Reference Gene Expression in Adipose-Derived Stromal Cells Undergoing Adipogenic Differentiation.

Carla Dessels,Michael Sean Pepper

doi:10.1089/ten.tec.2019.0076

Abstract

Adipose-derived stromal cells (ASCs) are becoming increasingly attractive as cellular therapy products. Their differentiation potential, the secretion of growth and differentiation factors, and the ability to cryopreserve the cells over extended periods are important features. Changes in experimental conditions result in changes in gene expression, and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) has become an important tool for measuring these changes. There is, however, the potential to introduce technical bias in the process, which can be diminished through the selection of stable reference genes (RGs). Using geNorm software, in this in vitro study we explore the effects that adipogenic differentiation for a 21 day induction period, cryopreservation (freshly isolated ASCs or previously cryopreserved/frozen ASCs), and culture medium supplementation (fetal bovine serum vs. pooled human platelet lysate) have on the stability of 11 RGs. We found that RG stability is markedly affected by the different experimental conditions. Of the RGs assessed, YWHAZ, HPRT, TBP, and ACTB were stably expressed genes under all experimental conditions. We recommend that a panel of stable RGs should be selected before studying gene expression during adipogenesis, and that this is based on the experimental condition(s) being investigated.Impact StatementAs the use of adipose-derived stromal cells (ASCs) in clinical trials increases, so does the amount of experimental data from research groups, many of which use human ASCs to study adipogenesis in obesity. Different conditions are constantly being applied to ASCs in vitro, to obtain a therapeutic product for potential downstream applications. Few articles have looked at the effect of different conditions on ASC reference gene (RG) expression and stability, which was the aim of this research, as such this article will assist other researchers to make an informed decision about RG selection for gene expression studies using ASCs including those for adipogenesis.

Highlights

Adipose-derived stromal cells (ASCs) are being assessed for their therapeutic potential in clinical trials.[1,2,3] To be successful in adipose-derived stromal cells (ASCs)’ downstream applications, production needs to adhere to good manufacturing processes (GMPs).[4]
Few articles have looked at the effect of different conditions on ASC reference gene (RG) expression and stability, which was the aim of this research, as such this article will assist other researchers to make an informed decision about RG selection for gene expression studies using ASCs including those for adipogenesis
The least variable Cq values were seen for RPLP0 (0.82) and the most variable Cq values were seen for PPIA (2.19)

Summary

Introduction

Adipose-derived stromal cells (ASCs) are being assessed for their therapeutic potential in clinical trials.[1,2,3] To be successful in ASCs’ downstream applications, production needs to adhere to good manufacturing processes (GMPs).[4] The latter can be achieved by replacing animal and chemical products with xeno-free and clinical-grade alternatives.[5] Altering experimental conditions could alter the ASC product and this will be reflected by changes in gene expression.[6,7,8,9] Attractive features of ASCs include the fact that they can be cryopreserved and stored for prolonged periods of time with minimal loss of their characteristics[10,11,12]; they remain undifferentiated during expansion[13]; and they have the potential to form adipocytes, chondrocytes, and osteocytes.[14,15] Reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is the preferred method for measuring gene expression during adipogenesis,[15,16,17] and as such requires the selection and validation of a panel of internal controls or reference genes (RGs) for normalization

Methods

Results

Discussion

Conclusion