Abstract
Long-term expansion of mesenchymal stem cells (MSCs) under defined culture conditions is necessary in human stem cell therapy. However, it alters the characteristics of MSCs. Since quantitative real time polymerase chain reaction (qRT-PCR) is widely used as one of the key analytical methods for comparative characterization, the validation of reference genes (RGs) for normalization under each experimental condition is important to achieve reliable qRT-PCR results. Therefore, the most stable RGs for long-term expanded bone marrow- and umbilical cord blood-derived MSCs (BM-MSCs and UCB-MSCs) under defined culture conditions for up to 20 passages were evaluated. The more apparent alterations in characteristics such as differentiation capacity, proliferation, senescence, and the expression of RGs were noted in BM-MSCs than UCB-MSCs during long-term expansion. The RG validation programs (GeNorm and NormFinder) suggested that PPIA, HPRT1, and YWHAZ were suitable for normalization in qRT-PCR regardless of MSC types and long-term culture expansion, and the traditional RGs (ACTB and GAPDH) were less stable in long-term expanded MSCs. In addition, the use of these RGs for normalization of OCT4 expression in long-term expanded BM-MSCs showed that a less stable RG (GAPDH) showed contrasting data compared to other RGs. Therefore, the use of RGs such as PPIA, HPRT1, and YWHAZ for normalization in qRT-PCR experiments is highly recommended for long-term expanded MSCs to generate accurate and reliable data.
Highlights
Since gene expression studies have enabled us to understand the gene-regulatory network of cellular mechanisms, the quantitative analysis of gene expression is essential in cellular biology [1]
In quantitative real time polymerase chain reaction (qRT-PCR), the normalization of genes of interest (GOI) against a reference gene (RG) is imperative to investigate the relative quantification of gene expression, since it is believed that reference genes (RGs) are stably and constantly expressed in cells regardless of experimental conditions [4,5,6]
In consistent with the results obtained from geNorm, NormFinder showed that YWHAZ, Hypoxanthine phosphoribosyltransferase 1 (HPRT1), and PPIA were the most stable RGs during long-term expansion of BM-mesenchymal stem cells (MSCs) and UCB-MSCs while traditional RGs were less stably expressed
Summary
Since gene expression studies have enabled us to understand the gene-regulatory network of cellular mechanisms, the quantitative analysis of gene expression is essential in cellular biology [1]. Mesenchymal stem cells (MSCs) are being highlighted as promising candidates for stem cell therapy and have made a significant impact in the field of regenerative medicine due BioMed Research International to their self-renewal property, wide accessibility, multilineage differentiation potential, and immunomodulation properties [12]. In this context, bone marrow-derived MSCs (BMMSCs) have been extensively studied in the past decades. The present study aims to investigate the most stable RGs to provide an accurate evaluation of relative mRNA fold change of GOI in longterm expanded human MSCs (BM-MSCs and UCB-MSCs) under defined culture conditions. Ten commonly used RGs (18S, B2M, EEF1A1, GAPDH, HPRT1, PPIA, RPLP0, TBP, ACTB, and YWHAZ) were studied and compared to determine the appropriate RGs for long-term expanded BMMSCs and UCB-MSCs under defined culture conditions through widely used RG validation programs: GeNorm and NormFinder
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