title: What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes.
abstract: In the last years, mesenchymal stem cells (MSCs) have been identified as an attractive cell population in regenerative medicine. In view of future therapeutic applications, the study of specific differentiation-related gene expression is a pivotal prerequisite to define the most appropriate MSC source for clinical translation. In this context, it is crucial to use stable housekeeping genes (HGs) for normalization of qRT-PCR to obtain validated and comparable results. By our knowledge, an exhaustive validation study of HGs comparing MSCs from different sources under various differentiation conditions is still missing. In this pivotal study, we compared the expression levels of 12 genes (ACTB, Β2M, EF1alpha, GAPDH, GUSB, PPIA, RPL13A, RPLP0, TBP, UBC, YWHAZ and 18S rRNA) to assess their suitability as HGs in MSCs during adipogenic, osteogenic and chondrogenic differentiation. We demonstrated that many of the most popular HGs including 18S rRNA, B2M and ACTB were inadequate for normalization, whereas TBP/YWHAZ/GUSB were frequently identified among the best performers. Moreover, we showed the dramatic effects of suboptimal HGs choice on the quantification of cell differentiation markers, thus interfering with a reliable comparison of the lineage potential properties among various MSCs. Thus, in the emerging field of regenerative medicine, the identification of the most appropriate MSC source and cell line is so crucial for the treatment of patients that being inaccurate in the first step of the stem cell characterization can bring important consequences for the patients and for the promising potential of stem cell therapy.
authors.affiliation: Cell Factory Franco Calori, Center for Transfusion Medicine, Cellular Therapy and Cryobiology, Department of Regenerative Medicine, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milano, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Ragni
authors.forename: Mariele
authors.initials: M
authors.lastname: Viganò
authors.forename: Paolo
authors.initials: P
authors.lastname: Rebulla
authors.forename: Rosaria
authors.initials: R
authors.lastname: Giordano
authors.forename: Lorenza
authors.initials: L
authors.lastname: Lazzari
mesh_terms: D048948:14-3-3 Proteins; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism*; D000273:Adipose Tissue / Q000166:cytology / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D020043:Genes, Essential; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D010011:Osteocytes / Q000166:cytology / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D035181:TATA-Box Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D015415:Biomarkers; D035181:TATA-Box Binding Protein; C509817:TBP protein, human; C572235:YWHAZ protein, human; D005966:Glucuronidase