Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity during in vitro Expansion

Guniz Bas,Kali Woods,Julia T. Oxford,Gunes Uzer,Stacie Loisate,Eric J. Hayden,Stephanie F. Hudon,Richard Beard,Xinzhu Pu

doi:10.1038/s41598-020-66055-0

Abstract

A primary component of exercise, mechanical signals, when applied in the form of low intensity vibration (LIV), increases mesenchymal stem cell (MSC) osteogenesis and proliferation. While it is generally accepted that exercise effectively combats the deleterious effects of aging in the musculoskeletal system, how long-term exercise affects stem cell aging, which is typified by reduced proliferative and differentiative capacity, is not well explored. As a first step in understanding the effect of long-term application of mechanical signals on stem cell function, we investigated the effect of LIV during in vitro expansion of MSCs. Primary MSCs were subjected to either a control or to a twice-daily LIV regimen for up to sixty cell passages (P60) under in vitro cell expansion conditions. LIV effects were assessed at both early passage (EP) and late passage (LP). At the end of the experiment, P60 cultures exposed to LIV maintained a 28% increase of cell doubling and a 39% reduction in senescence-associated β-galactosidase activity (p < 0.01) but no changes in telomere lengths and p16INK4a levels were observed. Prolonged culture-associated decreases in osteogenic and adipogenic capacity were partially protected by LIV in both EP and LP groups (p < 0.05). Mass spectroscopy of late passage MSC indicated a synergistic decrease of actin and microtubule cytoskeleton-associated proteins in both control and LIV groups while LIV induced a recovery of proteins associated with oxidative reductase activity. In summary, our findings show that the application of long-term mechanical challenge (+LIV) during in vitro expansion of MSCs for sixty passages significantly alters MSC proliferation, differentiation and structure. This suggests LIV as a potential tool to investigate the role of physical activity during aging.

Highlights

The aging phenotype of mesenchymal stem cell (MSC) is associated with decreased proliferative and differentiative capacity[7,8], which delays healing and impairs the repair of wear and tear in musculoskeletal tissues
The cell area of the late passage (LP) group was 5.97×104 px[2] and was not different than the P6 group while remained 51% and 41% larger compared to both the early passage (EP) and EP + low-intensity vibration (LIV) groups, respectively (p < 0.0001)
As DAVID analysis identified changes in the focal adhesions, actin binding and stress fibers pathways, we further investigated the changes in the cytoskeleton related proteins in both the LP and LP + LIV groups, we used P6 MSCs as a baseline and only reported significantly changed proteins in either LP or LP + LIV MSCs (p < 0.05)

Summary

Introduction

The aging phenotype of MSCs is associated with decreased proliferative and differentiative capacity[7,8], which delays healing and impairs the repair of wear and tear in musculoskeletal tissues. Another study investigated the effect of a 4-month-long daily ladder climbing regimen, started at 17-months of age, on MSCs isolated from rats: compared to adult MSCs from 5-month-old rats, aged MSCs showed decreased osteogenesis which was partially restored by 4-month-long exercise intervention[13]. While these studies highlight the benefits of exercise at the level of MSC, mechanical signals that are produced during exercise are accompanied by changes in cardiorespiratory fitness and caloric expenditure at a systemic level that may confound these outcomes[14,15]. Changes in MSC proliferation, differentiation, senescence markers, mechanosensitivity and proteome were compared using immunofluorescence, qPCR, western blotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Fig. 1b)

Methods

Results

Conclusion