Identification of differentiation-stage specific molecular markers for the osteoblastic phenotype

Natalie Twine, Li Chen, Marc Wilkins, Moustapha Kassem

Publikation: Konferencebidrag uden forlag/tidsskriftPosterForskningpeer review

Abstrakt

The phenotype of osteoblastic (OB) cells in culture is currently defined using a limited number of markers of low sensitivity and specificity which belong mostly to extracellular matrix proteins. Also, for clinical use of human skeletal (mesenchymal) stem cells (hMSC) in bone regeneration, there is a need to identify predictive markers for in vivo bone forming capacity.
Thus, we employed Illumina RNA sequencing (RNASeq) to examine changes in gene expression across 8 time points between 0-12 days of ex vivo OB differentiation of hMSC. We identified a subset of expressed genes as potentially sensitive markers of ex vivo differentiating OB (n=123) based on: i) identified in literature and/or had the Gene Ontology (GO) category: ‘osteoblast differentiation’ ii) exhibited significant changes in expression (> 2 Fold Change (FC), FDR <0.1) at any one time point during OB differentiation.
Pearson’s correlation of the 123 expression profiles, followed by hierarchical clustering, resulted in 4 groups of markers: early differentiating OB markers (n= 28), which had a peak expression during 0-24 hours and were enriched for GO ’extracellular matrix organisation’ e.g. COL1A1, LOX, SERPINH1; middle stage differentiating OB markers (n=20), which had a peak expression during 3-6 days and were enriched for GO ’extracellular matrix’ and ‘skeletal system development’ e.g BMP4, CYP24A1, TGFBR2; late stage differentiating OB markers (n=27), which had a peak expression 9-12 days and enriched for GO ‘bone development/osteoblast differentiation’ e.g.BMP2 ,IGF2 and a pleitropic group of marker genes (n=13) with peak expression during early and late time points that included growth factors e.g. VEGFA, PDGFA, FGF2.
In addition, a subset of these OB markers (n=21) e.g. CLEC3B, SMAD6, BMP4, NRP3, DLX5 were able to predict hMSC in vivo bone forming capacity as they were upregulated by more than 1.5 FC in hMSC-derived clones with high bone forming capacity compared to low bone forming clones. Interestingly, 33 OB markers were significantly up-regulated (>2FC, FDR<0.1) in cultured hMSC obtained from patients with osteoporosis (n=5) compared to age-matched control (n=4).
Using RNA-seq and cluster analysis, we identified a set of stage-specific molecular markers that define the progression of OB phenotype during ex vivo culture of hMSC, predict in vivo bone formation capacity of hMSC and can be employed to study the mechanisms of impaired bone formation in patients with osteoporosis.
OriginalsprogEngelsk
Publikationsdato7. okt. 2013
Antal sider1
StatusUdgivet - 7. okt. 2013
BegivenhedASBMR - Baltimore Convention Centre, Baltimore, USA
Varighed: 4. okt. 20137. okt. 2013

Konference

KonferenceASBMR
LokationBaltimore Convention Centre
LandUSA
ByBaltimore
Periode04/10/201307/10/2013

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