Human mesenchymal stem cells (hMSCs) can differentiate into different cell types, nevertheless the heterogeneity among hMSCs isolates is an important hurdle preventing routinely and robust use and thereby success of hMSCs in regenerative therapies. Gene expression profiling studies in our lab have identified 135 cell surface expressed proteins that were specifically upregulated within 4 days of osteogenic differentiation hMSCs. Here, we characterise one of these cell surface receptors, i.e. CD24, during osteogenic differentiation of hMSCs. Bone marrow-derived hMSCs were differentiated into osteoblasts using dexamethasone and beta-glycerophosphate. The cells were analysed by FACS analysis, biochemical assays, qPCR, KI-67 and EdU incorporation. The percentage of cells expressing CD24 in non-differentiated hMSCs ranged from 2-10% depended on the bone marrow donor and significantly increased 2-3 fold upon osteogenic differentiation within 7 days (P<0.001). FACS sort experiments illustrated the existence of 3 different populations within undifferentiated hMSCs based on CD24 expression. 1) CD24 negative cells, 2) CD24 positive cells, and 3) CD24 negative cells that express CD24 upon osteogenic differentiation. Interestingly, CD24 positive cells have significantly reduced alkaline phosphatase expression (1.8 fold) after 7 days of osteogenic differentiation (P<0.001) and have strongly reduced mineralisation capacity after 21 days. Moreover, KI-67 staining illustrated a significant lower percentage of proliferating cells among the CD24 positive population than among the CD24 negative population (P<0.05) in bone marrow and after 6 days of non-differentiated and osteogenic differentiation. Furthermore, EdU incorporation showed less CD24 positive cells went through S-phase (24.7%) than CD24 negative cells (35%) in the non-differentiated hMSCs (P<0.05), however after 7 days of osteogenic differentiation there is no significant difference between the two populations. CD24 positive cells have a reduced proliferation, differentiation, and mineralisation capacity in hMSCs differentiated into osteoblasts. This suggests that cells expressing CD24 are a subset that have a reduced osteogenic differentiation capacity in vitro and may have other yet unknown functions. Currently we are further characterising the CD24 positive cells, their role in interaction with other bone marrow cells, and analysing the effects of CD24 knockdown on osteoblast differentiation.
Disclosure: The authors declared no competing interests. This work was supported by NIRM- Netherlands Institute of Regenerative Medicine (grant number FES0908).