The CCAAT-binding transcription factor NF-Y, consists of three subunits, NF-YA, NF-YB and NF-YC, all necessary for DNA binding. NF-YA is characterized as the regulatory subunit of the complex because of its differential expression during cell cycle. NF-YA appears to have a complex role in hematopoietic stem cell (HSC) biology. So far, however, there are no available data on the role of NF-Y in mesenchymal stem cell (MSC) biology. The aim of this study is the evaluation of NF-YA expression levels during the ex vivo expansion of human bone marrow (BM)-MSCs and the assessment of its possible role for the BM-MSC differentiation process towards the osteoblastic and adipocytic lineages.
BM-MSCs were isolated from posterior iliac crest aspirates of hematologically healthy individuals undergoing orthopedic surgery after written informed consent. BM-MSCs were ex vivo expanded until passage 10 (P10). Total RNA was isolated from BM-MSCs at P2 and P10 for the evaluation of NF-YA mRNA levels. P3 BM-MSCs' ex vivo differentiation into adipocytes and osteoblasts was induced using the appropriate culture media. Mineralization was evidenced via Alizarin Red and Von Kosa staining and lipid droplets were revealed with Oil Red O staining. Total RNA and proteins were isolated from undifferentiated and differentiated BM-MSCs at various time-points. Protein levels of NF-YA were immunodetected by Western blot. The relative gene expression of NF-YA, as well as that of lineage-specific markers was evaluated by real-time PCR. All PCR results are expressed as 2−ΔCt. Adipocytic and osteoblastic differentiation of BM-MSCs was also induced following knockdown of NF-YA expression after BM-MSC transduction with lentiviral particles carrying either shRNA against NF-YA or nonsense shRNA. Total RNA was isolated from transduced BM-MSCs at several time-points during differentiation.
A statistically significant (p=0.04) reduction in the mRNA levels of NF-YA was found in P10, as compared to P2 BM-MSC cultures (n=12).
During adipogenic differentiation, NF-YA significantly increased (p=0.0478) at day 16, as compared to the onset of differentiation induction (day 0), (n=14). Similarly, protein levels of NF-YA also increased during adipogenesis Furthermore, NF-YA expression levels strongly correlated with the mRNA levels of adipogenesis-associated genes PPARG (r=0.74, p<0.0001), C/EBPA (r=0.76, p<0.0001) and LPL (r=0.74, p<0.0001). In contrast, during osteogenic differentiation no statistically significant change was detected in NF-YA mRNA or protein expression. (n=9). In order to evaluate if NF-YA is functionally related to adipogenic and osteogenenic differentiation of BM-MSCs, NF-YA knockdown was performed and adipocytic as well as osteoblastic differentiations were induced. Throughout adipogenesis, the relative mRNA levels of NF-YA in shNF-YA-transduced BM-MSCs were reduced to 51.03% compared with control (non-transduced) cells. At day 21 of adipogenesis, a reduction of 31.22%, 70.06% and 74.3% was also observed in PPARG, C/EBPA and LPL mRNA levels, respectively. In addition, Oil Red O staining indicated a lack of lipid droplets in shNF-YA-transduced BM-MSCs compared with control cells. During osteogenesis, NF-YA mRNA levels in shNF-YA-transduced BM-MSCs were reduced to 52.37% compared to control cells. At day 21 of differentiation the mRNA levels of the osteogenesis- associated genes DLX5, RUNX2, OSX and ALP remained unaffected in shNF-YA-transduced cells compared to control cells. Furthermore, Alizarin Red and Von Kossa staining did not reveal any significant difference between shNF-YA-transduced BM-MSCs and control cells.
In conclusion we have shown for the first time, that BM-MSCs display decreased NF-YA mRNA levels after prolonged ex vivo expansion, a finding that might be associated with MSCs' senescence and/or loss of stemness., We have also shown that NF-YA does not have any major effect on BM-MSCs' osteogenic differentiation but displays a significant role in their adipogenic differentiation as was shown by (a) the increased NF-YA mRNA and protein levels during the ex vivo adipogenesis, (b) the positive correlation between the NF-YA mRNA expression levels and the adipocyte-associated genes PPARG, C/EBPA and LPL and (c) the impaired adipogenic differentiation following NF-YA knock down. Collectively our results imply a novel role for NF-YA in BM-MSCs' biology
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.