Phenotypic analysis of cell surface markers and gene expression of human mesenchymal stem cells and chondrocytes during monolayer expansion.
Both chondrocytes and mensenchymal stem cells (MSCs) are the most used cell sources for cartilage tissue engineering. However, monolayer expansion to obtain sufficient cells leads to a rapid chondrocyte dedifferentiation and a subsequent ancillary reduced ability of MSCs to differentiate into chondrocytes, thus limiting their application in cartilage repair. The aim of this study was to investigate the influence of the monolayer expansion on the immunophenotype and the gene expression profile of both cell types, and to find the appropriate compromise between monolayer expansion and the remaining chondrogenic characteristics. To this end, human chondrocytes, isolated enzymatically from femoral head slice, and human MSCs, derived from bone marrow, were maintained in monolayer culture up to passage 5. The respective expressions of cell surface markers (CD34, CD45, CD73, CD90, CD105, CD166) and several chondrogenic-related genes for each passage (P0-P5) of those cells were then analyzed using flow cytometry and quantitative real-time PCR, respectively. Flow cytometry analyses showed that, during the monolayer expansion, some qualitative and quantitative regulations occur for the expression of cell surface markers. A rapid increase in mRNA expression of type 1 collagen occurs whereas a significant decrease of type 2 collagen and Sox 9 was observed in chondrocytes through the successive passages. On the other hand, the expansion did not induced obvious change in MSCs gene expression. In conclusion, our results suggest that passage 1 might be the up-limit for chondrocytes in order to achieve their subsequent redifferentiation in 3D scaffold. Nevertheless, MSCs could be expanded in monolayer until passage 5 without loosing their undifferentiated phenotypes.
UMR 7561 CNRS, Université Nancy I, F 54505 Vandoeuvre-les-Nancy, France.
Christel
C
Cournil-Henrionnet
Céline
C
Huselstein
Yun
Y
Wang
Laurent
L
Galois
Didier
D
Mainard
Véronique
V
Decot
Patrick
P
Netter
Jean-François
JF
Stoltz
Sylvaine
S
Muller
Pierre
P
Gillet
Astrid
A
Watrin-Pinzano
D000368:Aged; D000369:Aged, 80 and over; D015703:Antigens, CD / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism*; D001853:Bone Marrow; D018929:Cell Culture Techniques / Q000379:methods; D054337:Cell Dedifferentiation / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D020219:Chondrogenesis / Q000502:physiology; D003094:Collagen / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D008875:Middle Aged; D055758:SOX9 Transcription Factor / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods
D015703:Antigens, CD; D015415:Biomarkers; D055758:SOX9 Transcription Factor; D003094:Collagen