Abstract
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) enables the monitoring of changes in cell phenotype via the high-throughput screening of numerous genes. RT-qPCR is a fundamental approach in numerous research fields, including biomaterials, yet little attention has been given to the potential impact of 3D versus monolayer (2D) cell culture and to the requirement for a constant validation of the multiple steps of gene expression analysis. The aim of this study is to use high-quality RNA to identify the most suitable reference genes for RT-qPCR analysis during the osteogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs). BM-MSCs are cultured under osteogenic conditions for 28 days in 2D or within hyaluronic acid hydrogels (3D). RNA is subject to quality controls and is then used to identify the most stable reference genes using geNorm, NormFinder, and the ∆Cq method. The effect of the reverse transcriptase is investigated, as well as the expression of osteogenic-related markers. This study shows marked differences in the stability of reference genes between 2D (RPLP0/GAPDH) and 3D (OAZ1/PPIA) culture, suggesting that it is critical to choose appropriate reference genes for 3D osteogenic cell cultures. Thus, a thorough validation under specific experimental settings is essential to obtain meaningful gene expression results.
Highlights
Bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) are certainly among the most studied cell type in the musculoskeletal research field [1]
We have demonstrated that the 3D osteogenic culture of human BM-MSCs within methacrylated HA (MeHA) hydrogels requires an improved RNA extraction method and the use of difffferent sets of reference geness ccoommppaarreeddttooccoonnvveennttiioonnaallmmoonnoolalayyeer/r2/2DDoostsetoeoggeneniciccucultlutruerse.sT
Accurate gene expression analysis can only be performed following the validation of a broad range of reference genes involved in different aspects of cellular activities to minimize the effect of variation induced by the experimental conditions [10]
Summary
Bone marrow-derived mesenchymal stem/stromal cells (BM-MSCs) are certainly among the most studied cell type in the musculoskeletal research field [1]. Their multilineage potential is the continuous object of various investigations, as it suggests the possibility of cell-based therapies to regenerate tissues such as bone and cartilage. The osteogenic differentiation of BM-MSCs was described for the first time in monolayer culture by Jaiswal and colleagues in the late 1990s [2]. Monolayer cell cultures are a simple method to gain a first perception of the mechanisms underlying cell differentiation, but they lack the three-dimensional characteristic of tissues. Tissue engineering makes use of natural and synthetic materials to more faithfully recreate the 3D microenvironment and increases the complexity of in vitro culture systems
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