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Transplantation of Allogeneic PW1pos/Pax7neg Interstitial Cells Enhance Endogenous Repair of Injured Porcine Skeletal Muscle

Visual Abstract

S keletal muscle is a dynamic, highly plastic tissue that is capable of undergoing repair and regeneration in healthy individuals (1). However, this regenerative capacity can become impaired in muscle diseases, such as muscular dystrophy, and as a result of disuse and ageing (2,3). This leads to decreased proliferation, delayed fusion, and reduced differentiation of muscle progenitors, as well as a gradual replacement of muscle fibers by fat and fibrosis with poor vascularization (4,5). The mechanisms that underpin impaired skeletal muscle regeneration are still unclear; however, recent findings suggest that the microenvironment and/or systemic factors (6,7) affect the response of various endogenous skeletal muscle progenitor cells to repair and regenerate after injury (8). This has led us to focus upon therapeutic strategies that target resident muscle progenitors and stimulate endogenous repair mechanisms.
Autologous stem cell approaches are attractive from a theoretical and biological standpoint; however, for skeletal muscle where the muscle satellite cells cannot be effectively propagated to large numbers in vitro, and like other tissue-specific stem/progenitor cells, such as cardiac, are affected by age and disease, they are impractical. Moreover, administration of cells can induce therapeutic responses by indirect means, such as secretion of growth factors and interaction with endogenous repair processes, represented by the resident stem/progenitor cells (9,10). This has been termed the paracrine effect. Therefore, there seems to be little advantage in the use of autologous cells because a similar, and perhaps enhanced, effect can be obtained by the administration of a cell type isolated from allogeneic sources, which could be made readily available at reduced costs (11). We have previously shown that intracoronary injection of allogeneic porcine cardiac stem cells (CSCs) after myocardial infarction (MI) activates the endogenous, resident CSCs through a paracrine mechanism, resulting in improved myocardial cell survival, function, remodeling, and regeneration (12).
Skeletal muscle satellite cells, although capable of muscle fiber regeneration, have a limited migration capacity with an inability to cross the endothelial wall (13). Moreover, cultured satellite cells ex vivo have decreased stemness, proliferation, and myofiber differentiation, which has subsequently hampered their translation (14). In recent years, skeletal muscle has been shown to harbor other stem/progenitor cells, such as the PW1 pos /Pax7 neg interstitial cells (PICs), which are bipotent, giving rise to new skeletal muscle fibers and vasculature-associated cell types in vitro and in vivo (15,16). Unlike the satellite cells, we have shown that porcine PICs (pPICs) can be successfully isolated and propagated in vitro up to passage 40 (P40) while maintaining a stable phenotype/genotype (16). Therefore PICs represent an attractive and alternative cell source for regenerative therapies.
The pig represents an excellent biological model for human biomedical preclinical research due to its anatomic, physiological, pathological, and genomic similarities to humans (17). In terms of porcine skeletal muscle biology, considerable correlation between contractile, metabolic, and morphological features to human skeletal muscle exist (18)(19)(20). Although small animal models are a valuable resource, they do not always accurately reflect the clinical manifestation, with many cell therapies that have been shown to be highly effective in small animal models (21,22) yielding only modest therapeutic value in human clinical trials (23,24). Combined with the 3 orders of magnitude difference in mass between rodent models and humans, there is an obvious need to test cell therapies in more illustrative animal models. Developing a porcine preclinical model for skeletal muscle regeneration can provide a useful link between the widely used rodent models and humans so that an improved prediction of therapeutic efficacy of therapies can be achieved and successfully translated to patients. Moreover, unlike other organs, skeletal muscle is an efficient regenerative tissue yielding adequate resident progenitor cells, which give rise to new myofibers and vasculature following injury.
Altogether, this makes the porcine skeletal muscle an appropriate model system to test proof-of-concept regenerative therapeutic approaches, such as the allogeneic cell approach, described here for the first time to our knowledge. Allogeneic stem cell therapy is conceptually and practically different from any presently in clinical use and could be applied in the repair and regeneration of other tissues, such as the heart.
Here, we tested the therapeutic reparative effect of delivering allogeneic pPICs to the injured pig skeletal muscle. We hypothesized that allogeneic pPICs transplanted following skeletal muscle injury would supply regenerative bioactive factors capable of activating the endogenous stem/progenitor cells, enhancing and accelerating the regeneration of damaged skeletal muscle tissue. It was also hypothesized that the allogeneic pPICs survive long enough in the allogeneic host to produce their paracrine effect activating the endogenous target cells before being eliminated by the host immune system. Therefore, the cells transplanted are allogeneic, but the regeneration is completely autologous.  surgery room and secured to the surgical table with limb bindings. The lower hind limb was shaved and disinfected using iodine before surgery.

METHODS
The first phase of the in vivo study used 6 pigs. In order to assess 2 different injury models, either an open freeze/crush (n ¼ 3) or cardiotoxin (CTX) injury (n ¼ 3) of the tibialis anterior (TA) muscle was subsequently performed. Briefly, a 5-cm longitudinal incision of the skin and underlying fascia was made along the anterior aspect of the distal limb. The skin and fascia were retracted, the TA muscle located, nonabsorbable Prolene sutures (Ethicon, Somerville, New Jersey) were used to mark the injury site (1 cm Â 1 cm 2 ), and the injury was induced. In the case of freeze/crush injury, the muscle was crushed with forceps (Fine Science Tools, Foster City, California), pre-cooled in liquid nitrogen. This procedure was repeated 7 times for 5 s each, in direct continuity with the respective distal crush in the defined area. In the case of CTX injury induction, a 10 mmol/l CTX solution, Naja mossambica mossambica (Sigma-Aldrich, St. Louis, Missouri), was filtered and brought to room temperature before injection. The CTX was dispersed in a series of 5 intramuscular injections using a 25-ga needle to deliver a total volume of 500 ml to the predefined area. The contralateral leg served as a sham control (CTRL) and was treated identically; however, no injury was applied in the case of freeze/crush, or phosphate buffered saline (PBS) alone was injected in animals receiving the CTX injury. The injury area was identified through marking with sutures. After injury induction, the superficial skin was sutured; animals Allogeneic pPICs Enhance Repair of Injured Muscle were allowed to recover and then sacrificed by anesthetic overdose at either 14 or 21 days post-injury.
In the second phase of the in vivo study, 10 pigs were randomly assigned to receive either saline vehicle (PBS) or allogeneic GFP pos pPICs, 15 min post-CTX injury. GFP pos pPICs were propagated and cryostored between P3 and P12. Before transplantation, GFP pos pPICs were pre-mixed, brought into suspension, centrifuged, washed twice with PBS, and then counted. A total of 20 Â 10 6 GFP pos pPICs were resuspended in 500 ml of PBS and injected intramuscularly into the injured TA through a 25-ga needle (n ¼ 5). Control animals were treated identically; however, 500 ml of PBS alone was injected (n ¼ 5).
Both treatments were distributed across 5 injection sites to the injury site. The contralateral control leg of each animal served as a sham CTRL and received no injury, only PBS, using the same protocol.
In separate pigs, local delivery of human recombinant insulin-like growth factor (IGF)-1 (8 mg) and hepatocyte growth factor (HGF) (2 mg) (Peprotech, Rocky Hill, New Jersey) was achieved by diluting both growth factors in a total volume of 500 ml of PBS before being dispersed in a series of 5 intramuscular injections using a 25-ga needle to deliver the total volume to the pre-defined injured area (n ¼ 5). In the case of ureido-pyrimidinone (UPy)þIGF-1/HGF treatment, the UPy hydrogelators were synthesized by SyMO-Chem BV (Eindhoven, the Netherlands), as described previously (25). To prepare the hydrogel, polymer solutions were dissolved at 10% by weight in PBS by stirring at 70 C for 1 h and were subsequently cooled to room temperature. To liquefy the polymer solution, the pH was increased to pH 8.5 by adding 2-ml aliquots of a 0.1 mol/l NaOH stock solution. The hydrogel was then sterilized with ultraviolet light for 1 h, and human recombinant IGF-1 and HGF were added before use, yielding a final concentration of 8 mg and 2 mg, respectively. A total volume of 500 ml of UPy hydrogelþIGF-1/HGF was administered as per the method described in the preceding text (n ¼ 5).
In order to track newly formed cells post-injury, we used the thymidine analogue, 5-bromo-2 0 -deoxyuridine (BrdU). In order to deliver BrdU to the animals over the course of the regeneration period, we used an IV delivery system. This involved making a channel through the pig's neck musculature and feeding an IV line through, which was subsequently connected to the jugular vein. This enabled us to access a cannula situated on the dorsal aspect of the pig's neck, which was directly linked to circulation system. This method allowed daily administration of BrdU at a dose of 10 mg/kg/day without the need to sedate the animals. Animals were sacrificed by anesthetic overdose at 14 days post-injury.
Taken together, these data suggest that allogeneic pPICs exert their action via a paracrine effect, activating endogenous progenitors before being cleared by the host immune system post-injury.

PRO-REGENERATIVE PARACRINE
FACTORS. To identify the mechanism through which pPICs were able to promote regeneration in skeletal muscle, we looked to their secretome. Porcine PICs were found to express a range of cytoprotective/proregenerative growth factors and cytokines ( Figure 5A), similar to that observed for stem/progenitor cells, such as mesenchymal stem cells (29) and satellite cells (30).
To enrich for a highly paracrine secreting pPIC population, we identified whether pPICs expressed differential levels of factors depending on their differentiation status. This was necessary because following pPIC transplantation into injured skeletal muscle, the microenvironment could facilitate pPICs to differentiate into a myogenic precursor-like cell type, which could then express differential levels of proregenerative factors, compared with its undifferentiated counterpart. Therefore, a comparative qRT-PCR analysis was performed between undifferentiated pPICs and those that had undergone 24 h of myogenic differentiation in vitro (differentiated pPICs). Overall, there were distinct differences between the differentiated and undifferentiated pPICs ( Figures 5B and 5C, Supplemental Table S1).    Figures 6A and 6B). Next, we showed that factors that were expressed by pPICs, such as IGF-1, HGF, and NRG-1, when supplemented to pPICs in vitro, significantly (p < 0.0001) increased their proliferation (Figure 6C), and for TGF-b1, increased pPIC differentiation (Figures 6D and 6E)  Figure S7).
To determine whether IGF-1 and HGF could improve regeneration to a similar degree as pPIC transplantation, we injected IGF-1 and HGF into CTXinjured skeletal muscle, and we also administered the same growth factors through a UPy hydrogel, as we have done previously in the porcine chronic MI model (47). We found that IGF-1/HGF administration improved muscle regeneration, increasing the number of BrdU pos myofibers (8 AE 2%/total myofibers vs. 2 AE 1%/total myofibers CTX-PBS-treated) and the BrdU pos regenerating myofibers were larger (33.28 AE 6.3 mm vs. 23.8 AE 7.3 mm CTX-PBS-treated) (Figure 7). However, these effects were not further enhanced if the growth factors were administered through the UPy hydrogel (BrdU pos myofiber 8 AE 3%/total myofibers and BrdU pos regenerating myofibers diameter 35.58 AE 4.98 mm), and more importantly, the improvements were not as great as transplantation of pPICs ( Figure 1). Therefore, pPICs are superior to targeted growth factor administration because they release a plethora of growth factors and cytokines, which activate multiple endogenous repair mechanisms, contributing to accelerated and effective repair and regeneration in vivo.

DISCUSSION
The present study documents that in a porcine model of skeletal muscle damage, which is applicable to Moreover, the assessment of satellite cell activation is hampered by lack of markers to identify activated satellite cells in the interstitial space (30). Also, the endogenous PICs might have maintained an activated state for longer due to their role in vascular repair. differentially regulated by at least 2-fold were selected and are illustrated in a volcano plot. Red indicates those genes that were up-regulated, black represents genes whose expression levels did not change, whereas green indicates genes that were down-regulated in differentiated pPICs compared with undifferentiated pPICs, n ¼ 3. (D) Western blots confirmed that pPIC exposure to a myogenic permissive environment led to an increase in IGF-1, TGF-b1, and NRG-1, and a decrease in HGF protein expression, n ¼ 3. Diff ¼ differentiated; pPIC ¼ porcine PW1 pos /Pax7 neg interstitial cell; Undiff ¼ undifferentiated.