Abstract
Adipose-derived stromal/stem cells (ASCs) are multipotent in nature that can be differentiated into various cells lineages such as adipogenic, osteogenic, and chondrogenic. The commitment of a cell to differentiate into a particular lineage is regulated by the interplay between various intracellular pathways and their resultant secretome. Similarly, the interactions of cells with the extracellular matrix (ECM) and the ECM bound growth factors instigate several signal transducing events that ultimately determine ASC differentiation. In this study, RNA-sequencing (RNA-Seq) was performed to identify the transcriptome profile of osteogenic induced ASCs to understand the associated genotype changes. Gene ontology (GO) functional annotations analysis using Database for Annotation Visualization and Integrated Discovery (DAVID) bioinformatics resources on the differentially expressed genes demonstrated the enrichment of pathways mainly associated with ECM organization and angiogenesis. We, therefore, studied the expression of genes coding for matrisome proteins (glycoproteins, collagens, proteoglycans, ECM-affiliated, regulators, and secreted factors) and ECM remodeling enzymes (MMPs, integrins, ADAMTSs) and the expression of angiogenic markers during the osteogenesis of ASCs. The upregulation of several pro-angiogenic ELR+ chemokines and other angiogenic inducers during osteogenesis indicates the potential role of the secretome from differentiating ASCs in the vascular development and its integration with the bone tissue. Furthermore, the increased expression of regulatory genes such as CTNNB1, TGBR2, JUN, FOS, GLI3, and MAPK3 involved in the WNT, TGF-β, JNK, HedgeHog and ERK1/2 pathways suggests the regulation of osteogenesis through interplay between these pathways. The RNA-Seq data was also validated by performing QPCR on selected up- and down-regulated genes (COL10A1, COL11A1, FBLN, FERMT1, FN1, FOXF1, LAMA3, LAMA4, LAMB1, IGF1, WNT10B, MMP1, MMP3, MMP16, ADAMTS6, and ADAMTS14).
Highlights
extracellular matrix (ECM)’s regulatory ability is compromised by imbalances in ECM remodeling enzymes, ECM injury, or biological aging and senescence of the cells that deposit ECM11
To assess the overall gene changes following osteogenic differentiation, three pooled donor Adipose-derived stromal/stem cells (ASCs) were induced with osteogenic differentiation media for 21 days and generation RNA sequencing was performed as described in the methods
The differentially expressed genes were subjected to gene ontology functional analysis that revealed the enrichment of ECM and angiogenesis along with ERK1/2 and JNK pathways
Summary
ECM’s regulatory ability is compromised by imbalances in ECM remodeling enzymes, ECM injury, or biological aging and senescence of the cells that deposit ECM11. Integrins are heterodimeric transmembrane receptors that act as molecular bridges between the cytoplasm and the ECM to facilitate the transmission of mechano-transductory signals stimulated by binding of ECM components to the receptor portion of integrins. This interplay between the cytoplasm and ECM via the integrins regulates proliferation, cell-migration, survival, and differentiation[14]. Based on the enriched pathways, we studied the ECM genes associated with the matrisome, ECM remodeling enzymes (MMPs, and ADAMTSs), integrins, and secretomic genes to gain insight into the regulatory roles of osteo-ECM and the secretome during osteogenesis to enhance bone formation and vascularization. The RNA-Seq data was analyzed to correlate and contrast the relationship between angiogenic and osteogenic genes and their transcription factors
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