Terminal differentiation is not a major determinant for the success of stem cell therapy - cross-talk between muscle-derived stem cells and host cells

We have found that when muscle-derived stem cells (MDSCs) are implanted into a variety of tissues only a small fraction of the donor cells can be found within the regenerated tissues and the vast majority of cells are host derived. This observation has also been documented by other investigators using a variety of different stem cell types. It is speculated that the transplanted stem cells release factors that modulate repair indirectly by mobilizing the host's cells and attracting them to the injury site in a paracrine manner. This process is loosely called a 'paracrine mechanism', but its effects are not necessarily restricted to the injury site. In support of this speculation, it has been reported that increasing angiogenesis leads to an improvement of cardiac function, while inhibiting angiogenesis reduces the regeneration capacity of the stem cells in the injured vascularized tissues. This observation supports the finding that most of the cells that contribute to the repair process are indeed chemo-attracted to the injury site, potentially through host neo-angiogenesis. Since it has recently been observed that cells residing within the walls of blood vessels (endothelial cells and pericytes) appear to represent an origin for post-natal stem cells, it is tempting to hypothesize that the promotion of tissue repair, via neo-angiogenesis, involves these blood vessel-derived stem cells. For non-vascularized tissues, such as articular cartilage, the regenerative property of the injected stem cells still promotes a paracrine, or bystander, effect, which involves the resident cells found within the injured microenvironment, albeit not through the promotion of angiogenesis. In this paper, we review the current knowledge of post-natal stem cell therapy and demonstrate the influence that implanted stem cells have on the tissue regeneration and repair process. We argue that the terminal differentiation capacity of implanted stem cells is not the major determinant of the cells regenerative potential and that the paracrine effect imparted by the transplanted cells plays a greater role in the regeneration process.

cartilage (AC), and explore the possibility that the repair is induced by host cell recruitment, angiogenic and/or anti-infl ammatory activities, and not necessarily restricted to the diff erentiation of the implanted cells in host tissue.
Our group has used MDSCs in cardiac transplantation experiments, which display an improved transplantation capacity when compared with myoblasts. MDSC engraftment was 25-fold higher than myoblasts in diseased hearts (with a mean of 53 donor MDSCs versus 2 donor myoblasts found within the injected hearts at 12 weeks post-injection) [54]. Importantly, they elicited signifi cant improvements in cardiac function by exhibiting superior cell survival and improving angiogenesis [54][55][56], most likely due to their secretion of vascular endothelial growth factor (VEGF) [57].
Although the exact origin of mouse MDSCs remains to be determined, these cells express the endothelial cell marker von Willebrand factor and can spontaneously participate in blood vessel formation after being injected into skeletal and cardiac muscle [58]. Th eir participation in blood vessel formation appears to be due to their expression of VEGF and by the fact that they can also diff erentiate toward an endothelial cell lineage [2,54]. Th e latter results suggest that a relationship exists between mouse MDSCs and endothelial cells. Other types of stem cells derived from the walls of blood vessels, including mesoangioblasts and perivascular cells, appear to share similarities with MDSCs, which also supports our hypothesis that a relationship exists between MDSCs and endothelial cells. We have also isolated several populations of muscle-derived cells from human skeletal muscle by fl uorescence-activated cell sorting (FACS) that coexpress myogenic (CD56 and Pax7) and endothelial cell markers (CD34, von Willebrand factor, ulex, and VEcadherin) both in vitro and in vivo [59][60][61]. We observed that certain types of cells that constitute the walls of blood vessels in adult human muscle (endothelial cells, myo-endothelial cells, and pericytes) appear to be very early myogenic progenitor cells that have high myogenic potential, and regenerative capacities in both skeletal and cardiac muscles [59,[61][62][63], much like that exhibited by murine MDSCs [2]. We have recently observed that a greater improvement in left ventricular function was observed after the intramyocardial injection of myoendothelial cells when compared to hearts injected with myoblasts [63]. Transplanted myo-endothelial cells generated relatively good engraftments within the infarcted myo cardium and also stimulated angiogenesis, attenuated scar tissue formation, and supported the proliferation and survival of endogenous cardiomyocytes more eff ectively than transplanted myoblasts or endothelial cells [63]. In a diff erent set of preliminary studies, we also observed that the injured hearts injected with skeletal muscle-derived pericytes displayed signifi cant improvements in cardiac contraction, greater neoangio genesis, and a signifi cant reduction in scar area formation when compared with hearts injected with phosphate-buff ered saline [64].
Th ese latter fi ndings suggest that myo-endothelial cells and pericytes likely represent the human counterpart to murine MDSCs and consequently comprise a potential therapeutic cell source that could provide valuable benefi ts for patients suff ering from myocardial infarction [64]. We have observed that after the implantation of murine MDSCs and human muscle-derived cells (myo-endothelial cells and pericytes), the induction of heart repair is mediated mostly by the host cells. Indeed, we have observed that only a small fraction of the donor cells can actually be found within the regenerated heart tissue, indicating that the host cells must be largely contributing to the cardiac repair process [54,63]. Th ese results indicate that the injected cells may act as a reservoir of secreting molecules that play a role in the repair process without actively diff erentiating toward a cardiomyocyte lineage or by fusing with host cardiac cells.
Th e cell-mediated paracrine and bystander eff ects on cardiac repair have also been observed with other stem cell types, including BM-derived cell populations [65][66][67][68][69], hematopoietic cells [70], adipose-derived stem cells [71], endothelial progenitor cells [72], human blood endo thelial cells [73], and kidney-derived MSCs [74]. Th ese reports support the hypothesis that the benefi cial eff ect seen with MDSCs is likely due to the increased secretion of paracrine factors and not primarily due to the diff erentiation capacity of the donor cells toward a cardiac phenotype (for reviews refer to [28,75]), especially since the cardiac diff erentiation of these stem cells after implantation remains extremely low (Table 1).
In support of this paracrine eff ect hypothesis, we previously reported that inhibiting angiogenesis by inject ing genetically manipulated MDSCs that express the anti-angiogenic protein sFlt-1 reduces the regeneration capacity of MDSCs in injured heart. Th e fi ndings of this study demonstrated that most of the cells contributing to the repair process were indeed chemo-attracted to the injury site by the injected cells [57]. Although the paracrine action of the donor stem cells is widely accepted, the origin of the host cells that participate in the repair process remains largely unknown. Further experiments are underway to determine the type of cells and their origins. Likely candidates for the host cells involved in the repair process include, but are not limited to, BM-derived cells, vascular-derived endothelial progeni tor cells, infl ammatory cells and resident tissue stem cells. Since it is well established that the vascular supply plays a major role in cardiac tissue repair, it is logical to speculate that blood vessel-derived cells are also involved in the repair process after cardiac injury. Indeed, it has already been shown [76][77][78] in other animal injury models that tissue repair induces the mobilization and incorporation of BM-derived endothelial progenitor cells, suggesting that some of the chemo-attracted host cells are perhaps deri ved from endothelial progenitor cells. It is important to bear in mind that promoting angiogenesis will bring more blood vessel-derived cells to the injury area, but also cells derived from the BM and bloodstream; therefore, caution needs be taken when reporting these research fi ndings.
Th ese results with MDSCs and other stem cell types (Table 1) strongly support the fact that a stem cell's multipotent capacity -in this case the ability to diff erentiate into a cardiac lineage -is not a pre-requisite for the cell's ability to readily aid in the repair of an injured heart. We cannot exclude that a stem cell's ability to readily undergo cardiac diff erentiation does not provide additional benefi cial eff ect for cardiac repair; however, this still remains to be verifi ed experimentally. More importantly, we have observed that the ability of MDSCs to resist environmental stresses, including oxidative and infl ammatory stresses, through the cells' high antioxidant capacity (via glutathione and superoxide dismutase), plays a major role in the high regenerative capacity of MDSCs in various tissues, including the heart [79,80]. Moreover, we have observed that treating MDSCs with the reducing agent glutathione signifi cantly reduced their ability to repair the heart, supporting our driving hypothesis that a cell's ability to survive within the injured tissue is more important than its terminal diff erentiation potential [79]. A population of stem cells that could survive better in this type of harsh environment could enhance the regeneration process, potentially through an increase in their paracrine eff ect (for example, increased angiogenesis).
A recent study showed that preconditioning BMderived MSCs prior to their transplantation enhanced their capacity to repair infarcted myocardium, which was attributable to an improvement of donor cell survival and an increase in angiogenesis/vascularization and proangiogenic factors [81]. In addition, Pasha et al. [82] reported that preconditioning cells with the chemokine stromal-derived factor 1 alpha (SDF-1) could signifi cantly enhance BM-derived MSC survival, vascular density, engraftment, and myocardial function [82]. MSCs derived from adult BM genetically modifi ed with the antiapoptotic gene Bcl-2 enhanced cell survival, engraftment, revascularization, and functional improvement in a rat left anterior descending ligation model of myocardial infarction via an intracardiac injection [83]. Taken together, these results suggest that a cell's ability to survive the harsh microenvironment within the injured heart represents a major determinant for its regenerative capacity. Surviving cells can eventually promote the repair process primarily through a paracrine eff ect that involves angiogenesis, especially for cardiac repair ( Figure 1).
Finally, it is important to state that stem cells with cardio myocyte properties (such as cardiac stem cells, embryonic stem cells or genetically engineered cells with cardiomyocyte inducers) are perhaps more likely to terminally diff erentiate into cardiomyocytes and participate in heart repair than stem cells incapable of diff erentiating toward a cardiac lineage. It is possible that cardiac diff erentiation of stem cells prior to their transplantation could improve cardiac function due to their ability to integrate more eff ectively with the host myocardium. It will also be important to determine whether diff erentiating the cells toward a cardiac lineage prior to their implantation could infl uence the paracrine eff ect of the stem cells and how this would infl uence their action in the cardiac repair process. Th us far, however, from our observations and reports in the literature (Table 1), very few post-natal stem cells have been shown to adopt a cardiomyocyte phenotype, yet the vast majority of transplanted stem cell types have been shown to improve cardiac function.

Stem cell therapy for articular cartilage repair
In this section, we investigate the paracrine eff ect of stem cell therapy in the AC repair process of osteoarthritis (OA), where the cells used (MDSCs) have the ability to undergo chondrogenic diff erentiation but the paracrine eff ect of the donor cells on the promotion of angiogenesis is not required, but, in fact, needs to be inhibited. Th erefore, it is important to determine, in this situation, whether the paracrine eff ect of the stem cells on the local microenvironment also plays a major role in the regenerative capacity of the stem cells for AC repair, without relying on angiogenic-related cells (blood vesseland circulation-derived stem cells). OA is a chronic degenerative joint disorder with worldwide impact that is primarily characterized by AC destruction and osteophyte formation. One of the chief mediators of OA is infl ammation and angiogenesis. Yin and Pacifi ci [84] demonstrated that VEGF treatment during early limb bud development in chick embryos causes excess vascularization and consequently reduced condensation of the chondrogenic mesenchyme. In the growth plate, VEGF has been reported to play an essential role in cartilage vascularization and absorption of hypertrophic chondrocytes, which together lead to endo chondral ossifi cation [85][86][87]. On the other hand, when VEGF was blocked with the soluble receptor protein (sFlt-1), it led to the expansion of a zone of hypertrophic cartilage and the inhibition of cartilage resorption [86]. Similar to endochondral ossifi cation, osteophyte formation during OA development has been reported to involve VEGF signaling [88]. For an extensive review on the relationship between angiogenesis and OA in humans and animal model studies, we refer our readers to the review by Ashraf and Walsh [89], who outlined the complexity of OA and the interrelationship between angio genesis, infl ammatory processes, damage, innervation, and pain perception in the joint.
Recent data reveal that the expression of VEGF and its receptors (Flt1 and Flk1) in osteoarthritic cartilage refl ects the ability of VEGF to enhance catabolic  pathways in chondrocytes by stimulating matrix metalloprotein ase activity and reducing tissue inhibitors of matrix metalloproteinases (TIMPs) [90][91][92][93]. Th ese data suggest that, apart from the eff ect of VEGF on cartilage vascularization and proliferation of cells in the synovial membrane, chondrocyte-derived VEGF promotes catabolic pathways in the cartilage itself, thereby leading to a progressive breakdown of the AC extracellular matrix. Since AC is a tissue type that is poorly supplied by blood vessels (avascular), nerves, and the lymphatic system, it has a very limited capacity for repair after injury. Although several therapies have been used to treat OA, no widely accepted treatments have been established, with the exception of arthroplasty. For this reason, tissue engineering techniques aimed at repairing AC have been extensively studied and chondrocyte transplantation is currently performed in clinics [94][95][96]. Th e current most eff ective OA treatment, besides arthroplasty, is the use of autologous chondrocyte transplantation. However, this treatment has several limitations, including the use of neighboring healthy donor cartilage, diffi culty in treating large-scale defects, limited expansion capacity of the primary chondrocytes, the need for a periosteal patch to maintain engineered cartilage, and the fact that, in most cases, only 30 to 40% of the defect regenerates AC, with the remaining defect being fi lled with fi brocartilage [97][98][99]. In light of these limitations, it is important to fi nd other sources of cells that are abundant and capable of chondrogenic diff erentiation. Stem cells are more attrac tive than primary chondrocytes because of their After implantation in injured tissue, cell survival plays a major role in the repair process. The cell's ability to survive consequently leads to better long-term proliferation, self-renewing ability, and multipotent diff erentiation capacity, but the main eff ect within the injured tissue appears to be as a reservoir for secreting molecules that can induce a variety of paracrine eff ects, especially chemo-attraction. The paracrine eff ect may have a major infl uence on the local microenvironment (blocking angiogenesis in articular cartilage) or on the systemic environment that involves the recruitment of host cells from the systemic circulation (increasing angiogenesis in the heart). The systemic eff ect appears to be primarily involved with angiogenesis, which is responsible for bringing a multitude of stem cells to the injured tissues.
superior capacity for self-renewal, proliferation, and survival follow ing microenvironmental stress. Recently, stem cell-based therapies have been used clinically for cartilage repair [100,101]. Several studies have suggested that stem cells can undergo chondrogenesis and repair AC in experi mental cartilage injury models (osteochondral lesions), including studies using MDSCs [102]. We have already reported that bone morphogenetic protein (BMP)4-transduced MDSCs improve cartilage regenera tion in in vitro pellet cultures and in an in vivo cartilage defect model (osteochondral defect) [102]. We have also shown recently that human muscle-derived cells (myo-endothelial cells and pericytes) can undergo chondro genic diff erentiation in vitro, albeit to a diff erent extent [61,62].
Since the expression of VEGF by chondrocytes in the osteoarthritic joint has been related to cartilage destruction [88,90,91,93,[103][104][105] and the induction of arthritis (especially when the dosage reached a certain threshold) [106][107][108], it is likely that blocking VEGF would prove to be a benefi cial means of preventing or delaying the progression of OA. Th is hypothesis was recently supported by the fact that treatment with sFlt-1 (a VEGF antagonist) decreased the severity of arthritis in a murine model [86,109,110] and our recent observation that the injection of MDSCs expressing both BMP4 and sFlt-1 improved AC repair in a more eff ective manner than MDSCs expressing just BMP4 [111,112]. Our results suggest that sFlt-1/BMP4-transduced MDSCs, which were transplanted intra-capsularly into an OA rat model, enhanced AC regeneration via BMP4's autocrine/ paracrine eff ects, and contributed to an appropriate environment that prevented chondrocyte apoptosis by blocking the intrinsic VEGF catabolic pathway and by extrinsic VEGF-induced vascular invasion. Treatment of MDSCs with sFlt-1 and BMP4 combined is potentially an eff ective therapy for OA repair that may improve the quality and persistence of regenerated AC [111]. Since the cells were injected into the joint fl uid, most of the injected cells do not primarily contribute to the regeneration of the AC through their diff erentiation into chondrocytes; instead, they chemo-attract host cells to the injury site, which are the cells primarily seen in the regenerated tissue. We are performing experiments to determine the origin of these host cells that participate in the repair process by testing the role of infl ammatory/immune, BM and synovial cells. Since we have observed that the injection of muscle-derived cells (isolated from rabbit skeletal muscle) in the joint fl uid of rabbits leads to a massive attachment of the injected cells in the synovium [113], we posit that synovium-derived cells are implicated in the repair process.
Although the mechanism behind the benefi cial eff ect of blocking angiogenesis in AC repair is still not fully elucidated, these results highlight the importance of controlling the local microenvironment by reducing angio genesis. Th erefore, the reduction of angiogenesis eliminates the mobilization of blood vessel wall-and circulation-derived progenitor cells and thus their recruitment and diff erentiation toward a chondrogenic lineage, which demonstrates the paracrine eff ect that the implanted stem cells exert on the local microenvironment for optimizing the AC repair process (Figure 1).
Recently, Gelse and colleagues [114] reported that transplanted rib chondrocyte spheroids could repair a cartilage defect in a miniature pig model by producing BMP2 and attracting the host's BM-derived cells. Th e transplanted chondrocyte spheroids provided a stimulatory paracrine eff ect that induced the in-growth and chondrogenic ability of the host BM-derived cells. Th is paracrine eff ect was observed to be far more important to the repair process than the direct diff erentiation of the transplanted cells into chondrocytes. Although the transplanted cells enhanced the tissue repair process, these experiments further validate our hypothesis that the AC repair process, even using stem cells other than MDSCs, also relies on the paracrine eff ect that the donor cells impart on the host cells.

The microenvironment infl uences the fate of stem cells
BMP4-transduced MDSCs can undergo osteogenesis and promote bone repair when injected into a bone defect [115][116][117], which is diffi cult to explain given that similar BMP4-transduced cells can promote the repair of AC when injected into an osteochondral defect. Th is phenomenon, however, is a good example of how the microenvironment infl uences the regenerative ability of the transplanted stem cells. After a bone or AC injury occurs, a multitude of signals are released at the site of injury. It is likely that the chemotaxis of host cells is enhanced by growth factors and cytokines produced by the donor cells, which are in turn aff ected by their interaction with the extra cellular matrix at the injury site. Speculatively, stem cells injected into the site could aid and enhance the mediation of the repair process; however, knowing that the repair process relies primarily on host cell participation, it is easier to understand why BMP4-expressing MDSCs have a benefi cial eff ect on both bone and AC repair since the repair process does not rely on the terminal diff er en tiation of the donor cells per se. Furthermore, Blanke et al. [118] have shown that successful repair of cartilagi nous tissue after transplantation of chondrocytes was associated with their production of thrombo spondin-1 and chondromodulin-I anti-angiogenic proteins. Th ey found that tissues resisted ossifi cation when the chondro cytes produced detectable levels of anti-angiogenic proteins, which counteracted the angiogenic activity of endothelial cells. It is very likely that MDSCs react in a similar manner to the local environmental cues and produce anti-angiogenic proteins similar to thrombo spondin-1 and chondromodulin-I when in a cartilaginous microenvironment, a hypothesis that will need to be further investigated in future studies.
Another example that demonstrates the infl uence of the microenvironment is the fact that the regenerative capacity of stem cells has been shown to be infl uenced by the age of both the host-and the donor-derived stem cells [119]. Th e results showed the rejuvenation of aged progenitor cells after their exposure to a young systemic environment where a young and aged mouse had their circulatory systems linked by heterochronic parabiosis. Th ese fi ndings highly implied that the proliferative property of satellite cells obtained from old mice is restored after incubation with the serum from a young animal. Th ese results further support our hypothesis that the regenerative process of given stem cells is strongly infl uenced by signals found within the microenvironment.
Furthermore, we have recently observed that the regenera tive capacity of stem cells also appears to be infl uenced by the gender of the donor cells and the host. In fact, we reported that female MDSCs are more myogenic and can promote muscle regeneration in a more eff ective manner than male MDSCs [120]. On the other hand, male MDSCs were shown to be more osteogenic and chondrogenic and promoted both bone and AC repair in a more eff ective manner than their female counterparts [111,121]. Although the mechanisms behind these gender diff erences are still unclear, we have shown that the host microenvironment is also infl uenced by the gender of the animal and plays a role in the effi ciency of the repair process. Indeed, we have reported that bone formation mediated by male MDSCs is superior in a male host versus that of a female host [121]. Interestingly, female MDSCs produce better bone when injected into a male host when compared to a female host. Th ese results further support our hypothesis that the microenvironment infl uences the fate and regenerative potential of the injected stem cells.

Conclusion
Although it has been speculated for numerous years that the high regenerative potential of stem cells is due to their terminal diff erentiation capacity, current fi ndings appear to indicate that very few donor cells actually diff erentiate and participate in the regeneration of these injured tissues; instead, the vast majority of the cells reconstituting the regenerated tissues are host-derived. Th ese fi ndings are further supported by recent results showing that when the paracrine signaling of the implanted stem cells is interrupted (that is, by blocking VEGF and angiogenesis), there is a reduction in the regeneration and repair capacity of the injured tissues, as is the case for cardiac muscle repair. Although it is still unclear which host cells are involved in the repair processes after stem cell transplantation, blood vessel cells, immune and infl ammatory cells and resident cells at the site of the injury (especially for AC damage), appear to play a role in the regeneration/repair process. It is quite clear, however, that the terminal diff erentiation of stem cells does not represent a major determinant for the success of stem cell therapy; instead, it appears that donor cell survival and the cells' paracrine eff ect play much more critical roles in the success of stem cell therapy. Th is fi nding challenges current dogma indicating that embryonic stem cells may have an advantage over adult-derived stem cells because of their higher level of multipotentiality. We therefore put forward the proposition that it is the stem cell's superior cell survival capacity that leads to its increased ability to chemoattract host cells through the secretion of certain growth factors and chemokines and this is the key important feature for successful stem cell therapy, more so than stem cells' terminal diff erentiation capacity.

Future directions
Th e paradigm shift in evaluating stem cell engraftment based on their terminal diff erentiation into host cells underscores the need to understand the biology of stem cells to fully utilize their potential. Furthermore, we are increasingly aware that stem cells alone are not suffi cient for a long lasting regenerative eff ect. Th e ultimate goal would thus be the generation of tissues incorporating stem cells, scaff olds and biological materials that permit communication with host tissues, allowing optimal remodeling and improved functionality. Future regenerative schemes may include cells and a complex milieu of factors based on a rigorous understanding of stem cells' paracrine secretions. Computer-aided bioreactors, bioprinters, artifi cial or decellularized organs and other biodevices could also benefi t from knowledge of stem cells' paracrine activities.
Competing interests JH has received remuneration as a consultant from Cook MyoSite Inc., Pittsburgh, USA over the past 5 years, the other authors declare that they have no competing interests. of Pittsburgh and the Orris C Hirtzel and Beatrice Dewey Hirtzel Memorial