Effect Of In Vivo And In Vitro Microenvironment On The Stemness Of Rat Adipose-derived Stromal Cells And Related Mechanism

Background: Adipose-derived stromal cells(ADSCs)represent an important source of mesenchymal stromal cells(MSCs)for tissue engineering and cell therapy.However,during in vitro culture,ADSCs quickly lose the proliferation and multilineage differentiation capacity due to the loss of local microenvironment,which largely restricts their application in clinical.It has been reported that one or more secreted factors in the microenvironment can effectively regulate the biological characteristics of ADSCs.Previously,a continuous expression of insulin-like growth factor 2(IGF2)in tissues was reported to maintain the self-renewal ability of several types of stem cells.However,its effects on ADSCs remain largely unknown.In addition,many studies that focus on mimicking in vivo microenvironment to some degree are demonstrated to maintain the stemness of ADSCs in vitro,but restrictions exist.Therefore,how to mimic the real microenvironment at most is urgent.Objective: To explore the effects of IGF2 on the stemness of rat ADSCs and its related mechanism,and investigate the effect of the in vivo culture condition on the stemness of rat ADSCs by culturing in adipose tissues.Methods: Section I.Rat ADSCs were isolated and identified.Section II.(1)IGF2 level in rat inguinal adipose tissue with different ages was detected by qRT-PCR and Western blot analysis.(2)MTT assay and Western blot were used to detect the proliferation and expression of stemness-related markers NANOG,OCT4 and SOX2 of ADSCs with different concentrations of exogenous IGF2.(3)Specific IGF-1R/IR inhibitor BMS-754807 and IGF-1R inhibitor picropodophyllin(PPP)were added to detect the effects of IGF2 on the proliferation,stemness-related markers expression,and adipogenic and osteogenic differentiation of ADSCs.Section III(1)In vivo cell culture model was constructed by injecting the fourth passage GFP-ADSCs into bilateral inguinal adipose tissue of nude mice.Then GFP-ADSCs from the in vivo group were collected 3 days,5 days and 7 days after injection and cells from the in vitro group were collected simultaneously.Therefore,ADSCs were divided into three groups: the in vivo group,the in vitro group and the control group(passage 4 ADSCs).(2)MTT assay,CFU-F and qRT-PCR were used to detect the proliferation ability of ADSCs;the expressions of stemness-related markers were dectected with qRT-PCR,Western blot and IF analysis;specfic chemical staining and qRT-PCR were used to measure the adipogenic,osteogenic and chondrogenic differentiation of ADSCs.(3)mRNA expressions in ADSCs derived from the three groups were determined by RNA-sequencing.Gene Ontology(GO)analysis and Pathway enrichment analysis were carried out for differentially expressed genes and verification of genes were conducted by qRT-PCR analysis.Results: Section I: Isolated and cultured ADSCs at lower passage exhibited the morphological characteristics and trilineage-differentiation ability of MSCs.The cells highly expressed MSCs-specific markers(CD90 and CD29)and rarely expressed hematopoietic cell markers(CD34 and CD45).Section II.(1)The expression of IGF2 in rat adipose tissue decreased after 10 days of age.(2)In the presence of 1% FBS,100 ng/mL IGF2 promoted the proliferation,and NANOG,OCT4 and SOX2 expression in ADSCs,which were blocked by BMS-754807.However,only the promotion effect on stemness-related markers expression was neutralized by PPP.(3)Similarly,adipogenic and osteogenic differentiation of ADSCs increased by 100 ng/mL IGF2.BMS-754807 blocked the up-regualtion effect while PPP only blocked the adipogenic differentiation increased by IGF2.Section III(1)The quantity of ADSCs decreased with increased culture time in vivo.Morphologically,ADSCs grown in adipose tissue were in three-dimensional growth pattern with pseudopodia,and then exhibited spindle-like shapes with higher diopter after 24 h adhesion in vitro.However,ADSCs from the in vitro group showed large flat-shaped morphologies with lower diopter.(2)ADSCs form the in vivo group on day 3,5 and 7 showed up-regualted proliferation by MTT assay and decreased P15 and P21 levels compared with the in vitro group,while the number of clonies was significantly higher than that in the in vitro group on day 5 and day 7.(3)The expressions of stemness-related markers of ADSCs from the in vivo group were not significantly different from those of the in vitro group until day 5 and day 7.(4)Compared with the in vitro group,the chondrogenic differentiation of ADSCs from the in vivo culture group significantly increased on day 3,the osteogenic differentiation significantly up-regulated on day 5,and the adipogenic differentiation significantly increased on day 7.(5)RNA-seq results indicated 1308 genes were differentially expressed between the in vivo and in vitro groups,and 419 were differentially expressed between the in vitro group and the control group.GO and KEGG enrichment analysis suggested that differentially expressed genes were mainly located in extracellular matrix and extracellular space.These genes were involved in regulating cell proliferation and extracellular matrix synthesis,in which signaling pathways regulating pluripotency of stem cells,Notch signaling pathway,Jak-STAT signaling pathway could be involved(P < 0.05).Conclusion: IGF2 expression in rat adipose tissue decreases after 10 days of age and that exogenous IGF2 supplementation can enhance the proliferation and multilineage differentiation of ADSCs from 8-week-old rats,indicating that IGF2 in the microenvironment may contribute to the stemness regulation of ADSCs.In addition,ADSCs cultured in adipose tissue in vivo have increased proliferation and pluripotency abilities,which are regulated by a variety of genes that are involved in many signaling pathways,including stem cell pluripotency regulatory,Notch,Jak-STAT pathways.