PPIA and YWHAZ Constitute a Stable Pair of Reference Genes during Electrical Stimulation in Mesenchymal Stem Cells

Lynsey Steel,David M Ansell,Sarah H Cartmell,Enrique Amaya

doi:10.3390/app12010153

Lynsey Steel, David M Ansell + Show 2 more

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https://doi.org/10.3390/app12010153

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Journal: Applied sciences	Publication Date: Dec 24, 2021
Citations: 1	License type: CC BY 4.0

Affiliation: Henry Royce Institute, University of Manchester

Abstract

Mesenchymal stem cells (MSCs) are multipotent adult stem cells with great potential in regenerative medicine. One method for stimulating proliferation and differentiation of MSCs is via electrical stimulation (ES). A valuable approach for evaluating the response of MSCs to ES is to assess changes in gene expression, relative to one or more reference genes. In a survey of 25 publications that used ES on cells, 70% selected GAPDH as the reference gene. We conducted a study to assess the suitability of six potential reference genes on an immortalized human MSC line following direct current ES at seeding densities of 5000 and 10,000 cells/cm2. We employed three methods to validate the most stable reference genes from qRT-PCR data. Our findings show that GAPDH and ACTB exhibit reduced stability when seeded at 5000 cell/cm2. In contrast, we found that the most stable genes across both plating densities and stimulation regimes were PPIA and YWHAZ. Thus, in ES gene expression studies in MSCs, we support the use of PPIA and YWHAZ as an optimal reference gene pair, and discourage the use of ACTB and GAPDH at lower seeding densities. However, it is strongly recommended that similar verification studies are carried out based on cell type and different ES conditions.

Highlights

In the field of regenerative medicine, changes in gene expression are often studied to provide evidence that stem cells are undergoing the process of differentiation
Analysis of Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) data using the ∆∆CT method requires a comparison to be made between the detection level of a gene of interest relative to one or more reference genes, commonly referred to as housekeeping genes, that remain stable during the experimental procedure [2]
A survey was undertaken of 25 published journal articles showing gene expression data in electrically stimulated cells, and 70% of these used Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the reference gene, with the second most popular (12%) being ACTB (Supplementary Table S1)

Summary

Introduction

In the field of regenerative medicine, changes in gene expression are often studied to provide evidence that stem cells are undergoing the process of differentiation. Of the techniques used to gather these data, one of the most common is real-time quantitative reverse transcription polymerase chain reaction, qRT-PCR [1]. Analysis of qRT-PCR data using the ∆∆CT method requires a comparison to be made between the detection level of a gene of interest relative to one or more reference genes, commonly referred to as housekeeping genes, that remain stable during the experimental procedure [2]. Mesenchymal stem cells (MSCs) are of particular interest to the research community due to their immunomodulatory characteristics and their multipotency, which enables differentiation down osteogenic, chondrogenic, adipogenic, and neurogenic lineages [3,4]. A promising method for solving this could be the application of electrical stimulation (ES) to cells in vitro or in vivo, which has been shown to stimulate cell proliferation [6,7,8,9], initiate cell migration [10,11,12,13] and accelerate differentiation [14,15,16,17]

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