Abstract
Investigating mesenchymal stromal cell differentiation requires time and multiple samples due to destructive endpoint assays. Osteogenesis of human bone marrow derived mesenchymal stromal cells (hBMSCs) has been widely studied for bone tissue engineering. Recent studies show that the osteogenic differentiation of hBMSCs can be assessed by quantifying the ratio of two important transcription factors (Runx2/Sox9). We demonstrate a method to observe mRNA expression of two genes in individual live cells using fluorescent probes specific for Runx2 and Sox9 mRNA. The changes of mRNA expression in cells can be observed in a non-destructive manner. In addition, the osteogenic hBMSCs can be prospectively identified and obtained based on the relative intracellular fluorescence of Sox9 in relation to Runx2 using fluorescence activated cell sorting. Relatively homogeneous cell populations with high osteogenic potential can be isolated from the original heterogeneous osteogenically induced hBMSCs within the first week of induction. This offers a more detailed analysis of the effectiveness of new therapeutics both at the individual cell level and the response of the population as a whole. By identifying and isolating differentiating cells at early time points, prospective analysis of differentiation is also possible, which will lead to a greater understanding of MSC differentiation.
Highlights
Typical methods for checking Human bone marrow derived mesenchymal stromal cells (hBMSCs) osteogenesis include immunostaining of a number of osteogenic differentiation markers, and detection of the mRNA expression of these markers using RT-PCR
There is a critical need for a new method to observe mRNA expressions in live cells and isolation of relative homogeneous stromal cells
Isolated mononuclear cells were seeded at a density of 50,000 cells/cm[2] in cell culture flasks, and cultured in α-modified essential medium (α-MEM; GIBCO), 10% fetal bovine serum (Sera Plus, PAN-Biotec), 1% penicillin and streptomycin (GIBCO), and 5 ng/ml recombinant human basic fibroblast growth factor
Summary
In order to confirm this, cells which cultured in OM for 6 days and tagged with probes specific either for Runx[2] or Sox[9] were separated by FACS in populations with different mfi levels (Fig. 1E,F) and mRNA levels of sorted cells were quantitatively assessed using RT-PCR. It confirms that osteogenically induced hBMSCs are not homogeneous, and cells have different proliferation rates and osteogenic differentiation potential These heterogeneous cells can be sorted into a relatively homogeneous population based on the mRNA expression of Runx[2] and Sox[9]. P1 has the highest osteogenic differentiation potential and lowest proliferation rate, a phenotype more associated with osteoblasts
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