Proteome analysis during chondrocyte differentiation in a new chondrogenesis model using human umbilical cord stroma mesenchymal stem cells.
Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies.
Rheumatology Division, Cellular Therapy Unit, INIBIC-Hospital Universitario A Coruña, 15006 A Coruña-Spain.
Alexandre
A
De la Fuente
Jesús
J
Mateos
Iván
I
Lesende-Rodríguez
Valentina
V
Calamia
Isaac
I
Fuentes-Boquete
Francisco J
FJ
de Toro
Maria C
MC
Arufe
Francisco J
FJ
Blanco
D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes; D020219:Chondrogenesis / Q000502:physiology*; D015180:Electrophoresis, Gel, Two-Dimensional; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D008954:Models, Biological; D020543:Proteome / Q000032:analysis*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism*; D014470:Umbilical Cord / Q000166:cytology / Q000378:metabolism*
D015415:Biomarkers; D020543:Proteome; D012333:RNA, Messenger