Interferon-γ and Hypoxia Priming Have Limited Effect on the miRNA Landscape of Human Mesenchymal Stromal Cells-Derived Extracellular Vesicles.

Juliette Peltzer,Sébastien Banzet,Marion Grosbot,Marie-Emmanuelle Goriot,Philippe Mauduit,Kyle Lund,Jean-Jacques Lataillade

doi:10.3389/fcell.2020.581436

Abstract

Mesenchymal stromal cell (MSC)-based cell therapy has received great interest in regenerative medicine. Priming the cells during the culture phase can improve their efficacy and/or survival after injection. The literature suggests that MSC extracellular vesicles (EV) can recapitulate a substantial part of the beneficial effects of the cells they originate from, and that micro-RNAs (miRNAs) are important players in EV biological action. Here, our aim was to determine if two classical priming methods of MSC, interferon-gamma (IFNγ) and hypoxia (HYP), could modify their EV miRNA content. Human bone marrow MSCs (BM-MSCs) from five healthy donors were cultured with IFNγ or in HYP or in control (CONT) conditions. The conditioned media were collected after 48 h in serum-free condition and EV were isolated by ultracentrifugation. Total RNA was isolated, pools of CONT, IFN, and HYP cDNA were prepared, and a miRNA profiling was performed using RT-qPCR. Then, miRNAs were selected based on their detectability and measured on each individual EV sample. Priming had no effect on EV amount or size distribution. A set of 81 miRNAs was detected in at least one of the pools of EVs. They were measured on each individual sample; 41 miRNAs were detected in all samples. The principal component analysis (PCA) failed to discriminate the groups. HYP induced a significant decrease in EV hsa-miR-34a-3p content and IFN induced a significant increase in five miRNAs (hsa-miR-25-3p, hsa-miR-106a-5p, hsa-miR-126-3p, hsa-miR-451a, and hsa-miR-665). Taken together, we found only limited alterations in the miRNA landscape of MSC EV with a high inter-individual variability.

Highlights

Mesenchymal stromal cells (MSCs) are adult, non-hematopoietic cells first identified in the bone marrow
The second priming by IFNγ was carried out under the following condition: MSCs were seeded at 4000 cell/cm2 in growth culture medium at T◦= 37◦C, O2 = 20%, humidity = 95% until they reach 70– 80% of confluence, the cells were washed with phosphate buffered saline (PBS), placed in MEMα without serum, and supplemented with 25 ng/ml of IFNγ for 48 h
To validate our extracellular vesicles (EV) isolation protocol, we performed a proteomic analysis of the control MSC-EV preparation

Summary

Introduction

Mesenchymal stromal cells (MSCs) are adult, non-hematopoietic cells first identified in the bone marrow. They are found in numerous other tissues such as adipose tissue, muscle, gingiva, as well as in perinatal tissues (umbilical cord or placental membranes). 2013; Galipeau and Sensébé, 2018). They were first thought to differentiate in other cell types, it is widely accepted that, except for bone, most of their beneficial effects are due to their paracrine function. EV contain proteins, mRNAs, micro-RNAs (miRNAs) that reproduce a substantial part of the biological effects of the cells they originate from. EV have been proposed as a surrogate for MSC therapy, easier to produce and store, and immediately available

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