Abstract
Mesenchymal stromal cells (MSCs) have been intensively researched in the orthopaedic field since they hold great promise for aiding the regeneration of musculoskeletal tissues. While there are a range of postulated surface markers to identify MSCs, currently there are no known cell markers that predict in vivo osteochondral potency. Runt-related transcription factor 2 (Runx2) is considered as an essential transcription factor in osteoblast differentiation [1] and has been shown to physically interact with retinoblastoma protein (pRb), which leads the loss of osteoblast proliferation and the activation of genes concerning terminal differentiation of osteoblasts [2]. The aim of this study was to use adenoviral-mediated gene overexpression/knockdown to investigate the interplay between Runx2 and pRb during in vitro osteogenic differentiation of human bone marrow (hBM)-MSCs.
A first generation human adenovirus (hAd) serotype 5 dE/E3 carrying the gene of interest (Runx2 or shRNA-Runx2) were propagated and amplified in AD-293 cells, and purified over successive CsCl gradients. A second generation hAd serotype 5 carrying the gene of interest (Rb1) was generated. High efficiency single or double transduction of undifferentiated hBM-MSCs was achieved using lanthofection [3]. The transduced hBM-MSCs were then differentiated in osteogenic medium (OM) and osteogenic potency was assessed by quantification of alkaline phosphatase (ALP) activity (day 14) and Alizarin red staining (day 28). In addition, cell cultures were assessed for absorbance at OD 450nm, correlating to the refractive index of calcified areas, at days 0, 7, 14, 21 and 28 [4]. Quantitative RT-PCR was used to confirm expression of target genes following viral transduction. Basal medium was used as a control.
Untransduced hBM-MSCs cultures grown in OM demonstrated peak calcium deposition at day 28, while the overexpression of either Runx2 or Rb1 accelerated peak calcium deposition to day 21. Consistent with this, Runx2 overexpression increased ALP activity of hBM-MSCs cultured in OM, while Rb1 overexpression enhanced ALP activity of hBM-MSCs cultured in both basal and osteogenic conditions. Co-expression of Runx2 and Rb1 did not further increase ALP activity compared to cells transduced with Runx2 or Rb1 alone.
Alizarin red staining revealed that overexpression of either Runx2 or Rb1 increased mineral deposition in hBM-MSCs under basal conditions, although mineralisation was not enhanced above that of untransduced cells when cultured in OM. However, mineralisation was markedly enhanced above levels in untransduced cells when Runx2 and Rb1 were co-expressed in hBM-MSCs grown under both basal and osteogenic conditions.
This study demonstrates an important stimulatory role of pRb in enhancing ALP activity of hBM-MSCs in the absence of osteogenic clues. However, pRb overexpression alone is insufficient to enhance mineralisation, requiring the co-expression of Runx2 in hBM-MSCs. The crucial nature of Runx2 for osteogenic differentiation of hBM-MSCs was demonstrated since knockdown of Runx2 prevented both mineral deposition and the increased ALP activity observed in untransduced cells grown in OM. Interestingly, overexpression of Rb1 could not compensate for the knockdown of Runx2 since Rb1 overexpression did not recover either mineral deposition or ALP activity in hBM-MSCs where Runx2 expression was inhibited.
