Exercise and Stem Cells - Part II, Haematopoietic Stem, Endothelial Progenitor, and Mesenchymal Stem

INTRODUCTION

In today’s post in the “Exercise and Stem Cells” mini-series, we will be focusing upon work by Boppart, De Lisio, and Witkowski published in 2015.(1) I would like to start by thanking Dr Marni Boppart (Associate Professor, Department of Kinesiology and Community Health, University of Illinois, Urbana; full-time faculty, Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign) for kindly having shared this important manuscript with me. Many of the concepts that we will discuss in future weeks flow from this manuscript. Also, I would like to again thank Dylan Davies for his excellent photography!

As we saw in last week’s blog, there are differences between acute exercise and exercise training in general and in their respective effects on stem/progenitor cells. “Exercise can dramatically alter strain sensing, extracellular matrix composition, and inflammation, and such changes in the niche likely alter ASC [adult stem cell] quantity and function postexercise” (p. 423). Regarding adult stem cells (ASCs), we will take the definition to be what Drs. Melton and Cowen offered - ASCs “are a single cell (clonal) that self-renews and generates differentiated cells.”(2, p. xxvii) ASCs must be able to make an exact copy (a clone) and also asymmetric division (makes a different type of cell). We will avoid any lengthy discussions of differentiation potential (i.e., totipotent, pluripotent, multipotent, unipotent) because of the contentiousness of the discussion as well as the fact that it would be a distraction from the topic of focus today.

We now know that there are multiple tissues, and perhaps all tissues, of adult organisms that have resident stem/progenitor cells. The cells listed in the title of this blog, haematopoietic stem cells and medicinal signaling cells (HSCs and MSCs, respectively) are found in bone marrow, blood, and multiple tissues throughout the body. The endothelial progenitor cells (EPCs) are circulating cells with angiogenic potential first described in 1997 by Asahara et al.(3) There can be difficulties in distinguishing between EPCs and HSCs because there seems to be overlap and because we have no know cellular surface markers that are completely unique to stem cells only.

Last week we focused on haematopoietic stem and progenitor cells (HSPCs), which could also include EPCs. While we will not be repeating the blog from last week, this week we focus upon as much delineation as possible between HSCs and EPCs with regard to the effects of exercise (acute and training). Additionally, in this post, we begin discussion on the effects of exercise (acute and training) on MSCs.

BACKGROUND

A. Haematopoietic Stem Cells

Acute Exercise

We discussed HSCPs in last week’s blog post, today we continue to focus upon HSPCs and work to separate out effects of exercise on EPCs from their discussion. Boppart and colleagues point out that the manuscript under discussion in last week’s blog (De Lisio and Parise, 2013) had recently been published, but that Boppart et al updates the information specifically with regard to HSCs defined by colony forming units (CFUs), as well as phenotypic markers LSK, CD34, CD34+/CD38-, or side population (SP) “as well as speculate on their physiological function following exercise-stimulated mobilization. Due to the heterogeneity of HSC populations, these cells will be described as hematopoietic stem/progenitor cells (HSPCs)” (p. 427). Therefore, it seems most appropriate to have the manuscript by Boppart and colleagues serve as the foundation for this blog post as it further elaborates on discussions from last week and lays the foundation for topics the will be discussed in future blog posts.

An increase in the HSPC quantity in peripheral blood following a bout of acute exercise was first published in 1978 by Barret et al.(4) This early study brought about much discussion and excitement; however, further publications found that the rise in HSPCs in peripheral blood from an acute bout of exercise “was increased immediately following cessation of exercise, but returned to baseline after 15 min” (p. 428). Additional work for that following the sudden increase in HSPC quantity in peripheral blood, there is a rapid decline through a 60 minute period following conclusion of the bout of acute exercise. This window was considered to be too short to be of clinical utility for bone marrow transplantation because aphaeresis for HSPC harvesting can take several hours. More importantly, as we saw last week, it took maximal exercise to very transiently raise the HSPC quantity in peripheral blood and that submaximal exercise lasting an hour did not raise HSPC quantity. Acute exercise, even in maximal intensity (confirmed in several subsequent studies), led to an extremely brief increase in HSPCs. The primitive HSPC population (CD34+CD38- cells) have been investigated in only a few studies in acute exercise; however, it was revealed that these cells are not impacted by acute exercise. HSPCs in low-intensity exercise (defined as cycling at 70% of VO2max for 4 hours), as we saw last week, are not changed much and any change occurred only after 3 hours into the exercise and quickly returned to baseline directly after exercise was halted. The transient mobilization increase is “consistent between both men and women and young and elderly populations” (p. 429). Hence we must continue to investigate exercise training rather than simply acute exercise to see if training could be of benefit in cellular therapies used in the practice of regenerative medicine.

Exercise Training

In a study on young, trained, marathon runners, a three- to fourfold increase in circulating CD34+ cells was found compared to sedentary individuals; however, the increase in these cells in the periphery was not related to volume of training. As we discussed last week, the intensity of exercise is a crucial element to be considered rather than simply the length of the exercise. More studies exist utilizing animal models rather than humans. “Overall, these animal data suggest that a well-designed training program under controlled conditions can increase the quantity of HSPCs both with the bone marrow and in the extramedullary sites such as peripheral blood and spleen” (p. 431).

B. Endothelial Progenitor Cells

With regard to endothelial progenitor cells (EPCs), there are three types of in vitro assays used to isolate them and three distinct EPC populations observed - (a) endothelial colony-forming cells (ECFCs or “late” EPCs), (b) circulating angiogenic cells (CACs or “early” EPCs), and (c) colony-forming unit-Hill (CFU-Hill or CFU-CACs). “Exercise can restore endothelial function and decrease CVD [cardiovascular disease] risk…. Increased EPC appearance in circulation and EPC functional improvements via exercise are hypothesized to constitute novel mechanisms by which physical activity promotes cardiovascular health” (p. 434). Because of the various EPC populations (and that some may/may not have HSCs in them), Boppart and colleagues refer to CACs (as opposed to EPCs) to cover the heterogeneous cell populations.

Acute Exercise

In general, acute exercise does increase CACs in the peripheral blood of healthy volunteers, patients with risk for CVD, and in those with established CVD. However, the acute response to acute exercise is variable based upon CAC-subtype, exercise intensity, exercise duration, type of exercise, and disease status, consistent with what has been discussed previously regarding HSPC quantity and exercise. Moreover, the various populations of CACs as well as the differences in exercise may also explain much of the variation that exists in data on acute exercise.

Further information was revealed on the pathways active in mobilizing CACs that has helped further develop our understanding of acute exercise. “Endothelial nitric oxide synthase (eNOS) is the enzyme responsible for the production of the vasodilator nitric oxide (NO) by endothelial cells” (p. 438). Increased shear stress from increased blood flow due to exercise leads endothelial cells to release NO leading to both vasodilation and smooth muscle cell relaxation. As such, eNOS is a critical regulator of CAC mobilization. In fact, if eNOS is blocked, the benefits of high-intensity cycling exercise was also found to be blocked.

“CACs from patients with disease are not only reduced in circulation, but are dysfunctional compared with those from healthy individuals (i.e., they show increased senescence and apoptosis with decreased migratory capacity), which may contribute to poor vascular outcomes and prognosis” (p. 439). In patients with disease, acute exercise can be of benefit to CAC function and increased migration. However, the limitations of the positive effects of acute exercise suggest that exercise training may be a better solution.

Exercise Training

In a surprising way, exercise training effects quantity of CACs in healthy versus diseased patients (e.g., CVD - heart disease, coronary artery disease, and peripheral artery disease) in a different way. Diseased patients had a greater benefit from exercise training than healthy volunteers with regard to CAC quantity. In these patients, a significant increase in resting CACs were noted with exercise training. Most of these exercise training programs were focused on aerobic training (70-85% of max heart rate or 60-70% VO2max) rather than high intensity training. Nevertheless, blood draw revealed elevated CACs often more than 24 hours after the final exercise session (this was done to eliminate possibility of acute exercise influence). In a study of 8 weeks of aerobic training in patients with heart failure, there was a significant increase in CAC quantity and function; however, 8 weeks after discontinuing the training, the CAC numbers returned to baseline. It seems necessary to have continual exercise training; moreover, it further demonstrated that physical exercise training does indeed impact CACs in these populations. “Exercise training appears to improve CAC more consistently in patient populations than healthy individuals, as both training studies and cross-section comparisons in healthy participants have reported no effect of exercise on CACs” (p. 440). In a recent study of heart failure patients, it was revealed that “6 months of training may have been sufficient to elicit sustained CAC improvements in these patients to levels comparable to healthy controls. A similar finding was reported in a comparison of regularly active and inactive young but healthy men…” (p. 440).

Exercise training should be engaged and not abandoned, whenever possible. In a different study when highly active, older men with a history of long-term endurance training stopped training for only 10 days, “the number of CACs fell to the level of sedentary men of the same age” (p. 441). In other words, commence exercise training and continue training to maintain CACs at healthy levels.

“Currently, mechanistic studies evaluating the effect of exercise on the release of angiogenic factors relating to angiogenesis and endothelialization are lacking. This is an important area for future research in order to optimize cell-based therapies for disease” (p. 441). That said, there are data of indirect evidence to reveal an association between CACs and improved vascular function with exercise training. Over the past 20 years, it has “become clear that physical activity greatly influences mobilization and function of CAC populations, particularly in patients with CVD risk or disease, and they may be important mechanisms by which exercise promotes vascular health” (p. 442). Unfortunately, women have been poorly represented in these studies. Age-related conditions (e.g., menopause) should be investigated with regard to CACs and exercise training.

C. Medicinal Signaling Cells (aka, Mesenchymal Stem Cells)

Dr. Arnold Caplan first coined the term “mesenchymal stem cells” in 1987. Recently, he has coined a new term for these cells - “medicinal signaling cells.” According to Caplan, this new name reflects the fact that there are no data demonstrating that when transplanted these cells behave in vivo as they do in vitro [i.e., differentiating into osteoblasts, adipocytes, and chondroblasts - these in vitro differentiations are a part of the minimal criteria for defining MSCs(5)]. Even cells that meet the minimal criteria set by the International Society for Cellular Therapy (ISCT) do not all represent MSCs. Additionally, MSCs are not a homogeneous cell population, but like HSCs and EPCs, are heterogeneous.

Acute Exercise and Exercise Training

MSCs have been identified in peripheral blood and localized to the vascular niche in numerous adult human tissues (e.g., bone marrow, adipose tissue, skeletal muscle, heart, tendon, dental pulp, and other tissues). “Current studies have investigated the effect of injury on MSC migration, proliferation, and function, yet to our knowledge, few studies exist which have evaluated the MSC response to either acute exercise or exercise training” (p. 443). Recently, a study following 5 weeks of progressive treadmill running found an increased bone formation rate and a decrease in the marrow adipocyte volume; this is likely due to suppression of MSC adipogenesis as a result of mechanical loading. Another study of mice running found that a 78% decrease in bone marrow adipose content was observed after 10 weeks of running 3 days/week. “Despite these intriguing results, studies that evaluate the tissue-specific MSC response to exercise do not exist outside of skeletal muscle” (p. 443). Therefore, we now move our discussion from MSCs to focus on skeletal muscle.

Resident Progenitor Cells in Skeletal Muscle

The predominant resident progenitor cell in skeletal muscle is referred to as a “satellite cell” and is responsible for repair and/or regeneration of myofiber in response to injury. The quiescent (think of a bear hibernating in winter) satellite cells can become activated through injury, leading to migration to sites of injury as well as asymmetric cell division. These cells “are increased in skeletal muscle following acute and repeated bouts of strength training, with preferential enhancement observed in the niche surrounding Type II fibers, the fiber type most susceptible to contraction-induced injury and age-related atrophy” (p. 445). Enhancement has also been observed in healthy, young, active men 1-3 days after acute maximal unilateral isolated eccentric exercise or traditional resistance-type exercise. Resistance exercise training can also increase satellite cell content in young and aged men and women (p. 445). As we have discussed in previous blogs, the environment in which a cell lives is crucially important. “Multiple intrinsic and extrinsic factors, including growth factors, mechanical strain, hypoxia, inflammation, ECM [extracellular matrix] composition, and topography, are altered by exercise and ultimately dictate the satellite cel response to muscle contraction” (p. 446). That is, the focus is once again upon the microenvironment and the paracrine effect factors in satellite cell activation postexercise. Some of these satellite cells are haematopoietic and could reconstitute the haematopoietic system in mice; they are CD45+. Other cells are CD45- and have no haematopoietic lineage markers and also display myogenic capacity.

Platelet-derived growth factor receptor-α (PDGFRα) expression seems to be a key cell surface marker for myogenic potential. Non-satellite PDFGRα cells demonstrate potential for myogenesis and muscle repair (indirectly). Such cells have been demonstrated to “increase twofold at 24 h post exercise and cells were localized to large vessels and nerves in the interstitium” (p. 447). Laboratory culture after isolating such cells from muscle have revealed that they express MSC and pericyte markers and negative for endothelial cell markers. These cells increased satellite cell number and new fiber synthesis following exercise, most likely through release of paracrine factors. However, much more remains to be learned about these cells and their response to both acute exercise and exercise training.

DISCUSSION

Again, we see the limited benefit that acute exercise brings to these various cells with high regenerative potential, but we also see a subset of these cells (CACs) in which exercise training is more beneficial to diseased patients, unless healthy individuals cease exercise training, at which point such cells quickly decline to sedentary individual levels.

Simply having a patient quickly perform a maximal exercise routine prior to cellular therapy aspirations may be of minimal benefit. Perhaps, in patients that are able to perform this, a maximal effort exercise could be performed prior to blood draw in platelet-rich plasma (PRP) which is easier to perform than aspiration of adipose tissue or bone marrow. As we saw last week, bone marrow cells did not change with acute exercise and as bone marrow aspiration would most likely occur outside of the 15-60 minute window of exercise, it seems of little benefit to incorporate acute exercise.

Future studies must include more women. Menopause leads to reduction in the quantity of immature cells in circulation as pre-menopausal women tend to have higher amounts of these cells in peripheral blood than age-matched men. Investigating exercise in such cases would be helpful. Additionally, the increase of satellite cells in the elderly may lead to a beneficial effect of training to counteract age-related muscle loss and sarcopenia. We will be discussing these topics in a future blog in this series of “Exercise and Stem Cells.”

Exercise training continues to have the data to support its incorporation not only for its general health benefits, but also because of its beneficial effects upon cells with high regenerative potential, and probably of even greater importance, the beneficial effects exercise training has upon cellular microenvironment. “The extent to which these stem cells [HSCs, CACs, and MSCs] change in response to exercise and are involved in the commonly recognized beneficial adaptation to exercise is not fully understood” (p. 448). That said, data do support the idea of incorporating exercise training for beneficial effects of cellular therapies, but more data are necessary to conclusively determine all of the impact exercise training brings.

REFERENCES

Boppart MD, De Lisio M, Witkowski S. (2015). Chapter 18 - Exercise and Stem Cells. Progress in Molecular Biology and Translational Science; 135:423-56.
Melton DA and Cowen C. (2009). “Stemness”: Definitions, Criteria, and Standards. In R Lanza, J Gearhart, B Hogan, D Melton, R Pederson, ED Thomas, J Thomson, I Wilmut (Eds.), Essentials of Stem Cell Biology (2nd ed) (pp. xxiii-xxix). Oxford, UK: Elsevier.
Asahara T, Murohara T, Sullivan A, et al. (1997). Isolation of putative progenitor endothelial cells for angiogenesis. Science; 275:964-7.
Barrett AJ, Longhurst P, Sneath P, Watson JG. Mobilization of CFU-C by exercise and ACTH induced stress in man. Exp Hematol; 6:590-594.
Dominici M, Le Blanc K, Mueller, I, et al. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy; 4:315-7.