The Chondrogenic Differentiation Of Human Umbilical Cord Mesenchymal Stem Cells In The Co-culture System

Objective: to observe the chondrogenic differentiation of human umbilical cordmesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUC-MSCs)by co-culturing the rabbit knee cartilage chondrocytes and hUC-MSCs in directco-culture system and Transwell co-culture system, probe into the feasibility ofchondrogenic diffferentiation of hUC-MSCs, and compare the effect of the twococulture system on the chondrogenic differentiation of hUC-MSCs, therefore, seekthe most suitable coculture methods, and provide the perimental basis for the sourceof seed cells of cartilage tissue engineering.Methods:Isolation and enaluation of hUC-MSCs---Primary hUC-MSCs were isolated bytype Ⅱcollagen. MTT method was used to test the proliferative activity ofhUC-MSCs at passage P3、P6、P9and P12; The cells surface antigens and cell cyclewere measured by flow cytometry; Adipogenic, osteogenic and chondrogeneticcapability were evaluated respectively by Oil red O staining, Alizarin red staining andAlcian blue staining.Primary Rabbit knee cartilage chondrocytes were isolated by type Ⅱcollagen, andwere digested by trypsin for continuous passage culture. The cell growth curve ofRabbit knee cartilage chondrocytes at passage P1、P2、P3、P4、P5and P6wasanalyzed by MTT assay; the chondrocytes were identified by Alcian blue staining andtoluidine blue staining; COL2-A1and GAG mRNA expression level ofchondrocytes at passage P1、P2、P3、P4、P5and P6were assayed by ordinaryPCR and real-time fluorescence quantitative PCR.The coculture of hUC-MSCs and Rabbit knee cartilage chondrocytes---hUC-MSCsand Rabbit knee cartilage chondrocytes were cocultured in direct coculture systemand Transwell coculture sysytem as the Table3-1showed. while, hUC-MSCs with thesame number in the DMEM/F12medium served as negative group, and hUC-MSCswith the same number in the basic chondrogenic differeentiation medium served as positive group. In the Transwell coculture system, Rabbit knee cartilage chondrocyteswere in the upper rooms, and hUC-MSCs were in lower rooms.The morphology andthe proliferation of the cells before and after coculture were observed under theinverted phase contrast microscope;21days later, Type Ⅱcollagen (COL2A1) andglycosaminoglycan (GAG) were analyzed qualitatively by toluidine blue andimmunofluorescence technique, respectively, the contents of COL2A1and GAG wereestimated from the determination of hydroxyproline content and Alcian Blue methodseparately. The mRNA expressions of GAG and COL2A1were assayed by real-timefluorescence quantitative PCR.Results:The shapes of growth curve of hUC-MSCs at passage P3、P6、P9and P12weresimilar; hUC-MSCs all expressed highly CD29, CD44and CD105, but CD31, CD34,CD40, CD45and HLA-DR were not expressed; the Flow cytometry result showed that(77.08±0.84)%hUC-MSCs were in the G0/G1phase, and the rest of hUC-MSCs werein the proliferative and divisive stage; the positive Oil red O staining, Alizarin redstaining and Alcian blue staining indicated that hUC-MSCs could be differentiated intoadipose tissue, bone and cartilage.The Alcian blue staining and toluidine blue staining of Rabbit knee cartilagechondrocytes were positive, demonstrating that chondrocytes were obtained. Rabbitknee cartilage chondrocytes at passage P1、P2and P3had the same growth curves.However, the growth curve of Rabbit knee cartilage chondrocytes at passage P4had atendency to decline, and the growth curve at passage P6declined more apparently,showed that cell proliferation of rate Rabbit knee cartilage chondrocytes from4thgeneration began falling significantly. Ordinary PCR and real-time quantitative PCRrevealed that the expression level of GAG and COL2-A1gene of Rabbit kneecartilage chondrocytes at passage4began to decrease, that of GAG of chondrocytes atpassage6reached minimum, and that of COL2-A1of chondrocytes at passage6didnot basicly express.After21days, the toluidine blue staining and immunofluorescence reaction of the positive control group and all experimental group were positive, indicating that therehad secretion of GAG and COL2-A1; the contents of GAG and COL2-A1in1:4group and their mRNA expression level were higher than other experimental groupand positive control group, and their had significant statistical difference (P＜0.05).Conclusions:1. There existed no obvious change and proliferation rate in phenotype ofhUC-MSCs within10generation, and hUC-MSCs could be used to coculture withother cells.2. The mRNA expression level of GAG and COL2-A1of Rabbit knee cartilagechondrocytes from passage4had begun decreased significantly; the chondrocytes atpassage6had transformed into osteoblast.3. The co-culture of rabbit chondrocytes and hUC-MSCs at defined ratios canpromote the ability of induction of hUC-MSCs into the chondrocyte. The optimal cellratio appears to be4:1(chondrocytes: hUC-MSCs). Compared with the directcoculture system, Tanswell coculture could significantly increase expression level ofGAG and COL2-A1mRNA and it was conducive to the chondrogenic differentiationability of hUC-MSCs. Therefore, Transwell co-culture system could better promotethe hUC-MSCs induce into chondrocytes-like cells.