Differential Protein Expression between Chondrogenic Differentiated MSCs, Undifferentiated MSCs and Adult Chondroctyes Derived from Oryctolagus cuniculus in vitro.
"This preliminary study aims to determine the differentially expressed proteins from chondrogenic differentiated multipotent stromal cells (cMSCs) in comparison to undifferentiated multipotent stromal cells (MSCs) and adult chondrocytes (ACs). Methods: ACs and bone marrow-derived MSCs were harvested from New Zealand White rabbits (n = 3). ACs and cMSCs were embedded in alginate and were cultured using a defined chondrogenic medium containing transforming growth factor-beta 3 (TGF-β3). Chondrogenic expression was determined using type-II collagen, Safranin-O staining and glycosaminoglycan analyses. Two-dimensional gel electrophoresis (2-DE) was used to isolate proteins from MSCs, cMSCs and ACs before being identified using liquid chromatography-mass spectrometry (LC-MS). The differentially expressed proteins were then analyzed using image analysis software. Results: Both cMSCs and ACs were positively stained with type-II collagen and safranin-O. The expression of glycosaminoglycan in cMSCs was comparable to AC at which the highest level was observed at day-21 (p>0.05). Six protein spots were found to be most differentially expressed between MSCs, cMSCs and ACs. The protein spots cofilin-1 (CFL1) and glycealdehyde-3-phosphate dehydrogenase (GAPD) from cMSCs had expression levels similar to that of ACs whereas the others (ie. MYL6B, ALDOA, TAGLN2, EF1-alpha), did not match the expression level of ACs. Conclusion: Despite having similar phenotypic expressions to ACs, cMSCs expressed proteins which were not typically expected."
"TAGLN2 was significantly down-regulated in cMSC. Absence of TAGLN2 has shown to increase the activity of Rho GTPase that phosphorylates MYL6B and induces formation of stress fibres"
"Activity of GAPD in cMSC and ACs has increased three folds as compared to MSCs."
"ALDOA expression was lower in cMSCs than both the MSCs and ACs. ALDOA is a glycolytic enzyme for energy generation in chondrocytes, particularly marked at cell maturity. Reduction of energy modulated by ALDOA may limit the ability of ACs to restore GAG matrices. Recently, both GAPD and ALDOA have been found to interact with S100A1 and S100B proteins that suppress hypertrophic differentiation and mineralization of chondrogenic cells"
"[EIF-1A] was significantly down-regulated in cMSC"
"Phosphorylated EF1-alpha by type-I TGFβ receptor kinase at Serine 300 exerted a direct inhibitory on protein synthesis, therefore reducing cell proliferation"
"MYL6B, ALDOA, TAGLN2 and EF1-ALPHA were not as highly modulated during MSC chondrogenesis as compared to ACs."
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