluorescent labeledIRDye800secondary antibody diluted in TBS-T in the dark for2h at roomtemperature. After thorough washing in TBS-T, the blots were scanned usingthe LI-COR Odyssey Infrared Imaging System using the appropriate channels.The integral optical density of bands was detected with Gelpro Analyzer. Theexpression levels of AE1/AE3were determinated with GAPDH as innercontrol.Results: The morphology of MSCs changed from spindle shapes to apolygonal epithelium-like shape when co-cultured with VECs fordifferentiation on day7and day14. Immunocytochemical staining of MSCsco-cultured with vaginal epithelial cells for AE1/AE3revealed expression ofepithelial marker in induced cells. Western blot analysis showed atime-dependent increase in protein expression of epithelial marker AE1/AE3in induced MSCs for0,7, and14days (1.00±0.00;36.28±1.49,7days vs.0day, P<0.01;48.80±3.17,14days vs.0day, P<0.01). Cells at14days showedhigher levels of AE1/AE3protein than cells at7days (48.80±3.17vs.36.28±1.49, P=0.0232).Conclusions: VECs can trigger epithelial differentiation of MSCs byindirect co-culture. The expression level of epithelial marker AE1/AE3increased with the co-culture time prolonged.Part four: Role of Wnt/β-catenin signaling pathway in the differentiationof bone marrow mesenchymal stem cells into epithelial cellsObjective: To explore the role of the Wnt/β-catenin signaling pathway inthe process of differentiation of bone marrow mesenchymal stem cells (MSCs)into epithelial cells.Methods: For differentiation, VECs in the top chamber and MSCs in thebottom chamber were co-cultured using0.4μm cell culture transwell inserts.Then,7–14days after the initial plating, Western blot was conducted to detectthe expression of β-catenin，GSK-3β，pi-GSK-3β, TCF-3, and pan-cytokeratin AE1/AE3. To evaluate the role of Wnt/β-catenin signaling, the co-culturemedium of MSCs and VECs were devided into three groups:(1) control group,KBM supplemented with3%FBS;(2) Wnt signaling activator group, KBMsupplemented with3%FBS and20μM lithium chloride (LiCl);(3) Wntsignaling inhibitor group, KBM supplemented with3%FBS and20ng/mlDickkopf-1(DKK-1). Western blotting was used to show the expression levelsof β-catenin, GSK-3β, TCF-3, and pan-cytokeratin AE1/AE3under co-cultureconditions with LiCl and DKK-1for7days. GAPDH was used as the innercontrol.Results: Western blot analysis demonstrated more expression ofβ-catenin (1.50±0.11vs.1.00±0.00, P=0.009), pi-GSK-3β (1.50±0.23vs.1.00±0.00, P=0.09) and TCF-3(1.25±0.16vs.1.00±0.00, P=0.19) and lessexpression of GSK-3β (0.69±0.04vs.1.00±0.00, P=0.002) in co-culturedMSCs on day7than on day0. After14days of co-culture, the expressionlevels of β-catenin (1.85±0.23vs.1.50±0.11, P=0.24) and pi-GSK-3β (2.31±0.33vs1.50±0.23, P=0.114) were higher than after7days, and GSK-3βlevels were lower (0.34±0.03vs.0.69±0.04, P=0.0021). However, theexpression level of TCF-3was lower than at7days (0.98±0.17vs.1.25±0.16, P=0.3144). The expression levels of GSK-3β in co-cultured MSCspresented trend opposite that of β-catenin levels. In addition, thephosphorylation levels of GSK-3β in induced MSCs tended to oppose thelevels of GSK-3β, corresponding to their each protein levels.20μM LiClincreased the expression levels of β-catenin (1.24±0.07vs.1.00±0.00, P=0.02)and TCF-3(2.15±0.24vs.1.00±0.00, P=0.14) and decreased expression ofGSK-3β (0.60±0.07vs.1.00±0.00, P<0.01) after7days of co-culture.Conversely, β-catenin (0.92±0.08vs.1.00±0.00, P=0.38) and TCF-3(0.77±0.09vs.1.00±0.00, P=0.06) expression decreased after DKK-1exposure, and GSK-3β (1.11±0.03vs.1.00±0.00, P=0.02) expressionincreased. Moreover, LiCl caused an increase in the levels of AE1/AE3expression relative to controls (1.20±0.18vs.1.00±0.00, P=0.32). Cellsexposed to DKK-1showed less AE1/AE3expression than those in control group (0.83±0.11vs.1.00±0.00, P=0.19).Conclusions: Wnt/β-catenin signaling might participate in the regulationof MSC differentiation into epithelial cells under co-culture conditions andthat up-regulation of Wnt/β-catenin with LiCl might speed up thedifferentiation process.