As noted through studies of animal models, MSC are present in the perivascular compartment of many skeletal muscles [23] -[25] , with some sex differences in numbers and activity also noted (discussed in [23] -[25] ). These MSC can be differentiated into the usual three lineages (osteogenic, chondrogenic, adipogenic) after isolation, as well as possibly others (e.g. myogenic, neural).

Injection of skeletal muscles of the rabbit hind leg with repeated doses of BoToxA leads to the rapid atrophy of the muscles with loss of function [26] [27] . Subsequent to the atrophy, there is a “fatty infiltration” of the muscles which persists for some time (discussed in [28] ). The source or origins of the fatty infiltrate has not been characterized, and the findings are more descriptive than mechanistic thus far. Therefore, based on the temporal relationships, following BoToxA injection the overt atrophy occurs rapidly, and then in response to prolonged loss of mechanical integrity of the muscle, a fatty infiltrate occurs somewhat later, but which can be protracted. The fate of the muscle-associated MSC has not been addressed in this situation based on a search of the literature. In addition, the fate of MSC within existing fat pads of the knee, such as Hoffa’s fat pad, during such BoToxA-induced atrophy has not been described, but these issues are currently under investigation (unpublished).

Rapid atrophy of muscle and bone upon exposure to micro-gravity through space flight has long been known (reviewed in [29] -[31] ; and others). Such loss occurs throughout the body, but appears to be focused on a subset of muscles of the lower extremities which are apparently the most influenced by the 1G-mediated ground reaction forces on earth. While the muscle loss can be at least partially prevented by counter measures, bone loss is less influenced by existing modalities (reviewed in [32] ). Interestingly, the extent of bone and muscle loss by those experiencing micro-gravity in space flight is quite individualized, with some people losing tissue mass at rates several times faster than others (e.g. 2%/month vs 0.2%/month) (discussed in [33] ). Thus, there appears to be a genetic component to the rate of loss that is not overtly expressed when in the 1G environment (possibly silent mutations that are masked by the 1G environment). Some authors have suggested that the responses to microgravity and space flight are actually an accelerated aging scenario [34] -[36] . If the analogy is correct, then finding solutions to responses to microgravity may have application for aging earth-bound populations as well.

Interestingly, with prolonged exposure to micro-gravity, there is a reported “fatty or lipid infiltration” of the muscle (reviewed in [37] ). Where such fatty infiltrates come from, or why they appear has not been addressed, the phenomenon merely described. The recovery from such fatty infiltrates after a return to a 1G environment is apparently slow, but has not been described well in the literature (but likely could using improved ultrasound methodology or others). With six team members on the International Space Station presently for ~6 month stays, the number of individuals who could be monitored for onset and recovery is quite large by space-study standards. Similarly, one could perform a limited number of muscle biopsies to evaluate fat-associated MSC in such astronauts.

A surrogate for exposure to micro-gravity is “head down-tilt bed rest” (reviewed in [38] [39] ; and many others). Individuals subjected to this type of bed rest develop rapid bone loss indicators [40] , and subsequently muscle atrophy, overt bone loss, and other characteristics of space flight (reviewed in [31] [38] [39] ; and many others). Over time, individuals exposed to this environment also develop “fatty infiltrates” in their skeletal muscle and other tissues. And again, the condition has been described, but the source and causes have not been addressed. While this surrogate has been studied in relation to micro-gravity aspects, it is also clear that with the aging population, many of whom are confined to prolonged bed rest conditions, there is also risk for atrophy and other complications, including “fatty infiltration” of their skeletal muscles and other tissues [41] , and some counter measures prevent such accumulation [42] .

Therefore, in the Botulinum toxin studies, the space flight/microgravity studies, and the bed rest studies, as well as other animal studies of weightlessness (e.g. hind limb elevation to mimic loss of ground reaction forces) (reviewed in [37] [43] ), an accumulation of fat/lipid infiltrates occurs during prolonged exposure to the altered environment. It is clear that it happens, but the why and how it happens still remains to be determined. One possibility that could explain at least some of this response to weightlessness or loss of mechanical integrity is that mesenchymal stem cells in muscle and bone marrow are maintained in a meta-stable state by mechanical loading of the tissues, directly (via the MSC themselves) or indirectly via loading effects on the neuronal state of the tissues which when compromised, leads to a loss of the maintenance of the MSC in such a meta-stable state. Thus, alterations in these influences on MSC regulation may lead to the preferential differentiation of the MSC towards the adipose lineage (the default response). This may be due to an enhanced responsiveness to lineage-specific signals in the environment, or to an intrinsic loss of stability in the MSC.

While there is not much in the literature regarding this possibility, certainly the report of Meyers et al. [44] indicates that in a model of microgravity, there is enhanced adipogenesis and reduced osteoblastogenesis by human MSC. Interestingly, recent studies from the author’s laboratory have indicated that a subset of cloned MSC/MPC from porcine synovial fluid are restricted to differentiating towards either the osteogenic or adipogenic lineages ([3] ; JJ Kutcher, MSc Thesis, University of Calgary, 2012; Kutcher et al., in preparation). While not the dominant phenotype of MSC from porcine synovial fluid, the prevalence of this unique subset of MSC in other compartments such as muscle, bone marrow, and adipose tissue (e.g. abdominal fat, subcutaneous fat, Hoffa’s fat pad of the knee, brown fat) is unknown. Relevant to this point is also the fact that the studies by Meyers et al. [44] used mixed populations of human MSC, so it was not possible to make conclusions regarding whether only a subpopulation of MSC were affected by the microgravity conditions employed. Thus, it would be relevant to repeat those studies with well characterized cloned cells to allow stronger conclusions to be made. Such studies are currently underway (unpublished).

While the above question has not been answered directly, there are reports that mechanical loading can influence MSC lineage specificity even when the subjects are in the 1G environment of earth [45] [46] . Thus, low magnitude mechanical stimulation of C57BL/6 male mice promoted bone formation and depressed/inhibited diet-induced obesity. There were also increases in bone marrow MSC numbers following such stimulation. A pilot study with 48 young females also indicated that this low magnitude mechanical stimulation enhanced bone formation and kept visceral fat at baseline levels. Thus, unique mechanical loading regimens in a 1G environment may also be a methodological approach to interfere with obesity development [45] [46] . This concept has also been postulated recently by Gutin [47] who hypothesized that vigorous physical exercise may contribute to MSC differentiation preference.

MSC have been derived from a variety of human and preclinical model adipose tissues (e.g. subcutaneous fat, fat pads in the knee [e.g. Hoffa’s fat pad, infrapatellar fat pad], brown fat, and abdominal fat] (reviewed in [8] [48] -[50] ; and many others). In particular, MSC from subcutaneous fat via “liposuction” have been promoted as a ready source of cells for cartilage repair (reviewed in [8] [51] ), and a number of clinical trials are on-going to evaluate their efficacy.

Interestingly, MSC derived from obese mice are reported to be altered in their differentiation potential and they are altered irrespective of tissue source (bone marrow, infrapatellar fat pad, subcutaneous fat), and the alterations appeared to be lineage-specific with the chondrogenic lineage specifically affected (discussed in [55] ). Certainly obesity can lead to a metabolic disease or syndrome (reviewed in [52] -[54] ; and others), so this is finding is not surprising at some levels. This metabolic syndrome also likely contributes to the altered inflammatory state observed in obesity, directly via adipokine secretion ([53] [54] ; and others) or via subsequent amplification at systemic sites (discussed in [55] -[57] ; and others). Obesity may also influence the proliferative potential of adipose-derived MSC [58] , and therefore could alter regulation of these stem cells.

Acute joint inflammation due to an injury can also lead to alterations in the infrapatellar fat pad of the knee in ovine models [59] , and transient alterations in adipokine expression [59] and MSC levels (Solbak et al., in preparation). As yet, it is not yet clear as to whether such inflammation leads to altered lineage differentiation.

Finally, merely culturing adipose-derived MSC in monolayer culture is also reported to alter lineage-specific differentiation [60] , so care must be taken when making conclusions regarding adipose-tissue derived cells. Certainly culturing cells on plastic, in the presence of specific growth mediators, or serum has the potential to impact the MSC and influence the ability to translate in vitro findings to the in vivo condition.

These findings with MSC and other cells in adipose tissues are likely also relevant to the above discussion on the “fatty infiltration” of muscle, and bone marrow in response to an altered environment of muscle weakness, microgravity and prolonged bed rest. That is, these MSC populations are dynamic and respond to altered environments (e.g. loss of mechanical integrity, diet-induced obesity). In the case of diet-induced adiposity, it is clear there are still a large number of MSC in the tissues. Whether the MSC are contributing to the obesity (differentiation to adipocytes), as well as maintaining proliferation of the MSC compartment is possible, but not proven at this point. What is also unknown is whether the MSC compartment in muscle and bone marrow is also maintained in the tissues as the fatty infiltrate occurs. Thus, this should be a focus for future studies. The findings from such studies should either support or refute the premise that the adipose lineage for MSC differentiation is a “default” lineage when the mechanical environment is compromised.

As discussed above, it is clear that MSC/MPC are present in a variety adipose tissues. The numbers decline with age, and obesity can apparently influence the functionality of such cells. Also with age, the incidence of lipid deposition in various compartments (e.g. subcutaneous fat vs abdominal fat in males vs females with age) increases. All of the above beg the question, why are there so many MSC/MPC in adipose tissues, and could their dysregulation be a contributing factor in obesity development? If so, then MSC/MPC leading to the adipose lineage may be a potential target for interventions (e.g. new drug therapies) to regulate MSC/MPC in both prolonged space flight, prolonged bed rest, and in some forms of obesity (discussed above and in [45] ).

This concept is not entirely new as a number of reports have indicated that perhaps interfering with MSC differentiation towards the adipocyte lineage [61] -[64] , or even replacement of neural MSC in the hypothalamus [56] [65] -[67] could modulate obesity-related events. Some reports have even postulated certain molecules could be potential molecular targets for limiting such pathways [61] -[64] [68] [69] .

While performed prior to the onset of most information on MSC, a set of studies from the author’s laboratory using (NZB x NZW) F1 mice (referred to as NZB/W mice) may also provide some clues in this regard. This strain of mice develop large deposits of subcutaneous and intra-abdominal fat as they mature and age, reaching weights that are nearly double that of parental strain animals or younger animals. Treatment of females of the NZB/W strain with daily injections of lithium chloride (LiCl) leads to a nearly complete inhibition of the development of fat deposits over months, and there was a “re-activation” of fat deposition after treatment was discontinued [70] -[72] . While MSC in these animals were not assessed, more recent studies have indicated that LiCl treatment leads to inhibition of expression of adipocyte lineage genes in vitro with human bone-morrow-derived MSC [73] . Therefore, as lithium ion is a known inhibitor of GSK-3 and a modulator of WNT/beta-catenin pathway components, such studies should be revisited to ascertain whether the outcomes were due to impact on MSC stability and differentiation. Furthermore, as lithium ion treatment of aged MSC restores some of their functionality [74] , possibly such treatment could allow for re-establishment of MSC regulation following compromise as discussed above. However, as lithium ions do have potential side-effects, studies in mice to follow up the above discussed studies may lead to identification of specific targets that can be modulated using new drugs and therapeutic modalities for application in human populations.

The author thanks Dr. Yamini Achari for her review of the manuscript, as well as colleagues and trainees in the McCaig Institute for Bone & Joint Health (Calgary), the Centre for Hip Health and Mobility (Vancouver), the Technical University of Dresden (Germany), the University of Waterloo and the University of Toronto for discussions and interactions over the past decade which helped frame the above discussions. Studies from the author’s laboratory were supported by operating grants from the Canadian Space Agency (030SR.9F007-02-216), Alberta Innovates Health Solutions (2008170 and 200700596), and a salary award to DAH (Calgary Foundation-Grace Glaum Professorship).