Dataset Information

Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation.

ABSTRACT: BACKGROUND: The potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors that trigger osteoblast differentiation in MSCs is therefore critical to promote the osteogenic potential of human MSCs. In this study, we used microarray analysis to identify signalling molecules that promote osteogenic differentiation in human bone marrow stroma derived MSCs. RESULTS: Microarray analysis and validation experiments showed that the expression of IGF2 and IGFBP2 was increased together with integrin alpha5 (ITGA5) during dexamethasone-induced osteoblast differentiation in human MSCs. This effect was functional since we found that IGF2 and IGFBP2 enhanced the expression of osteoblast phenotypic markers and in vitro osteogenic capacity of hMSCs. Interestingly, we showed that downregulation of endogenous ITGA5 using specific shRNA decreased IGF2 and IGFBP2 expression in hMSCs. Conversely, ITGA5 overexpression upregulated IGF2 and IGFBP2 expression in hMSCs, which indicates tight crosstalks between these molecules. Consistent with this concept, activation of endogenous ITGA5 using a specific antibody that primes the integrin, or a peptide that specifically activates ITGA5 increased IGF2 and IGFBP2 expression in hMSCs. Finally, we showed that pharmacological inhibition of FAK/ERK1/2-MAPKs or PI3K signalling pathways that are enhanced by ITGA5 activation, blunted IGF2 and IGFBP2 expression in hMSCs. CONCLUSION: The results show that ITGA5 is a key mediator of IGF2 and IGFBP2 expression that promotes osteoblast differentiation in human MSCs, and reveal that crosstalks between ITGA5 and IGF2/IGFBP2 signalling are important mechanisms that trigger osteogenic differentiation in human bone marrow derived mesenchymal stromal cells.

SUBMITTER: Hamidouche Z

PROVIDER: S-EPMC2901205 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation.

Hamidouche Zahia Z Fromigué Olivia O Ringe Jochen J Häupl Thomas T Marie Pierre J PJ

BMC cell biology 20100623

<h4>Background</h4>The potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors that trigger osteoblast differentiation in MSCs is therefore critical to promote the osteogenic potential of human MSCs. In this study, we used microarray analysis to identify signalling molecules that promote osteogenic differentiation in human bone marrow stroma derived MSCs.<h4>Results</h4>Micro ...[more]

PMID: 20573191

Similar Datasets

Project description:Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red stain. Collectively, these results demonstrate a great potential of PRC alone in inducing proliferation of hMSCs without any influence from other lineage-specific growth media. PRC alone has similar capacity to enhance hMSC osteogenic differentiation as a standard OM, without changing the temporal profile of the differentiation process. Thus, PRC could be used as a substitute medium to provide sufficient pool of pre-differentiated hMSCs for potential clinical application in bone regeneration.

Project description:Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) +5 ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB + FGFlow)] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB +5 ng/ml FGF-2 [ACK(AB + FGFlow)] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB +5 ng/ml FGF2 [Lympho(AB + FGFlow)] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL) + heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB + FGFlow) or 10% AB +10 ng/ml FGF2 [Direct(AB + FGFhigh)] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB + FGFhigh) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB + FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB + FGFhigh) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1α and IL-1β expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB + FGFhigh) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.

Dataset Information

Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation.

Publications

Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets