What is beyond a qRT-PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes

Authors

  • Enrico Ragni,

    1. 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
    Search for more papers by this author
    • These authors contributed equally to this work.
  • Mariele Viganò,

    1. 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
    Search for more papers by this author
    • These authors contributed equally to this work.
  • Paolo Rebulla,

    1. 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
    Search for more papers by this author
  • Rosaria Giordano,

    1. 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
    Search for more papers by this author
  • Lorenza Lazzari

    Corresponding author
    • 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
    Search for more papers by this author

Correspondence to: Lorenza LAZZARI, 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.

Tel.: +39 02 5503 4053

Fax: +39 02 5503 2796

E-mail: lorenza.lazzari@policlinico.mi.it

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.

Ancillary