Proliferation Regulatory Mechanism And In Vitro Optimization Strategy About Human Mesenchymal Stem Cells

BackgroundMesenchymal stem cells(MSCs)are a kind of pluripotent stem cell which has self-renewal and differentiation ability.MSCs derived from the primal period of the mesoderm and ectoderm and it exists in the bone marrow,fat,synovial,umbilical cord,and muscle and so on.Under certain condition MSCs can be induced to cell from germ layers-ectoderm,mesoderm and endoderm.Compared with embryonic stem cells(ESCs),MSCs has advantage of easily obtained,non immune rejection,non ethical controversy.So it can be an ideal source in tissue engineering and regenerative medicine.MSCs can be obtained in vitro through primary culture.While in the process of subculture,MSC appears with irreversible senescence and it will lost the proliferation and self-renewal ability when it is cultured to about passage 8 to 10.Thus,the number of MSCs that can be used for cell therapy is very limited through primary culture.Bone mesenchymal stem cells(BMSCs)are the earliest discovered mesenchymal stem cells.There are many studies and reports on BMSC over the past few years.BMSC is earliest discovered in the clinical research as well.And it is expected to be the first MSC used in clinical treatment officially.BMSC can be cultured from bone marrow by means of attachment method or density gradient centrifugation,and then it can be isolated and screened by the specific expression of CD29,CD44,CD105 and so on.Multipotent adult progenitor cells(MAPCs)are kinds of pluripotent stem cells isolated from bone marrow mesenchymal stem cells.MAPCs are similar to early ESC in morphology that has showed clonal growth and totipotency.MAPCs have proved stronger proliferation ability and pluripotency than MSC and it can be passaged 80-120 times without signs of aging.The present studies suggest that MAPCs are subtype of MSCs.As its culture condition is consistent with the MSCs,and both of them co-express some markers likes CD105 and SSEA-3.In prophase,we screened a number of differentially expressed genes by ESCs-MAPCs-MSCs whole genome microarray.Among them,we selected prrx1 which is highly expressed in MSC as our research object.The gene encodes the mesenchymal marker paired mesoderm homeobox protein 1.Someone has indicated that prrx1 can directly participate in the epithelial-mesenchymal transition(EMT)and effect cancer cell proliferation ablilty.Umbilical mesenchymal stem cells(UMSCs)were isolated from umbilical cord.Compared with BMSCs,UMSCs has the advantages of better activity,stronger proliferation ability.It was investigated that the doubling time of UMSCs was significantly shorter than that of BMSCs,compared with the same culture passage.And cell cycle phase S and phase G1 of UMSCs was much higher than that of BMSCs.Exosomes are kind of small vesicles secreted by cells.The diameter of exosomes is about 40~100nm.Exosomes can carry cell specific active substances,including mi RNA,mRNA,protein and so on.The active substances in exosomes were shipped to other cells through exocytosis and endocytosis.As an important means of paracrine,exosomes can be involved in the process of cell proliferation,migration,differentiation and so on.At present,there have been reports of the proliferation of MSCs derived exosomes of other cells,suggesting that MSCs derived exosomes may contain a key substance that regulates the proliferation of MSCs.ObjectiveThe main purpose of this paper is to explore how the gene prrx1 and early generation UMSCs exosomes can affect the proliferation of MSCs,researching for an optimized culture system to solve the proliferation problem in vitro.Part Ⅰ Exploring the gene prrx1 knocked down on promoting the proliferation of MSCsMethod:(1)Screening the differentially expressed gene prrx1 through ESCs-MAPCs-MSCs whole genome microarray.(2)Obtain MSCs through primary culture.Carring out immuno histochemistry experiment to confirm the MSCs induced to osteoblast,chondroblast,adipose cells.(3)Utilizing siRNA to construct the prrx1 knocked down MSCs.Confirming the interference efficiency by Q-PCR.(4)Detecting the ki67 expression by Q-PCR and immunofluorescence.(5)Detecting the Edu incorporation ratio by Edu experiment.(6)Detecting the pluripotent stem cell related gene oct4、sox2、nanog expression by Q-PCR.(7)Detecting the epithelia gene cdh1,epcam,mesenchymal gene cdh2,vim expression by Q-PCR.Result:(1)We succeed to obtain MSCs which can be induced to osteoblast,chondroblast,adipose cells through primary culture.(2)Prrx1 knocked down can promote the proliferation of MSCs,promoting the expression of ki67 and the incorporation ratio of Edu.(3)Prrx1 knocked down can promote the self-renewal ability of MSCs,promoting the expression of oct4、sox2、nanog.(4)Prrx1 knocked down can promote the expression the epithelia gene cdh1,epcam.But mesenchymal gene cdh2,vim expressions remain unchanged.No morphology changed was happened as well.Conclusion: Prrx1 was the differentially expressed gene between MSCs and MAPCs,ESCs.Prrx1 knocked down can promote the proliferation and self-renewal ablity of MSCs,but it can induce MET happened.Part Ⅱ Exploring the exosomes secreted by early passage UMSCs in promoting the proliferation of MSCsMethod:(1)Obtain UMSCs through primary culture.Collecting the exosomes derived from UMSCs passage 1 and passage 2 through ultracentrifugation.(2)Using nanoparticle analysis system and micro-flow imaging system to identify the exosomes.(3)Check the proliferation ability of BMSCs changed by cell cycle experiment,Edu experiment and cell growth curve experiment.Result:(1)We use nanoparticle analysis system and micro-flow imaging system to prove that exosomed collected by ultracentrifugation fits the exosomes definition in size and expressing exosomes specific marker.(2)The exosomes derived from P2 USMCs exosomes can promote the proliferation of BMSCs,increasing the proportion of phase S of the cell cycle,improving the Edu incorporation ratio,increasing the number of cells.Conclusion: Ultracentrifugation provides a quick and simple way to collect the exosomes from medium supernatant.Exosomes derived from early passage UMSCs can promote the proliferation of BMSCs.Exosomes derived from P2 UMSCs may conatin some proliferation related substances.Part III Indentifying the proliferation related substances in P2 UMSCs exosomes and mechanism researchMethod:(1)Analysis the silver-stained P2 and P3 UMSCs exosomes SDS-PAGE gel.Cut the differentially luminance gel and find out the differentially expressed protein through mass spectrometric detection and bioinformatics analysis.(2)Use of protein chip and bioinformatics analysis to find out the differentially expressed protein between P2 and P3 UMSCs exosomes.(3)Confirming the differentially expressed protein through ELISA experiment.(4)Detecting the Edu incorporation ratio after adding CCR2 antagonist with P2 UMSCs exosomes to BMSCs.Result:(1)Mass spectrometric experiment indicates that the main component protein in UMSCs P2 and P3 exosomes is keratin and tubulin.(2)Protein chip indicate that CCR1 and CCR2 were differentially expressed between P2 UMSCs and P3 UMSCs exosomes.(3)ELISA confirms that there is more CCR1 and CCR2 in P2 UMSCs exosomes than in P3 UMSCs exosomes.There is more CCR2 in P3 exosomes than in P4 and P5 UMSCs exosomes.(4)CCR2 antagonist can resuce the promotion of Edu incorporation ratio by P2 UMSCs exosomes.Conclusion: Cytokine protein chip provide an effective way to detect the proliferation related protein in exosomes.CCR2 may result in the proliferation of BMSCs.