Mouse ES Conditioned Medium Promote Human Adipose Derived Stem Cells Proliferation

Objective：Collection of mouse embryonic stem cells conditioned medium，use for culturehuman adipose derived stem cells，detect the proliferation rate of hADSC and thedifferentiation capability of hADSC， trying to find a way to promote theproliferation of hADSC which does not affect cell differentiation.Method：Isolation and identification of hADSC—hADSC isolation，primary culture；MTTmethod was used to test the proliferative activity of hADSC at passage P3,P6and P9;The cells surface antigens and cell cycle were measured by flow cytometry;Adipogenic and osteogenic capability were evaluated respectively by Oil red Ostaining and Alizarin red staining.Culture of mouse embryonic stem cells,identification the undifferentiated state ofit,and collected conditioned medium. Mouse embryonic stem cells were cultured onfeeder cells(MEF),and were digested by trypsin for continuous passage culture,withLIF in the culture medium for sustain mES undifferentiated state.mES RNA wasextracted and identify the mES pluripotency factors expression by PCR.Culture ofundifferentiated mES,after24hours,collect the culture medium witch calledconditioned medium（CM）；hADSC cultured with conditioned medium,the biochemical marker weredetected.hADSC were divided into three groups,cultured by hADSC medium,40%CMmedium and40%ES medium,respectively.Cell surface markers were detected by flowcytometry. The expression of c-kit and Sca-1were detected by RT-PCR,to determinethe changes of cell properties of hADSC.Three group cells proliferation rate weredetected by MTT,three groups of cells for preparation of growth curve,cell cycle byflow cytometry to detect the proliferation of the cells; The expression cellpluripotency factors of nanog and OCT-4were detected by Q-PCR;The cell apoptosis marker of Caspase-3s was detected the by PCR,Q-PCR was used to detect theexpression of Caspase-3,also a very important relative cell apoptosis gene,flowcytometry was used to detect the P3,P15hADSC apoptosis;Also we use Q-PCR todetect of hADSC osteogenic differentiation marker genes of ALP,collagen type I,OPNand RUNX-2in the induction of0days,7days and14days,after21days ofinduction,hADSC were stained with Alizarin red; Adipogenic capability wereevaluated Oil red O staining after14days induction.Result:1.MTT results showed that,the proliferation of hADSC curve is in the shape ofS;Surface factor flow cytometry showed that CD29,CD44,CD73,CD90and CD105surface marker positive expression,and CD31,CD34,CD45and HLA-DR surfacemarkers negative expression;Detection of hADSC on cell cycle by flowcytometry,results show that (87.3±0.48)%of the cells in the G1phase,(5.3±0.84)%of cells in G2phase and (7.4±0.35)%of cells in S phase;Alizarin red S staining,oilred O staining showed that hADSC had osteogenic and adipogenic differentiationability,accord with the basic characteristics of mesenchymal stem cells.2.mES cell colonies were round,colony projections,full,smooth edge nucleocytoplasmic ratio and with good refractive index.High expression of pluripotencygene of nanog,OCT-4and sox-2in this mES cells.mES cells culture medium wascollected after24h culture,using the0.22um filter,filter remove mES.3.The result of three groups of cell surface markers detected by flow cytometryindcated that after cultured by CM there is no deference between three groups inpositive cell surface markers like CD29,CD34,CD73,CD90and CD105,but thenegative expression factors like CD31and CD45lower than hADSC group cells,andHLA-DR expression higher than hADSC group cells.PCR results showed that there isno difference in the expression of C-kit and Sca-1between three groups;MTT was used to detect the cell proliferation rate，40%CM group of MTT absorption value washigher than that in hADSC group (P=0.012<0.05),the result of cell counts of CMgroup showed that cell growth rate was significantly higher than the other twogroups.Cell cycle was detected by flow cytometry,CM groups in the percentage of Sphase was higher than that in other two groups,which indicated CM group cellproliferation higher than other two groups.Q-PCR results showed that the expressionof OCT-4,CM group，ES group,the expression was significantly higher than that inhADSC group，the expression of Nanog，the expression of CM was higher than that inES group,ADSC group，results showed that the CM medium could increase hADSCpluripotency;PCR results showed that the amount of CM group was significantlylower than that in ADSC group the expression of Caspase-3,P=0.003<0.01significantdifference，apoptosis was detected by flow cytometry，display ratio of apoptosis CMgroup than in ADSC group,CM medium could inhibit hADSC apoptosis;Theexpression of ALP and collagen type I,induced the highest expressed in the7day，theALP expression of7d and0d,14d have significant difference，and no difference in0d,7d,14d groups.OPN and RUNX-2are osteogenic differentiation late expressiongeneexpression induced by14d,ADSC and ES groups OPN and RUNX-2weresignificantly higher than that of CM group.Conclusion:1.Embryonic stem cells condition medium (ESC-CM) can enhance the proliferationof hADSC rate significantly which does not affect the cell properties of hADSC.2.Embryonic stem cells condition medium (ESC-CM) can increase the hADSCexpression of nanog and OCT-4gene，and can inhibit the apoptosis of hADSC.3.In osteogenic and adipogenic differentiation，osteoblast early expression gene hadno significant differences among groups. But late expression of genes such as OPN，expression of RUNX-2，CM group was significantly lower than the other two groups.