Stably expressed reference genes during differentiation ofbone marrow-derived mesenchymal stromal cells

Abstract

IntroductionMesenchymal stromal cells (MSCs) are multipotent cells that can differentiate to adipogenic, osteogenic, and chondrogenic lineages (Pittenger et al., 1999). MSCs play critical roles in coordinating tissue regeneration through their versatile differentiation capacity and immunomodulatory effects. Human MSCs can readily be isolated from different body sites (i.e. bone marrow, adipose tissue) and easily be cultured in vitro (Pittenger et al., 1999; Zuket al., 2002), making them an attractive cell source for biomedical research. Identifying the lineage commitment potential of MSCs is necessary to establish a deeper understanding of their therapeutic potential. Depending on culture conditions and their tissue of origin, MSCs have variable potency and molecular properties, including a global gene expression profile (Lee et al., 2004;Picchi et al., 2013;Wang et al., 2016).Because of its validity and sensitivity, quantitative polymerase chain reaction (qPCR) has been a technique of choice in assessing gene expression of MSCs derived from distinct body sites (Picchi et al., 2013;Wang et al., 2016). The reliability of qPCR results (i.e. target gene expression) can only be judged when optimum conditions are used. Using an accurate reference gene (RG) with a stable expression level is compulsory for normalizing target gene expression. Mostly genes that function in cellular integrity (e.g., beta-actin. ACTB) or metabolism (e.g., glyceraldehyde 3-phosphate dehydrogenase, GAPDH) are used as RGs. However, these frequently used genes have been reported to have variable expression levels under different experimental procedures that make them unsuitable to be used for normalization. Several studies showed that ACTB and GAPDH are improper RGs in MSC expansion and differentiation (

Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

“…In our specific experimental context, we use one cell type and ensure consistency in osteogenic differentiation conditions and experimental duration to specifically assess the impact of 2D vs. 3D culture conditions on reference gene expression. Previous work from Cagnan and colleagues [25] identifies RPLP0 and GAPDH as suitable reference genes during the differentiation of bone marrow-derived MSCs (both osteogenic and adipogenic). The stability of RPLP0 has also been confirmed in various studies of monolayer cultures across different MSC tissue sources and differentiation potentials [26][27][28], which is confirmed in our experimental settings.…”

Bone Marrow Mesenchymal Stem Cells Carrying FANCD2 Mutation Differ from the Other Fanconi Anemia Complementation Groups in Terms of TGF-β1 Production

“…Details of isolation and culture of BM-MSCs were described previously [27]. BM-MSCs were cultured in DMF10 medium containing 60% Dulbecco's modified Eagle's medium-low glucose (DMEM-LG; GIBCO, UK) and 40% MCDB-201 medium (Sigma-Aldrich, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBCO), 100 U/mL penicillin and 100 µg/mL streptomycin, and 2 mM L-glutamine (Biochrom AG, Germany).…”

“…Immunophenotyping of donors and patients was performed by flow cytometry using antibodies against mesenchymal and hematopoietic cell surface proteins, as described [27]. Directly labelled cell surface antibodies against CD29, CD44, CD73, HLA-ABC, CD90, CD105, CD106, CD140b, CD144, CD146, CD166, CD200, CD271 were used to detect cells from mesenchymal stromal cell origin.…”

“…BM-MSCs were cultured in adipogenic and osteogenic induction media to assess their differentiation capacity. Details of adipogenic and osteogenic differentiation protocols were described previously [27].…”

PKNOX2 expression and regulation in the bone marrow mesenchymal stem cells of Fanconi anemia patients and healthy donors

“…Details of RNA isolation, cDNA synthesis and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis were outlined previously [24,26]. cDNAs were synthesized from 260 ng RNA samples per 20 µl.…”