Ribosomal Protein L13a as a Reference Gene for Human Bone Marrow-Derived Mesenchymal Stromal Cells During Expansion, Adipo-, Chondro-, and Osteogenesis

Abstract

In the field of human mesenchymal stromal cell (MSC) research, quantitative real-time reverse transcription-polymerase chain reaction (qPCR) is the method of choice to study changes in gene expression patterns upon differentiation, application of stimuli, or of factors such as inhibitors or siRNAs. To reliably detect small changes, the use of a reference gene (RG) that is stably expressed under all conditions is essential. The large number of different RGs used in the field and the lack of validation of their suitability make the comparison between studies impossible. Therefore, this work aims to establish one single RG for mesodermal differentiation studies that use MSCs. Seven commonly used RGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal protein L13a [RPL13a], beta-actin [ACTB], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide [YWHAZ], eukaryotic translational elongation factor 1 alpha [EF1α], β2-microglobulin [B2M], and 18S ribosomal RNA [18S]) were investigated concerning their mRNA expression stability during expansion of bone marrow-derived MSCs up to four passages as well as during their adipo-, chondro-, and osteogenenic differentiation on days 9, 16, and 22 after induction. RPL13a was validated for qPCR studies of MSCs (bone marrow- and placenta-derived) and, additionally, for primary human bone cells (HBCs) and the osteosarcoma cell line MG-63. GAPDH and ACTB, the two most frequently used RGs, showed the highest expression variance. The superior performance of RPL13a should make it the RG of choice for all MSC studies addressing mesodermal differentiation.

Osteogenic differentiation of human mesenchymal stem cells in the absence of osteogenic supplements: A surface-roughness gradient study

“…Femur-derived bone marrow was used to isolate the mesenchymal stem cells used in this work, as previously described [51]. The human samples were collected, following informed written consent, from three independent donors (78-82 years old) with the approval from the ethics committee of the Kantonspital St. Gallen, Switzerland (EKSG 08/14).…”

GFOGER-Modified MMP-Sensitive Polyethylene Glycol Hydrogels Induce Chondrogenic Differentiation of Human Mesenchymal Stem Cells

“…Ribosomal protein L13 (RPL13) was used as a housekeeping gene as it has been recently demonstrated to be more stable than GAPDH. 44 The Livak method was used for analysis of quantitative real-time PCR data. 45 The primers used in this study are listed in Table 1.…”

Regional expression of the BCRP/ABCG2 transporter in term human placentas

“…The present study confirms minimal intraplacental variability in expression of these two genes. RPL13a was recently proposed as a superior reference gene in bone-marrow and placental-derived human mesenchymal stromal cells [26]. Although there is no literature on the use RPL13a as an endogenous control in placental tissue, our results demonstrate minimal site-to-site variability in its expression.…”