The Expression Pattern of Surface Markers in Canine Adipose-Derived Mesenchymal Stem Cells.

Nina Krešić,Andrija Musulin,Ivana Ljolje,Dragan Brnić,Boris Habrun,Marina Prišlin,Nenad Turk,Dunja Vlahović,Petar Kostešić

doi:10.3390/ijms22147476

Abstract

The influence of cultivation on the expression pattern of canine adipose-derived mesenchymal stem cells (cAD-MSCs) surface markers, contributing to, among others, the promotion of growth, proliferation, differentiation and immunomodulatory mechanisms of an excellent therapeutic, is still unknown. To fill the gap, we investigated CD90, CD44, CD73, CD29, CD271, CD105, CD45 and CD14 patterns of expression at the protein level with flow cytometry and mRNA level using a real-time polymerase chain reaction array. Gentle variations of expression occurred during cultivation, along with increased CD90, CD44 and CD29 expression, low and decreasing CD271 and CD73 expression and a decrease of initially high CD105. As expected, CD45 and CD14 were not expressed by cAD-MSCs. Interestingly, we discovered a significant decrease of CD73 expression, compared to early (P1–P3) to late (P4–P6) passages, although the CD73 gene expression was found to be stable. The percentage of positive cells was found to be higher for all positive markers up to P4. As CD73′s one important feature is a modulation from a pro-inflammatory environment to an anti-inflammatory milieu, the expression of CD73 in our conditions indicate the need to consider the time cells spend in vitro before being transplanted into patients, since it could impact their favourable therapeutical properties.

Highlights

The International Society for Cellular Therapy (ISCT) established minimum criteria for the definition of MSCs: adherence to tissue culture plastic; multipotency as demonstrated by in vitro differentiation into osteoblasts, adipocytes and chondroblasts; expression of surface markers CD73, CD90 and CD105 and negative for CD34, CD45, CD14 or CD11b, C79α or CD19 and HLA-DR [1]
The influence of cultivation on morphology, phenotype and gene expression changes is the subject of investigation in human MSCs [2,24,25]
The role of canines as companion animals is on the rise, and research on cAD-MSCs is important as it will provide basic knowledge about cAD-MSCs properties

Summary

Introduction

The International Society for Cellular Therapy (ISCT) established minimum criteria for the definition of MSCs: adherence to tissue culture plastic; multipotency as demonstrated by in vitro differentiation into osteoblasts, adipocytes and chondroblasts; expression of surface markers CD73, CD90 and CD105 and negative for CD34, CD45, CD14 or CD11b, C79α or CD19 and HLA-DR [1]. Many studies demonstrated that cells meeting the ISCT criteria possessed heterogeneous phenotypes and functionalities, heavily influenced by culture conditions [2]. The goal of successful expansion in vitro is to maintain stem cell phenotype and show repeated self-renewal, i.e., undergo extensive proliferation while maintaining their multipotent differentiation potentials [3]. MSCs properties, leading to discrepancies at the basic research level and adding biases to the evaluation of stem cell-based clinical study outcomes [2]. Further intensive research on the cell properties and application are certainly needed

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