Prolonged Strenuous Exercise Expands the Population of Developmentally Early Stem Cells in Bone Marrow (BM) and Mobilizes Them Into Peripheral Blood - Novel Evidence That Strongly Supports a Positive Effect of Physical Activity On Extension of Life Span At the Level of Stem Cells
Authors: Tarnowski Maciej , Piotrowska Katarzyna , Grymula Katarzyna , Suszynska Malwina , Poniewierska Agata , Ratajczak Mariusz Z 
Published: October 2019

Abstract
Abstract 584

Background.
Bone marrow (BM) contains a variety of stem cells, including hematopoietic stem/progenitor cells (HSPCs), endothelial progenitor cells (EPCs), mesenchymal stem cells (MSCs), and the developmentally most primitive very small embryonic-like stem cells (VSELs). Evidence has accumulated that HSPCs (Circ J. 2008;72:897) and EPCs (J Am Coll Cardiol. 2004;43:2314) are mobilized into PB during strenuous exercise. Therefore, we became interested in whether a pool of bone marrow (BM)-residing developmentally more primitive, highly quiescent, early stem cells (VSELs) would respond to stimuli related to prolonged, strenuous exercise. As we have demonstrated previously, these cells play an important role in tissue/organ rejuvenation and their number positively correlates with life span in experimental animals (Age 2012 in press doi:10.1007/s11357-011-9364-8).

Hypothesis.
We hypothesized that the positive effect of physical activity may be explained by the expansion of a developmentally early pool of highly quiescent stem cells that play a role in tissue/organ rejuvenation.

Experimental strategies.
To test the effect of exercise on the BM pool of stem cells, we employed C57Bl6 mice that were exposed to short (1 day), mid-term (14 days), and long- term (6 months) strenuous exercise in rotating wheels. These mice were subsequently sacrificed 1h after removal from the rotating wheels, and we i) measured changes in peripheral blood cell counts, ii) enumerated the number of VSELs and HSPCs both in BM and PB of sacrificed animals by FACS analysis, iii) performed clonogeneic in vitro methylocellulose assays to enumerate the number of CFU-GM and BFU-E mobilized into PB and the number of these cells residing in the BM microenvironment, iv) employed the in vivo bromodeoxyuridine (BrdU) incorporation assay to evaluate the number of VSELs and HSPCs in BM undergoing cell division, and finally v) performed a molecular analysis of the expansion and mobilization of VSELS by measuring the expression of genes regulating stem cell pluripotency at the mRNA (RQ-PCR) and protein levels (immunofluorescence of cells fixed on cytospin slides).

Results.
Our data confirmed that strenuous exercise mobilizes Sca-1+Lin–CD45+ HSPCs into PB, and, for the first time, we show that prolonged, enforced exercise of mice in rotating wheels is associated with expansion in BM and mobilization into PB of the most primitive population of stem cells, Sca-1+Lin–CD45– VSELs. The expansion of VSELs in BM and their mobilization into PB was confirmed by FACS analysis, immunohistochemical staining, and RQ-PCR analysis for expression of genes that regulate pluripotency (e.g., Oct-4 and Nanog) in small Sca-1+Lin–CD45–cells sorted from BM and PB. The proliferation of VSELs, which are a quiescent population of BM-residing stem cells, has been confirmed by BrdU incorporation. In contrast to VSEL expansion, we did not observe significant changes in the number of BM-residing HSPCs. As expected, mice exposed to prolonged exercise exhibited a significant increase in skeletal muscle and a decrease in abdominal fat.

Conclusions.
BM-residing stem cells respond to prolonged strenuous exercise by expansion and subsequent mobilization into PB of BM-residing VSELs, which, as proposed by us and others, may play an important role in tissue/organ rejuvenation and thus positively affect life span. Therefore, our data for the first time demonstrates a positive correlation between exercise and the expansion of a most primitive pool of stem cells in BM.
This work is supported by the EU Innovative Economy Operational Program POIG.01.01.02-00-109/09-01

Disclosures:
Ratajczak: Neostem Inc: Member of SAB Other.