| Background:Human mesenchymal stem cells(h MSCs)can be derived from bone marrow,umbilical cord,fat and many other tissues,which have been widely used in regenerative medicine,tissue engineering,cell therapy and other fields,but there are some problems such as limited sources,insufficient amplification ability in vitro,and replicative aging.Human induced pluripotent stem cells(hi PSCs)can be derived from patients’own cells and have unlimited proliferation and multidirectional differentiation ability,which can be induced differentiation in vitro to obtained human induced pluripotent stem cells derived mesenchymal stem cells(hi PSCs-MSCs).Unfortunately,existing methods of induced differentiation mostly use serum-containing mesenchymal medium directly,which has some problems such as unclear composition and low induction differentiation rate.More importantly,mesenchymal cells lack specific markers,which are prone to senescence due to continuous passage,and it is urgent to develop effective cell purification methods.Methods:In this thesis,hi PSCs were differentiated into mesenchymal-like cells in vitro using the widely used embryoid induction method.Using serum-containing embryoid medium as control,we transferred hi PSCs derived embryoid to ultra-low adhesion plate and cultured with E6 medium with definite composition for 5 days.We used CCK8,RT-PCR and flow cytometry to investigate the effects of TGF-βand RA on cell survival and mesenchymal differentiation efficiency at different stages.After determining the ideal suspension medium,EBs was cultured for 10 days with mesenchymal medium with a defined composition.Using Matrigel as control,the effects of different components(gelatin or peptide modified surfaces)on embryoid cell growth and mesenchymal differentiation efficiency were compared.Then,we prepared Polydimethylsiloxan(PDMS)of different widths(0μm,5μm,10μm,20μm,40μm)micropatterned surfaces,which were been modified with Poly(N-isopropylamide)PNIPAAm temperature sensitive coating and gelatin and characterize with X-ray photoelectron spectroscopy(XPS),atomic force microscopy(AFM)and water contact angle.Then,Then,the mesenchymal cells obtained from the above surface were inoculated into the micropatterned surface as single cells to study the purification effect of groove width on hi PSCs-MSCs.Finally,the obtained hi PSCs-MSCs were identified and compared with adult umbilical cord derived mesenchymal stem cells(h UMSCs)for their proliferative ability and multipotential differentiation.Results:We used E6 medium supplemented with 40ng/m L RA for the second 3days(D3-D5 stage)to promote mesodermal and mesenchymal differentiation after EBs suspension culture 5 days.At the stage of adherent culture,the expression of CD73,CD44,CD29,CD45,CD90 and CD105 genes on the surface of gelatin was high,and the positive expression of CD73 and CD44 on the surface of gelatin was the highest,so we confirmed the gelatin modified surface as the ideal surface.Then,AFM demonstrated successful acquisition of grooved patterned surfaces of different widths,and the characteristic absorption peaks of amide bond C=O stretching vibration observed by XPS spectra showed that the thermosensitive coatings were successfully prepared.Cell experiments confirmed that the thermosensitive coating could significantly improve cell survival compared with pancreatic enzyme digestion.More importantly,the cells cultured at 20μm width showed homogeneous mesenchymal cell morphology and high expression of CD73,CD44,CD29,CD45,CD90 and CD105mesenchymal markers.Finally,the CD73~+CD44~+hi PSCs-MSCs obtained showed similar in vitro amplification and multipotential differentiation ability as h UMSCs.Conclusions:In this study,the in vitro induction differentiation culture system of hi PSCs-MSCs was successfully constructed by using embryoid body method and composition specific medium.Moreover,mechanical"contact guidance"of the temperature-sensitive coating micropatterning surface was used to promote the purification of hi PSCs-MSCs,which can effectively obtain clinically usable mesenchymal cells.The results of this thesis are helpful to promote the application of hi PSCs-MSCs in the fields of tissue engineering,repair,gene therapy and cell transplantation,which have great scientific research significance and economic value. |
