Review
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Introduction
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Identifying a transplantable
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A transplantation method
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The survival of the graft
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Expert opinion
Intramuscular cell transplantation as a potential treatment of myopathies: clinical and preclinical relevant data Daniel Skuk† & Jacques P Tremblay †
CHUQ Research Center -- CHUL, Neurosciences Division -- Human Genetics, Quebec, Quebec, Canada
Introduction: Myopathies produce deficits in skeletal muscle function and, in some cases, literally progressive loss of skeletal muscles. The transplantation of cells able to differentiate into myofibers is an experimental strategy for the potential treatment of some of these diseases. Areas covered: Among the two routes used to deliver cells to skeletal muscles, that is intramuscular and intravascular, this paper focuses on the intramuscular route due to our expertise and because it is the most used in animal experiments and the only tested so far in humans. Given the absence of recent reviews about clinical observations and the profusion based on mouse results, this review prioritizes observations made in humans and non-human primates. The review provides a vision of cell transplantation in myology centered on what can be learned from clinical trials and from preclinical studies in non-human primates and leading mouse studies. Expert opinion: Experiments on myogenic cell transplantation in mice are essential to quickly identify potential treatments, but studies showing the possibility to scale up the methods in large mammals are indispensable to determine their applicability in humans and to design clinical protocols. Keywords: cell transplantation, clinical trials, myopathies, non-human primates, skeletal muscle Expert Opin. Biol. Ther. [Early Online]
1.
Introduction
Transplantation of myogenic cells (given the different possible meanings of the term ‘myogenic’, in the present review this is defined as corresponding to ‘giving rise to or forming muscular tissue’) is an experimental approach under study for the potential treatment of diseases of the skeletal muscle. Since myoblasts were the only myogenic cells identified for several decades (the earliest references to myoblast cultures date from at least 1915 [1]), they were the first cells to be proposed for this therapeutic use [2]. The term myoblasts defines the proliferating mononuclear myogenic cells that fuse among themselves to form myotubes, elongated and narrow syncytia that under appropriate conditions mature to form myofibers, the contractile multinucleated cells of the skeletal muscle parenchyma. This term applies to every instance in which this phenomenon takes place, that is, during histogenesis in utero, in postnatal myofiber regeneration and in in vitro culture of muscle cells. Partridge, Grounds and Sloper set the starting point of this approach in 1978, suggesting that ‘in subjects suffering from inherited recessive myopathies, muscle function might be restored if normal myoblasts could be made to fuse with defective muscle fibers’ [2]. Among all myopathies, Duchenne muscular dystrophy (DMD) has being the major target of myogenic cell transplantation. The reason is the relative frequency of this disease (a prevalence of 50 cases per million in the male 10.1517/14712598.2011.548800 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted
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Intramuscular cell transplantation as a potential treatment of myopathies
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The intramuscular transplantation of cells able to differentiate into myofibers is a potential therapeutic tool for the treatment of myopathies. For clinical purposes in myology, transplantable cells may accomplish at least one of the following: direct fusion with recipient myofibers to induce gene complementation, neoformation of myofibers and differentiation into satellite cells. The protocol of cell delivery to the target tissue must be defined in animal models allowing proper extrapolation to humans. The survival of the graft is mainly threatened by acute rejection, which is present when the grafted cells are allogeneic or express foreign epitopes. Current preclinical work may solve the main problems in the field, that is, restricted topographical participation of the grafted cells and acute rejection, by developing protocols that proven to be potentially useful in humans with an adequate balance between potential benefits and risks.
This box summarizes key points contained in the article.
population) and its severity: skeletal-muscle degeneration in the limbs and trunk, progressing during infancy and puberty, leading to severe to complete muscle loss, respiratory insufficiency and ultimately death from respiratory or cardiac complications. The early saga of clinical myoblast transplantation in myology is a good example of the relevance of having an appropriate preclinical basis to design clinical tests of innovative therapeutic procedures. The clinical trials conducted in the 1990s were undertaken after a few experiments in rodents, whose results were overstated and, in addition, gave no clues about the transplantation parameters to be used in humans (for a table summarizing these clinical trials see [3]). Using improvised transplantation protocols, these clinical trials reported scarce and limited results at the molecular level. With present knowledge, it is evident that these premature trials were designed with exaggerated expectancies about the properties of the transplanted cells. It was obviously believed that arbitrarily chosen amounts of myoblasts injected in a few sites of a skeletal muscle would diffuse spontaneously throughout the muscle to fuse with so many myofibers that a therapeutic effect would be achieved. Later research showed that this was optimistic speculation. Hence, a main lesson of that frustrated clinical saga is that it is crucial to know the actual behavior of the grafted cells under appropriate experimental conditions before planning clinical protocols able to give relevant information. This review aims to cover some information potentially relevant for clinical development of intramuscular cell transplantation in myology. For this reason, priority will be given to data obtained in humans and non-human primates. The profuse research in mice helped to define several properties 2
of myogenic-cell transplantation, but also yields much material without clear clinical significance. Conversely, preclinical studies in non-human primates helped to define technical and pharmacological parameters to improve the clinical protocols. 2.
Identifying a transplantable cell
Cell transplantation implies the implantation into the body of either differentiated cells that form the parenchyma and eventually the stroma of the organs, or precursor cells with the capacity to differentiate into the former. With regard to skeletal muscle, it seems technically difficult to transplant myofibers -- the differentiated elements of the parenchyma -- in order to reconstruct a tissue. Therefore, cell transplantation in myology was directed toward the second possibility: a precursor mononuclear cell. The task was facilitated by the fact that the skeletal muscle has specific committed stem cells, the satellite cells [4]. The niche of satellite cells is between the sarcolemma and the myofiber’s basal lamina, where they remain dormant until an event such as focal or total necrosis of a nearby myofiber is produced. Myofiber necrosis elicits the removal of myofiber debris by macrophages and the activation of satellite cells, which differentiate into proliferating myoblasts [5]. Satellite cells can completely regenerate myofibers without needing other sources of myogenic cells [6]. However, it is worth noting that other cells with the capacity to give rise to myoblasts were reported in mice and/or human muscles. These include ‘side-population cells’, ‘muscle-derived stem cells’, myoendothelial cells, mesoangioblasts/pericytes and CD133+ cells (for a review about these cells see [7]) and, more recently, a population of cells identified in human muscles that show aldehyde dehydrogenase activity [8]. It could be convenient to use the adjective ‘adult’ for myoblasts responsible of myofiber regeneration in postnatal life [9]. This would establish a difference from embryonic and fetal myoblasts that form skeletal muscles during histogenesis. This distinction is important because adult cells do not necessarily display the same gene expression profiles as embryonic or fetal counterparts [10]. One example of the different molecular dependence of myoblasts in the embryonic, fetal, neonatal and adult ages is the different relevance of myogenic determinants such as paired box proteins Pax3 and Pax7 [11]. Notably, adult myoblasts have different behavior and morphology from embryonic and fetal myoblasts in cell culture [12]. Satellite cells can be released from skeletal muscle fragments by standard enzymatic procedures of cell culture and can be easily sub-cultured as myoblasts, able to fuse to form myotubes that will eventually differentiate into myofibers [13]. The simplicity of obtaining and expanding these cells in vitro, facilitated the use of myoblasts for cell transplantation approaches not only in myology but, later, in cell-based therapies for heart pathologies [14].
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Properties needed in a cell to be candidate for transplantation in myology
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2.1
Lipton and Schultz reported in 1979 two of the three main properties that cell transplantation needs to be useful in myology [15]. They reported in rats and quails that intramuscularly grafted myoblasts fused with the recipient’s myofibers and, in a minor proportion, fused among themselves to form new small myofibers. Fusion of the grafted cells with the recipient’s myofibers gives rise to a phenomenon named gene complementation, meaning that the myofiber’s syncytia express proteins encoded by exogenous and endogenous nuclei [16]. Myofibers expressing exogenous and endogenous proteins are called mosaic or hybrid [16,17]. Gene complementation allows grafted myogenic cells to be vehicles of therapeutic genes. The typical example is the introduction of normal genomes in the genetically abnormal myofibers of patients with recessive genetic myopathies. The second property reported by Lipton and Schultz [15], the fusion of the grafted cells among themselves to form new myofibers, encourages research trying to restore functional contractile parenchyma in muscles in which too many myofibers were lost due to a degenerative or traumatic pathological condition. A third property was first reported by Yao and Kurachi in 1993 [18]: the possibility of myogenic cell transplantation creating a new pool of muscle precursor cells in the recipient muscle. Gene complementation The first report of gene complementation restoring a genetically missing protein in a myopathic animal was that of Partridge et al. in 1989 [19]. They transplanted normal mouse myoblasts in skeletal muscles of mdx mice, a mouse strain that develops a myopathy caused by the lack of dystrophin, the protein whose deficiency triggers DMD. They detected dystrophin in several myofibers after follow-ups of at least 3 weeks. Similar results were reported thereafter by other researchers [20,21] and this has become routine in myogeniccell transplantation research, including restoration of other proteins such as merosin in dy/dy mice (a model of merosindeficient congenital muscle dystrophy) [22] and dysferlin in SJL mice (a model of limb-girdle muscular dystrophy 2B) [23]. In humans, sporadic observations of enhanced dystrophin expression in DMD patients following normal-myoblast allotransplantation were reported during the clinical trials conducted in the 1990s (see [3] for a table summarizing these results). Nevertheless, most of the patients at that time gave negative results and only one study confirmed that the dystrophin detected after transplantation was of donor origin [24]. It was not until later clinical trials, using a protocol of cell injection and immunosuppression set up in non-human primates, which was shown that donor-derived dystrophin can be reliably detected in muscles of DMD patients allotransplanted with myoblasts from normal donors (Figure 1) [25-27]. 2.2
A key factor that makes myogenic-cell implantation difficult is the fact that intracellular proteins in myofibers remain near the nucleus of origin, in a region named the nuclear domain [28]. This is both because the mRNA diffuses only 100 µm from the nucleus [29] and because the proteins have also a limited spread [30]. Consequently, proteins of donor origin remain expressed in the myofiber regions in which the transplanted cells fused. The nuclear domain’s length varies for each protein, depending on factors such as if they remain soluble or anchored to stationary cellular components [30]. This factor conditions the analysis of transplantation results and a striking example was the comparison between green fluorescent protein and dystrophin in one study: following transplantation of cells expressing dystrophin and green fluorescent protein in mdx mice, dystrophin was detected through about 116 µm in myofibers, while green fluorescent protein diffused up to 1500 µm [31]. It is especially important to consider this factor, together with the differences in sensitivity among detection methods, when the results for a transgenic label in a transplantation experiment are extrapolated to a therapeutic protein, such as dystrophin in DMD. Dystrophin restoration in limited myofiber regions was remarked upon in some clinical trials of normal myoblast allotransplantation in DMD patients. Gussoni et al. [32] reported dystrophin expression ranging from 300 to more than 450 µm (the largest size of their analysis) in the myofibers, although without confirming whether this was actually donor-derived dystrophin. In another DMD patient in which donor-derived dystrophin was effectively detected, myofiber regions expressing donor-derived dystrophin were from at least 700 µm to more than 2 mm in size [26]. Restoration of myofibers In DMD, as in other myopathies, worsening of muscle weakness is produced by the progressive loss of myofibers. The optimal treatment for the advanced phases of these diseases must involve not only molecular correction but also restitution of functional myofibers. This second factor becomes even more important as the disease progresses. In mouse experiments, myoblast transplantation showed the capacity to completely or almost completely regenerate muscles destroyed by different acute treatments, recovering muscle mass and strength [33-36]. However, these acute experimental conditions are different from a chronic degenerative myopathy. In these mouse experiments, the damage was massive and acute, preserving the endomysial scaffold, in which the basal lamina seems important for an orderly muscle regeneration as observed in rats and rabbits [37]. In contrast, myofiber necrosis is insidious in chronic degenerative myopathies and the muscle is progressively replaced by adipose and/or fibrous tissue, losing the endomysial support. Mouse experiments showed that, under some conditions, myoblast transplantation could form new myofibers independent of a previous endomysial scaffold. This was the case of the fusion of the implanted myoblasts among themselves in 2.3
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Intramuscular cell transplantation as a potential treatment of myopathies
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Figure 1. Allotransplantation of normal myoblasts from an adult related donor in 1 cm3 of the Tibialis anterior of a DMD patient under tacrolimus immunosuppression, in a Phase IA clinical trial done by the authors. A. Several parallel intramuscular cell-injections were done with a 100-µl Hamilton precision syringe. The cells were injected homogeneously during the needle extraction, and the number and distribution of injections was monitored with the help of a sterile transparent dressing with a 5-mm grid. B. The whole cross-section of one of the biopsies done 1 month posttransplantation in a cell-grafted site is shown stained for fluorescent immunodetection of dystrophin. C. A schematic representation of B helps to illustrate the distribution of the dystrophin+ myofibers, represented in black on the gray background of the tissue.
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mdx mice in which muscle irradiation was done prior to transplantation [38]. Exogenous myoblasts were also able to neoform ectopical muscles after subcutaneous transplantation in mice [39]. Otherwise, whether myogenic-cell transplantation could form functional myofibers in skeletal muscles that degenerated to fibrosis and/or fat substitution remains insufficiently studied. One study showed that myotubes and regenerating myofibers developed across implants of adipose tissue inserted in regenerating skeletal muscles in mice, suggesting that perhaps adipose tissue in dystrophic muscle would not be an impediment for the neoformation of myofibers [40]. The presence of fat tissue in the muscle, however, could induce the transdifferentiation of some human muscle precursor cells into adipocytes, depending on whether they express or not CD34 [41]. Cell sorting could be useful to separate CD34+ human muscle-derived cells, which adipogenic potential after transplantation in immunodeficient mice, from CD34-negative cells, which did not transdifferentiate into fat cells [41]. Moving from these few observations in mice towards a useful procedure of muscle restitution in humans is still a challenge, among other factors because mice have inherently a better muscle regeneration competence than large animals [42]. Nevertheless, some clinical observations could be encouraging: in DMD patients allotransplanted with normal myoblasts, clusters of small dystrophin+ myofibers were interpreted as neo-formed by the fusion of the implanted myoblasts among themselves [25]. Formation of donor-derived satellite cells If some of the grafted cells remain in the muscle as mononuclear muscle-precursor cells, and especially as satellite cells, this would imply that the effect of transplantation may not be limited to the early fusion of the implanted cells but that it would provide also a source of normal muscle-committed stem cells to participate later in muscle hypertrophy and regeneration. Potentially, this could suggest that the amount of myofibers expressing dystrophin in DMD patients receiving transplants of normal myoblasts may increase over time, proven that a phenomenon similar to that reported in mdx mice is produced, that is, the expansion of clusters of myofibers expressing revertant dystrophin [43]. This property was extensively reported after transplanting mouse and human myoblasts in mice. The pioneer paper of Yao and Kurachi in 1993 reported that up to 2% of myoblast colonies obtained in culture from muscles grafted with mouse myoblasts were of donor origin, even after 5 months, and that these myoblasts were able to fuse with myofibers after a new transplantation, giving rise again to mononuclear cell precursors [18]. Gross and Morgan grafted mouse myoblasts into irradiated muscles and produced muscle necrosis four times at 3-week intervals using notexin, a substance that damages myofibers but not satellite cells [44]. Since there were regenerating donor-derived 2.4
myofibers after each muscle injury, this proved that some grafted myoblasts remained as muscle precursor cells competent to contribute to muscle regeneration after repeated muscle damages. Similar results were reported following the fate of fluorescent-labeled mouse myoblasts by in vivo fluorescence imaging [45]. Irintchev et al. were the first to report that some intramuscularly-transplanted myoblasts became satellite cells in the recipient [36,39]. They partially reconstituted destroyed muscles by mouse myoblast transplantation and they identified donor-derived mononuclear cells expressing M-cadherin in the border with myofibers, a characteristic of satellite cells [36]. In a second experiment, they formed ectopic muscles by subcutaneous myoblast implantation, and these neomuscles showed M-cadherin+ cells adjacent to myofibers, indenting the sarcolemma under the basal lamina in a typical satellite-cell position [39]. Heslop et al. [46] and Xu et al. [45] further identified donor-derived satellite cells in muscles that received transplants of mouse myoblasts, respectively by immunodetection of CD34 and Pax7. Similar results were reported with human myoblasts transplanted into immunodeficient mice. Abundant donorderived satellite cells were detected in mouse muscles one month after transplantation of myoblasts from adult humans (Figure 2) [47]. These human-derived satellite cells explain the presence of human donor-derived muscle-precursor cells evidenced in experiments of culture, re-transplantation and regeneration in immunodeficient mice [47] and confirmed preliminary observations suggesting that some human nuclei in isolated human/mouse hybrid myofibers were satellite cells [48]. Human fetal myoblasts also gave rise to mononucleated muscle-precursor cells following transplantation in immunodeficient mice [49]. Clinical observations suggest that formation of donorderived satellite cells could also occur following myoblast allotransplantation in DMD patients. Donor-derived mononuclear cells were detected in the muscles of DMD patients that received myoblast allotransplantations, and some of the donor-derived nuclei were in an anatomical position susceptible to correspond to satellite cells [25,47]. Other human-derived cells that were reported to form donor-derived satellite cells following transplantation in immunodeficient mice were muscle-derived CD133+ cells [50], and probably muscle-derived aldehyde-dehydrogenase+ cells [8]. 3.
A transplantation method
Identifying cells suitable for transplantation is the first step in developing a cell-based therapy. A crucial second stage is to deliver these cells to the target tissue in a way to reach a therapeutic result. Two major routes were studied in order to deliver myogenic cells to skeletal muscles: intramuscular injection and intravascular infusion. Since the subject of the present review
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Confocal microscopy
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Standard microscopy
Intramuscular cell transplantation as a potential treatment of myopathies
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Figure 2. Donor-derived satellite cells evidenced by fluorescent immunodetection in cross-sections of skeletal muscles of immunodeficient mice transplanted with myoblasts from an adult human donor 4 weeks before. A. A region of a mouse muscle composed almost entirely by human-derived myofibers, identified by a monoclonal antibody that reacts with human dystrophin but not with mouse dystrophin (red fluorescence). In a serial double-stained section, it can be seen that the region shown in A is filled with human nuclei (B., green fluorescence using an anti-human lamin A/C monoclonal antibody) and contains several nuclei expressing Pax7, a marker of satellite cells (C, red fluorescence). D to G show the region in the white rectangle in C, illustrating the co-detection of Pax7, human nuclei (human lamin A/C+) and basal lamina (laminin, blue fluorescence). Arrows indicate several human donor-derived nuclei that express Pax7 (a marker of satellite cells) and are placed in the periphery of myofibers and inside the basal lamina (i.e., in the anatomical position of satellite cells). Arrowheads indicate human Pax7+ nuclei outside the basal lamina. H to K show a single myofiber with the same triple co-detection, but with a higher magnification and in images taken with confocal microscopy. Confocal microscopy allows better discrimination of the intra-nuclear typical staining of Pax7, the peripheral nuclear labeling of lamin A/C and the anatomical position of the two donor-derived satellite cells in the periphery of the myofiber under the basal lamina. The lower magnification images taken by standard fluorescence microscopy aim to illustrate the abundance of donor-derived satellite cells in these conditions. Scale bars = 100 µm (A -- C) and 10 µm (D -- K).
is the intramuscular route, we will only mention that, up to now, a specific type of cells called mesoangioblasts was the only one that seems to be sufficiently efficient by intraarterial infusion in animal models of muscular dystrophies, including dogs with dystrophinopathy [51,52], although some positive results were also reported with CD133+ cells in mice [53]. Other cell types more recently reported as able to differentiate in muscle precursor cells following transplantation in mouse muscles require direct intramuscular injection (for a recent review about these cells see reference [54]). In the event that these cells prove to be useful for intramuscular transplantation in the clinics, they will be probably concerned by the same considerations made below. 6
Cell injection strategy: a crucial factor The major restriction of the intramuscular route is that the grafted cells fuse essentially with the myofibers near the injection trajectories. In non-human primates in which myoblasts are injected into normal muscles without pre-treatments, each myoblast-injection leads a strip of hybrid myofibers in muscle cross sections (Figures 3 and 4), as if the injected cells fused just with the myofibers damaged by the injection [55-57]. The pattern of donor-dystrophin expression in DMD patients receiving allotransplants of normal myoblasts was sometimes similar [24,25,27], but in other cases it was less defined (Figure 1) [25-27]. The local fusion of the implanted myoblasts would not be a problem if the donor-derived proteins were capable of 3.1
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Figure 3. Schematic representation of the mechanism allowing the incorporation of the grafted myoblasts in the recipient’s myofibers of normal skeletal muscles. The point of departure of the illustration is the cross section of a macaque skeletal muscle that was treated, one month before, with an injection of autologous myoblasts genetically modified ex vivo to express a micro-dystrophin under a MCK promoter and coupled to a peptide tag (I, peroxidase immunodetection of the peptide tag). A shows a needle traversing a muscle fascicle in this muscle and injecting the cells homogeneously during the needle extraction (orange arrow). B to G illustrate the process of injected-cell uptake in a myofiber isolated from this fascicle. This myofiber (B) is damaged by the needle and undergoes segmental necrosis (C). The necrotic region is invaded by macrophages (D) with two main functions: phagocytosis of the necrotic debris and release of factors helping myofiber regeneration. Myofiber regeneration is produced by the activation of the recipient’s satellite cells, which proliferate as myoblasts that fuse together (E). This regenerative process recruits some of the injected myogenic cells (E). The nuclei of the transplanted cells that participated in this regeneration are integrated in the myotubes that fill the gap lead by segmental necrosis (F). Later, these donor-derived nuclei will produce donor-derived proteins throughout a restricted length of the myofiber (G), leading to restricted regions of donor-protein expression in the fascicle (H). In histological cross sections of the cell-grafted muscle, this restricted fusion is observed as ‘strips’ of donor-derived protein expression (I).
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Figure 4. Intramuscular myogenic-cell transplantation in non-human primates using different injection devices. A. A precision Hamilton syringe operated manually. B. A repeating PB600-1 dispenser with a Hamilton syringe. C. A specific prototype for repetitive intramuscular cell injections. As in DMD patients, the density of cell injections is controlled with a sterile transparent dressing with a 5-mm grid. After at least one month, the fusion of the injected cells in the recipient’s myofibers is analyzed in cross sections of muscle biopsies using histological techniques to detect the transgenic proteins in the transplanted cells (D to F). The images correspond to cross sections of macaque muscles grafted with myoblasts labeled either with b-galactosidase (D, E) or a micro-dystrophin coupled to a peptide tag (F). Myofibers expressing donor-derived proteins are respectively detected by histochemical detection of b-galactosidase (D, E, dark staining) or fluorescent immunohistological detection of the peptide tag (F). The distribution of the myofibers expressing donor-derived proteins reproduces the pattern of the original cell-injection trajectories (indicated by the arrows). The density of b-galactosidase+ myofibers is higher in E than in D, because the density of cell injections was higher: 25 per cm2 in D and 100 per cm2 in E. Scale bars: 500 µm (D -- F).
diffusing throughout each myofiber. However, given that proteins are restricted to nuclear domains, which are very short in the case of dystrophin, myogenic-cell transplantation must insure a diffuse fusion of the implanted myoblasts throughout the muscle. If myoblasts are injected in a saline solution and without muscle pre-treatments, the only possibility to achieve a significant homogeneous expression of donor-derived proteins is to perform cell injections very close to each other and through the full muscle thickness [55]. With this technique, the amount of muscle expressing a donor-derived protein depends on the number of injections per muscle volume (Figure 4), that is, the more injections done, the better result is achieved in terms of donorprotein expression in the muscle, such as b-galactosidase or 8
dystrophin in monkeys [56,57] or dystrophin in DMD patients [25-27]. Looking to make a difference from the earlier clinical trials in which myoblast transplantation was performed by few injections away from each other, this strategy was called a high-density injections protocol [3], meaning by ‘density’ the amount of injections per volume of muscle. Is it possible to increase the efficiency of myoblast injections?
3.2
It is obvious that reducing the number of cell injections per volume of muscle will facilitate transplantation. To achieve that, the amount of muscle expressing a therapeutic protein (e.g., dystrophin in DMD) per cell injection must be increased.
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The factors reducing gene complementation to the vicinity of cell injections seems to be only two: i) the grafted cells fuse only with myofibers around the injection trajectory, and ii) proteins remain in nuclear domains. Therefore, the possibilities to reduce the number of injections are also two: i) to make grafted cells fuse more diffusely, and ii) to increase the nuclear domain of the therapeutic protein. Two factors may explain why the injected cells fuse mostly in the thin regions traversed by the cell injections: i) the transplanted cells lack the ability to move in the recipient muscled and/or ii) a muscle regeneration process is obligatory to draw the grafted cells into fusing with the recipient myofibers and, at least in a normal muscle, this occurs only in the regions damaged by the injections. In the belief that the first factor was the key one, several studies attempted to promote the intramuscular migration of the grafted myoblasts by inducing in them the secretion of enzymes that degrade the extracellular matrix and are crucial for the intra-tissular motion of cells [58-62]. Some of these studies reported an improved migration of myoblasts under particular experimental conditions in vitro and in vivo in mice, and even an improved transplantation success. Nonetheless, tests in non-human primates exposing myoblasts to motogenic factors (basic fibroblast growth factor and IGF-1) produced an enhanced migration in vitro and in some in vivo conditions in immunodeficient mice, but did not increase the success of allotransplantation in macaques [62]. Indeed, recent experiments in macaques indicate that transplanted myoblasts have by themselves the ability to migrate into the muscle, but that they migrate basically when there is a muscle damage and to fuse with regenerating myofibers [63]. More experiments addressed the second factor, with better transplantation results. They increased the uptake of the engrafted cells in the recipient muscle by increasing the number of regenerating myofibers. Intramuscular injection of myotoxins, like phospholipases derived from snake venoms [21,64] and local anesthetics [65,66], were efficient for myoblast transplantation in mice. Snake venom phospholipases like notexin and cardiotoxin are regularly used for studies of myogenic-cell transplantation in mice, and were used in other animal models such as rabbits [67]. Looking for an easier method applicable in humans, a study in mice increased myofiber regeneration in mdx mice by intense muscular exercise, almost doubling the success of myoblast transplantation [68]. In mice, hindering the recipient’s satellite cell proliferation favors the participation of the transplanted cells in muscle regeneration. The most frequent method to inhibit the proliferation of the recipient’s satellite cells is to submit skeletal muscles to high doses of ionizing radiation before transplantation [21,33,34,64]. Freezing the recipient muscles (cryoinjury) combines myofiber necrosis with killing the recipient’s satellite cells, and was used in mice as a pretreatment to increase the engraftment of the implanted myogenic cells [35,36,48,50].
Cryoinjury, which necroses also vessels and nerves, is appropriate for experiments in small muscles of mice but seems unthinkable for clinical use. Up to now, only the co-injection of myoblasts with notexin improved the success of cell transplantation in nonhuman primates, although this was observed only when the injections were highly concentrated in small volumes of muscle [55,69]. The possibility of increasing the nuclear domain of donorderived proteins was poorly studied. There is only one study in mdx mice, reporting a threefold expansion of dystrophin’s nuclear domain after transplanting transgenic myoblasts overexpressing dystrophin 50-fold [70]. Tools for intramuscular cell transplantation In clinical conditions, a protocol of myogenic cell transplantation involving many intramuscular injections makes the manual use of single precision syringes (Figures 1A and 4A) acceptable only for very small volumes of muscle [25,27]. Done in that way, the procedure is extremely slow and very demanding in terms of permanently ensuring cell delivery at the right depth. In a first attempt to partially improve the technique, some dispensers used for repetitive delivery of small volumes of liquid in serological analysis were tested [71]. The monosyringe dispenser PB600-1 from Hamilton was used for myoblast transplantations in monkeys (Figure 4B) [57,71], rabbits [72] and even an adult DMD patient [26]. Nevertheless, its clinical use seems restricted to small muscles or muscle regions and, yet again, the accuracy required to deliver cells specifically in the muscle through a thick skin could be a challenge. For this reason, it could be important to develop tools specialized for the percutaneous intra-tissular injection of cells, focusing on clinical use. As a first step, a semimanual prototype was developed that delivers very small volumes of cell suspension homogeneously through the intramuscular trajectory via several needles at the same time, avoiding as much as possible the delivery of cells into the skin and hypodermis (Figure 4C) [73]. 3.3
4.
The survival of the graft
Producing sufficient amounts of an optimal transplantable cell and delivering them adequately to the recipient must be completed by the survival of the graft. This survival should be analyzed at two periods: early and long-term. Early cell death: a poorly understood but not critical phenomenon
4.1
Several studies indicate that grafted myoblasts undergo a considerable mortality mostly within the first three days after transplantation. This was inferred from the post-transplantation loss of different markers used to label myoblasts in mice [74-79] and pigs [80]. Additionally, morphological evidence of apoptosis (by immunodetection of active caspase 3) and necrosis (by
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Intramuscular cell transplantation as a potential treatment of myopathies
histochemical detection of intracellular calcium deposition) was reported among the implanted myoblasts during this early post-transplantation period [79]. The importance of this early cell death is frequently overstated, that is it is fallaciously referred as a factor ‘limiting’ myoblast transplantation. In fact, this phenomenon does not affect the level of cell engraftment, which is actually dependent on the technique of cell implantation. Indeed, not all grafted myoblasts die [78-80] and, in mouse experiments, the proliferation of the surviving cells compensates partially [78] or totally [79] for the cell death. This process of cell death and proliferation is still not well understood. Some studies are contradictory, and, in some cases, this is due to methodological differences and even mistakes [79]. Different factors were blamed of this death, but experiments trying to control each of them never prevented the whole cell death and only minimal enhancements of survival were reported (understanding as ‘survival’ the extrapolation of quantification in the recipient muscle of a label present in the grafted cells). An exception was the administration of an anti- lymphocyte function associated antigen 1 (LFA1) antibody in specific experimental conditions in mice [76]. Initial studies in mice accused acute inflammation of killing the grafted myoblasts [76,81]. A specific study, however, did not find evidence that neutrophils, macrophages or NK cells deplete the grafted myoblast population [82]. Other causes suggested to induce apoptosis of the grafted myoblasts were hypoxia [68] and anoikis [83]. A research group raised the hypothesis that the survival of the myoblast graft is due to a minor subpopulation of cells with specific capacities to evade the early cell death and to proliferate to a great extent [78,84]. This hypothesis leaves unanswered the cause of the cell death but could explain the curious fact that, even if one or more factors could indiscriminately kill the grafted cells, the whole grafted-cell population is never eliminated. Considering that the early cell death never eliminates the cell graft and that the surviving cells proliferate to ensure graft success, the sole possible advantage of controlling this cell death would be, in theory, a reduction of the amount of cells needed for transplantation. A clear type of cell death observed after muscle-cell transplantation in non-human primates is the ischemic necrosis of the inner region of the intramuscular accumulations of injected cells (Figure 5) [85]. Since the implanted cells form avascular collections whose survival depends on the limited diffusion of oxygen and nutrients from the surrounding tissue, only a peripheral layer of around 100 -- 200 µm survives the first hours post-transplantation. Given that the extent of ischemic necrosis depends on the size of the cell graft, a massive ischemic death can be prevented or reduced by transplantation strategies in which formation of too large cell accumulations is avoided, ensuring that most cells stay within 100 -- 200 µm of the surrounding tissue [85]. 10
Long-term survival Immune rejection seems the sole factor menacing the longterm survival of myogenic-cell transplantation. The first description of lymphocyte infiltration with lost of the myoblast graft after allogeneic transplantation was done in mice as early as in 1979 [86]. Posterior reports identified CD8+ and CD4+ cells in these infiltrates [87-89]. Focal infiltrations of CD8+ lymphocytes and lymphocyte invasion of myofibers were observed in macaques that received myoblast allotransplantation with low immunosuppression [56,69,90]. In contrast with early cell death, acute rejection, if not controlled, prevents the success of cell transplantation. Nevertheless, this factor is well defined and there are strategies to control it, essentially pharmacological immunosuppression. Special attention should be given, however, to the choice of immunosuppressive drugs, since some of them can be detrimental for the transplanted cells (for a table about immunosuppressive drugs and myogenic cell transplantation see [3]). Several tests supported the usefulness of tacrolimus for myoblast allotransplantation in mice [21,22] and non-human primates [55,56,69,71,90,91]. For this reason, the most recent clinical trials of myoblast allotransplantation were carried out under tacrolimus monotherapy [25-27]. The first of them, involving nine DMD patients, had a follow-up of just one month [25,27], but in a DMD patient in which the follow-up was of 18 months, donor-derived dystrophin was preserved throughout this period: 27.5% of the myofibers were donor-dystrophin-positive one month post-transplantation and 34.5% at 18 months [26]. Considering the adverse effects of immunosuppression, a major goal in clinical transplantation is specific immune tolerance, that is long-term unresponsiveness to the graft in the recipient, preserving immune responses against pathogens or cancer. Transient immunosuppression allowed immune tolerance to myoblast grafts in some mouse strains [92], although not in other mouse strains [35,92] nor in non-human primates [55]. For myoblast allotransplantation in mice, central tolerance via mixed chimerism [93,94] was superior to anti-CD154 administration and donor-specific transfusion [95]. Nevertheless, central tolerance in the context of myogenic-cell allotransplantation would not include neoantigens in the hybrid myofibers which would need also peripheral tolerance [96]. A different experimental strategy to elude immunosuppression is the autotransplantation of cells genetically corrected ex vivo (for a recent review detailing this topic see [54]). Results in mice support a potential development of this strategy, and the viability of this approach was verified in non-human primates albeit for only one month post-transplantation [57]. This strategy avoids incompatibility of the MHC among the recipient and the transplanted mononuclear cells, but leaves the possibility of eliciting a specific immune response if the genetically-modified cells express epitopes not shared with the recipient [97]. 4.2
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c
Figure 5. Ischemic necrosis in a collection of grafted myoblasts one day after a unique injection of 20 106 myoblasts in the skeletal muscle of a macaque. The whole section of a muscle biopsy is shown (the epimysium is observed in the top of the biopsy), stained for histochemical detection of the oxidative enzyme nicotinamide adenine dinucleotide reduced diaphorase. The cell-suspension infiltrated the muscle in a polymorphic pattern, giving rise to cell accumulations of different sizes (a -- e). The arrowheads circumscribe these collections of transplanted myoblasts. Two regions are clearly delimitated in the largest accumulations of implanted cells (a -- c): a peripheral ring strongly stained (living cells with oxidative activity) and a central region almost devoid of oxidative reaction (necrosed cells without oxidative activity). The small accumulations of implanted cells (d, e) do not exhibit an ischemic central region. Scale bar: 500 µm.
5.
Expert opinion
Paraphrasing Brazelton and Morris [98], we realize that several ‘promising’ transplantable cells were not included in this review. These authors, in a review of pharmacology, made a distinction between a clinical drug and a molecule, stressing that ‘few molecules become drugs’ and specifying that they called molecule ‘a substance that, when injected into a rodent, results in abstracts, publications, grant funds, academic promotions and public stock offerings’ [98]. Since sometimes a similar concern could be pertinent in the context of cell transplantation, we kept some distance from reports claiming the discovery in mice of cells with huge myogenic potential (on the other hand, there are several reviews dealing with that topic). This does not imply a blind reluctance but rather a wary eye on that topic: there were already shooting stars in the sky of cell-based therapies in myology, such as the overvaluation of mouse observations about the potential of bone marrow transplantation to provide circulating myogenic progenitors able to restore dystrophin in DMD skeletal
muscles, which when analyzed in a human patient or tested experimentally in dystrophic dogs with dystrophinopathy showed to be nil or irrelevant [99,100]. It is therefore important to assess in the best animal model as possible the extent to which a ‘promising’ cell type has actually some clinical potential or, in contrast, to what extent the properties reported are only curiosities observed in specific experimental conditions and/or unique to the mouse biology. We also realize that the basis of the present review were studies in which the transplanted cells were the mononuclear precursor cells whose function is to fuse among themselves in order to form myotubes giving rise to myofibers, that is, myoblasts. In fact, regarding clinical transplantation, adult myoblasts were the only myogenic cells properly tested in myology, restoring dystrophin in variable amounts of myofibers in DMD patients and forming putative new myofibers. This was possible by using a method of intramuscular implantation previously developed in non-human primates and a quite suitable control of acute rejection with an immunosuppressant also tested in non-human primates. If other myogenic cells are finally tested clinically in the future for intramuscular transplantation, it is possible that they will be affected by the same challenges as adult myoblasts, that is, the restricted integration to the skeletal muscle only in the regions around the injection trajectories and the need to control their acute rejection with immunosuppression. These are thus the two important challenges in order to facilitate the clinical applicability of intramuscular cell transplantation in clinical settings with any benefit to myopathic patients: i) to expand the cell engraftment while, at the same time, reducing as much as possible the complications inherent to a protocol of cell delivery using a high density of cell injections, and ii) to reduce the toxicity of the approaches to control acute rejection. The last issue could be attained by refinements of the immunosuppression protocols or, ideally, through efficient strategies of immune tolerance. These are major goals in the global field of transplantation, and myogenic cell transplantation will be subsidiary to the progress in this research. Otherwise, cell transplantation potentially offers a specific alternative to avoid acute rejection or, at least, to control it with lower nuisances to the recipient, through the in vitro manipulation of cells. Regarding the promises of gene therapy or the intravascular delivery of myogenic cells, the difficulties associated with the intramuscular transplantation of cells seems to relegate this approach or to deny the difficulties through the quest for a ‘magic bullet’. However, as the treatment of cancer needs frequently the combination of different approaches such as surgery, radiotherapy and chemotherapy, the treatment of myopathies such as DMD could also need the combination of several approaches. It is logical to predict that effectively correcting the molecular problem in a disease such as DMD will stop its progression, but it also should be clear that this does not restore the lost muscle. Restoring muscle in patients in whom too many myofibers were lost, will inevitably need
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Intramuscular cell transplantation as a potential treatment of myopathies
strategies overlapping cell transplantation and tissue engineering. Thus, a third important challenge in the field of cell transplantation in myology is to address some research to clarify the factors that could contribute to forming new skeletal muscle in muscles in which myofibers were lost and replaced by fibrosis and fat tissue. Another weakness in the field of cell transplantation is that, when moving to clinical trials, some researchers seem to disregard the actual behavior of the transplanted cells. As in the early clinical trials of myoblast transplantation, there is a risk with proceeding as if the simple act of injecting cells into a tissue, no matter how, is ‘cell transplantation’. Something similar to putting a kidney from a donor into a recipient without performing vascular and ureterovesical anastomoses being called ‘kidney transplantation’: the term is semantically correct but the procedure, done that way, is a medical aberration. This obvious statement, however, does not seem so evident in the field of cell transplantation: often arbitrarily chosen amounts of cells are injected, no matter how, with no more rationale that the hypothetical way the cells are expected to act, and no more experimental basis than mouse experiments not designed to clarify the transplantation parameters which could be needed in the larger organs of a human. Therefore, a good knowledge of the behavior of the transplanted cells in the recipient is crucial for designing Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.
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Acknowledgements This work was supported by a grant of the Jesse’s Journey Foundation for Gene and Cell Therapy of Canada to Daniel Skuk.
Declaration of interest JP Tremblay has shares in CellGene Inc., a biotechnological company created to accelerate the development of cell therapies. D Skuk declares no conflict of interest.
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Affiliation
Daniel Skuk†1 & Jacques P Tremblay2 † Author for correspondence 1 CHUQ Research Center -- CHUL, Neurosciences Division -- Human Genetics, 2705 Boulevard Laurier, Quebec, Quebec G1V 4G2, Canada E-mail:
[email protected] 2 Centre de Recherche du Centre Hospitalier Universitaire de Que´bec, Sainte-Foy, Quebec, Canada
