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TISSUE-SPECIFIC STEM CELLS

Bone Marrow Transplantation Attenuates the Myopathic Phenotype of a Muscular Mouse Model of Spinal Muscular Atrophy

^aMolecular Neurogenetics Laboratory, Institut National de la Santé et de la Recherche Médicale, Inserm, U798, Evry, F-91057 France; University of Evry, Evry, F-91057 France;
^bInserm, U429, Paris, France;
^cDepartment de Pharmacologie Clinique, Hôpital de la Pitié-Salpêtrière, Paris, France

Key Words. Spinal muscular atrophy • Survival of motor neuron • Bone marrow • Transplantation • Skeletal muscle Hepatocyte growth factor

Correspondence: Judith Melki, M.D., Ph.D., Molecular Neurogenetics Laboratory, INSERM U798, 2 rue Gaston Crémieux CP5724, 91057 Evry, France. Telephone: 331-6087-4552; Fax: 331-6087-4550; e-mail: j.melki{at}genopole.inserm.fr

Received on March 23, 2006; accepted for publication on July 24, 2006.

First published online in STEM CELLS EXPRESS  August 3, 2006.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Bone marrow (BM) transplantation was performed on a muscular mouse model of spinal muscular atrophy that had been created by mutating the survival of motor neuron gene (Smn) in myofibers only. This model is characterized by a severe myopathy and progressive loss of muscle fibers leading to paralysis. Transplantation of wild-type BM cells following irradiation at a low dose (6 Gy) improved motor capacity (+85%). This correlated with a normalization of myofiber number associated with a higher number of regenerating myofibers (1.6-fold increase) and an activation of CD34 and Pax7 satellite cells. However, BM cells had a very limited capacity to replace or fuse to mutant myofibers (2%). These data suggest that BM transplantation was able to attenuate the myopathic phenotype through an improvement of skeletal muscle regeneration of recipient mutant mice, a process likely mediated by a biological activity of BM-derived cells. This hypothesis was further supported by the capacity of muscle protein extracts from transplanted mutant mice to promote myoblast proliferation in vitro (1.6-fold increase). In addition, a tremendous upregulation of hepatocyte growth factor (HGF), which activates quiescent satellite cells, was found in skeletal muscle of transplanted mutants compared with nontransplanted mutants. Eventually, thanks to the Cre-loxP system, we show that BM-derived muscle cells were strong candidates harboring this biological activity. Taken together, our data suggest that a biological activity is likely involved in muscle regeneration improvement mediated by BM transplantation. HGF may represent an attractive paracrine mechanism to support this activity.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Spinal muscular atrophy (SMA) is one of the most frequent genetic causes of death in childhood. SMA is a recessive autosomal disorder caused by mutations of the survival of motor neuron gene (SMN1) and characterized by degeneration of motor neurons associated with muscle paralysis [1, 2]. SMN is an ubiquitously expressed protein of 294 amino acids that forms a large multiprotein complex both in the cytoplasm and in the nucleus where it is concentrated in a structure called "gems" (gemini of coiled bodies) [3]. SMN is involved in and facilitates cytoplasmic assembly of small nuclear ribonucleoprotein into the spliceosome, a large RNA-protein complex that catalyzes the splicing reaction [4]. In the nucleus, SMN appears to be directly involved in pre-mRNA splicing, transcription, and metabolism of ribosomal RNA [5–7].

The most frequent mutation found in SMA patients is a homozygous deletion of SMN1 exon 7 [1]. Murine Smn exon 7 was flanked by two Lox P sequences (Smn^F7), and deletion of this exon (Smn⁷) has been directed to either neurons or skeletal muscle by using the Cre recombinase system (neuronal or muscular mutants, respectively) [8–11]. To direct homozygous deletion of Smn exon 7 to skeletal muscle only, the Cre recombinase was placed under the control of the human -skeletal actin (HSA) gene promoter (HSA-Cre) [12]. Characterization of the HSA-Cre transgenic line revealed a strong and specific Cre recombinase activity in skeletal muscle fibers leading to deletion of Smn exon 7 in myofibers only [11, 12]. In addition, the Cre recombinase is expressed in fused myotubes but not in myoblasts nor in other cell types [11]. Mutant mice (HSA-Cre, Smn^F7/F7), called mild muscular mutant, display a progressive myopathic phenotype characterized by necrosis of muscle fibers associated with variation in the size of the myofibers and infiltration of interstitial tissues [11]. A high proportion of myofibers with central nuclei was observed and immunolabeled with embryonic myosin heavy chain antibody, indicating that they correspond to regenerating myofibers and do not reflect an aspect of the pathology (data not shown; available on request). The regenerating myofibers compensate for the progressive loss of mature myofibers within the first 6 months after birth. Then, a progressive decline in the muscle regeneration process no longer counterbalances the muscle degeneration leading to a dramatic loss of myofibers associated with a progressive motor defect [11]. The mechanisms by which Smn⁷ results in myofiber necrosis and loss remain unclear. The same Smn mutation directed to the liver results in a severe liver phenotype characterized by marked rarefaction of hepatocytes and liver dysfunction [13]. These data indicate that Smn⁷ was not able to compensate for the lack of full-length Smn in various mammalian cell types [13].

Several reports clearly demonstrate that bone marrow-derived cells (BMDCs) contribute to skeletal muscle fibers. Bone marrow transplantations (BMTs) using unfractionated marrow have been performed in mice and showed for the first time that bone marrow contains cells able to be recruited to a damaged muscle [14]. In a transplanted mdx mouse, a mouse model of Duchenne muscular dystrophy (DMD), only 1%–3% of myofibers with detectable marker nuclei coming from donor cells were observed [15]. In mdx4cv mutant, another mouse model of DMD in which almost no revertant myofibers were observed, transplantation of unfractionated bone marrow did not result in more than 1% of total muscle fibers [16]. These results have shown a low contribution of BMDCs into muscle fibers. In addition, fusion instead of trans-differentiation of BMDCs has been recently reported in myofibers, hepatocytes, and Purkinje cells [17–19]. Moreover, it has been reported that circulating myeloid cells, including macrophages and granulocytes, were able to incorporate into muscle fibers [17], a feature that remains controversial [20]. These data have shown a limited capacity of BMDCs to replace damaged myofibers.

Multiple variables are involved in the incorporation of marrow cells to muscle fibers, including irradiation, donor-cell dose, and timing of muscle injury relative to the transplantation [21]. In addition, it has been shown that the proportion of muscle fibers incorporating adult BMDCs depends on the type of skeletal muscles or physiological stress applied to muscles [22, 23]. These data suggest that the recruitment of BMDCs into muscle fibers might depend on signals coming from host muscle. mdx mouse has been selected as a model for human muscular dystrophies, but it does not develop a motor defect nor loss of myofibers within the first year of life, which represents a major difference from the human disease [11, 24]. To determine whether BMT might have any beneficial effects on a model exhibiting a severe myopathic phenotype, unfractionated bone marrow cells were transplanted into mild muscular mutant mice (HSA-Cre, Smn^F7/F7).

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Mice
(HSA-Cre, Smn^F7/F7) mutant mice were generated by crossing homozygous (Smn^F7/F7) mice with those carrying (HSA-Cre, Smn^F7/+) genotype [11]. Animals were genotyped by polymerase chain reaction (PCR) amplification of DNA extracted from tail biopsies [11]. Green fluorescent protein (GFP) transgenic mice carrying wild-type Smn locus (Smn^+/+) were used as donors [25]. GFP transgene was also placed on (HSA-Cre, Smn^F7/F7) background, and (GFP, HSA-Cre, Smn^F7/F7) mice were selected as donors in a second set of experiments (described in Results). All BMT experiments were performed on non-GFP transgenic mice. Animal procedures were performed in accordance with institutional guidelines (agreements A91-228-2 and 3429).

Bone Marrow Transplantation
Bone marrow was sterilely isolated from 6–10-week-old isogenic wild-type (Smn^+/+) or (HSA-Cre, Smn^F7/F7) transgenic mice (H2b) that ubiquitously expressed enhanced GFP [25]. Femur and tibia were surgically removed and placed in Dulbecco's modified Eagle's medium (DMEM) culture medium with 10% fetal calf serum. Marrow was collected, and red blood cells were lysed in 0.75% NH₄Cl in 20 mM Tris, pH 7.2. To determine the rate of bone marrow (BM) cells expressing GFP, BM cells from three Smn^+/+ GFP transgenic mice were labeled with anti-mouse monoclonal antibody CD45.2 (clone 106, 1:200; BD Biosciences, Palo Alto, CA, http://www.bdbiosciences.com), Gr-1 (clone RB6-8C5, 1:500; BD Biosciences), and Sca-1 (clone D7, 1:500; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen), and labeled cells were analyzed together with GFP fluorescence using a FACSCalibur and CellQuest Software (BD Immunocytometry Systems, San Jose, CA, http://www.bdbiosciences.com). The rate of GFP⁺ CD45⁺, GFP⁺ Gr-1⁺, and GFP⁺ Sca-1⁺ was 44 ± 0.8%, 39 ± 0.3%, and 77 ± 1.5%, respectively. In bone marrow cells of GFP mutant mice (GFP, HSA-Cre, Smn^F7/F7; n = 3), the rate of GFP⁺ CD45⁺, GFP⁺ Gr-1⁺, and GFP⁺ Sca-1⁺ was 34 ± 7%, 27 ± 6.5%, and 65 ± 8%, respectively. No statistically significant difference was observed between wild-type (Smn^+/+) and mutant GFP mice (HSA-Cre, Smn^F7/F7; p > .15 for each comparison). For bone marrow transplantation, the cell pellet was resuspended in culture medium without fetal bovine serum and counted. Unfractionated BM cells (5 x 10⁶) were intravenously injected in the retro-orbital plexus of anesthetized 2-month-old recipient mice within 6 hours following irradiation. Recipient mice were subjected to irradiation at 6 Gy with an x-ray source and maintained in pathogen-free conditions. That dose prevented irradiation-related death of control or mutant mice within the first month, indicating that this dose was not sublethal. One and 3 months after irradiation and BMT, peripheral blood was collected and tested for chimerism by measuring GFP⁺ cells with a flow cytometer. Cells were labeled with biotin-conjugated anti-mouse monoclonal antibody CD45.2 followed by streptavidin-allophycocyanin (1:100; BD Biosciences) and R-phycoerythrin-conjugated rat anti-mouse anti-Gr-1. GFP fluorescence was measured at excitation and emission wavelengths of 489 and 508, respectively. Labeled cells were analyzed together with GFP fluorescence, and the rate of GFP⁺ CD45⁺ and GFP⁺ Gr1⁺ cells was determined.

Histological and Immunofluorescence Experiments on Muscle Tissue
Transverse sections (8–10 µm) of isopentane-frozen skeletal muscles including gastrocnemius and soleus of 8-month-old mice were stained with hematoxylin and eosin. To evaluate the total number of muscle fibers with or without central nuclei, serial 400-µm sections of the entire soleus were prepared. The highest number of myofibers per soleus muscle section was retained for statistical analysis. Double immunostaining of GFP and laminin was performed by using rabbit anti-GFP polyclonal (1:1,000; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) and rat monoclonal anti-laminin two antibodies (1:1,000; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). A mean of 1,000 myofibers per muscle was examined from each mouse group. Sections were mounted with Vectashield and 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and observed under a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com).

Single Muscle Fiber Isolation
Single muscle fibers were isolated from the extensor digitorum longus (EDL) muscle of 8-month-old mice according to Rosenblatt et al. [26]. Antibodies were incubated in the following concentrations: monoclonal rat anti-CD34 antibody (1:200; BD Biosciences) or monoclonal mouse anti-Pax 7 (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww). The nuclei were stained with DAPI. A total of 25 myofibers (representing around 1,000 nuclei) were examined per animal after CD34 or Pax7 immunolabeling. For GFP detection, 100 myofibers were examined per animal.

Rotarod Test
Tests started at 2.5 months of age and were performed every 2 days. The protocol consisted in placing mice on a rod placed 20 cm above the floor of apparatus and rotating at 5 rpm (Bioseb, Chaville, France, http://www.bioseb.com). The test was stopped after an arbitrary limit of 7 minutes. Mice were scored either positive (able to maintain their balance for 7 minutes) or negative (fell before 7 minutes). In the case of a positive test at 5 rpm, rotating speed was increased to 10 rpm for 20 minutes. For each session, two trials were performed, with a resting interval of 1 minute. Results are given as time mice were able to maintain their balance at 5 rpm (factor 1) and 10 rpm (factor 2).

Determination of Cre Recombinase Activity by DNA Analysis
Blood was collected by retro-orbital plexus puncture of anesthetized mice. Red blood cells were lysed in 0.75% NH₄Cl. Cells were then incubated in extraction buffer supplemented with 0.1 mg/ml of proteinase K. Detection of the Smn⁷, wild-type and Smn^F7 alleles, and Cre recombinase transgene was performed by PCR amplification as previously described [11].

RNA Analysis
Total RNA was extracted from skeletal muscle using Trizol reagent (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). cDNA was synthesized from total RNA by reverse transcriptase reaction. For Notch targets, primer sequences were as follows: HES-1, HES1F, 5'-CAA CAC GAC ACC GGA CAA AC-3'; HES1R, 5'-TCT TCT CCA TGA TAG GCT TTG ATG-3'; HEY-1, HEY1F, 5'-CTT GAG TTC GGC GCT GTG TTC C-3'; HEY1R, 5'-GAT GCC TCT CCG TCT TTT CCT-3'; HEY-L, HEYLF, 5'-CCC CTC ACC CTA CTC ACC A-3'; HEYLR, 5'-GCT TCA ACC CAG ACC CAA G-3'. For vascular endothelial growth factor a (VEGFa) transcripts, primer sequences were as follows: VEGFa-FOR1, 5'-AAG GAG AGC AGA AGT CCC ATG A-3'; VEGFa-REV1, 5'-AGC TTC GCT GGT AGA CAT CCA T-3'. For Cre recombinase transcripts, primer sequences were 5'-GCG GTC TGG CAG TAA AAA CTA TC-3' and 5'-GTG AAA CAG CAT TGC TGT CAC TT-3'. For hepatocyte growth factor (HGF) transcripts, primer sequences were as follows: HGF-FOR, 5'-GAG GTA CGC TAC GAA GTC TGT GA-3'; HGF-REV, 5'-GAT TCT GTG TGA TCC ATG GGA-3'. Real-time quantitative PCR was carried out using the SYBR-green master mix (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) and processed on an ABI Prism 7000 (Applied BioSystems). For each set of primers, first optimal primer concentration was determined and then a standard curve using increased dilution of cDNA was established in control tissues. Each reaction was performed in triplicate. Transcript level was normalized to aldolase (forward, 5'-TGAAGCGCTGCCAGTATGTTA-3'; reverse, 5'-GGTC-GCTCAGAGCCTTGTAGA-3') since there is no difference in aldolase transcript level between control and mutant skeletal muscle.

Immunoblotting Experiments
Frozen skeletal muscle (quadriceps) of 8-month-old mutant mice transplanted with wild-type bone marrow (^wtBM) (n = 5) and nontransplanted mutants (n = 5) were crushed in liquid nitrogen, transferred into a buffer containing 25 mM sodium phosphate (pH 7.2), 5 mM EDTA, and 1% SDS supplemented with protease inhibitor cocktail (Sigma-Aldrich) then boiled. For HGF and actin immunodetection, anti-HGF (1:100; mouse anti-rat HGF antibody; Institute of Immunology, Tokyo, http://www.tokumen.co.jp/english/index.html) and monoclonal anti-actin antibodies (1:50,000; clone AC-40; Sigma-Aldrich) were used.

Myoblast Proliferation Assay
Primary muscle cultures were prepared from newborn wild-type mice (Smn^+/+, 3–5 days old) as previously described [11]. Myoblasts were enriched by the preplate technique (95%) [11]. Proliferation medium consisted of DMEM (Invitrogen; Life Technologies, Rockville, MD, http://www.lifetech.com) supplemented with 10% horse serum, 10% fetal calf serum, 1.25% chick embryo extract, and 1% penicillin-streptomycin (Life Technologies). The nonadherent cells were collected, counted, and plated (10⁴ cells per dish) on 35-mm gelatin-coated dishes with grid (1% gelatin; Sigma-Aldrich). Before protein extracts were added, the number of cells was counted in 10 randomly chosen fields defined by the grids 2 days after myoblast purification. After the addition of protein extracts or phosphate-buffered saline (PBS) only, the same fields were selected for cell counting 1 day later. At day 2 or 3, no myoblast fusion occurred [11]. Quadriceps muscles of 8-month-old mice were isolated and crushed using a mortar and pestle. The muscles were incubated in cold PBS (500 µl/100 mg of tissue) for 2 hours with gentle shaking at +4°C. The crushed muscle extracts were centrifuged at 10,000g for 10 minutes, and the supernatant was collected and filtered through a 0.2-µm filter. Proteins were extracted from skeletal muscle of mutant mice irradiated and transplanted with wild-type (n = 6) or mutant BM cells (n = 3) or irradiated but not transplanted (n = 4). The myoblast culture was exposed to 0 (PBS as mock), 2, 5, and 10 µg of protein extracts for 1 day. At least three independent experiments were performed. In each experiment, muscle protein extracts of mutant mice transplanted with wild-type BM were run in parallel with those of mutant mice irradiated and transplanted with mutant BM or nontransplanted.

Statistical Analysis
Statistical comparisons were performed using the t test for all experiments except for comparing the number of GFP-positive and GFP-negative muscle fibers in transplanted mutant and control mice (²; Statview, Alsyd, France, http://www.statview.com). Data are presented as mean ± SE values.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
Transplantation of Wild-Type Bone Marrow Leads to Marked Improvement of Muscle Phenotype of Mutant Mice
Mice receiving irradiation followed by transplantation were designated transplanted, those receiving irradiation without transplantation were designated nontransplanted, and those receiving neither irradiation nor transplantation were designated untreated (Table 1). Depending on whether BM cells were isolated from wild-type (Smn^+/+) or mutant mice (HSA-Cre, Smn^F7/F7), BM was designated ^wtBM or mutant bone marrow (^mutBM), respectively (Table 1). In the first set of experiments, ^wtBM was used as donor cells. Two-month-old mutant mice were transplanted with ^wtBM cells (5 x 10⁶ cells; n = 39) from Smn^+/+ GFP mice or nontransplanted (n = 15). Control mice of the same age (n = 10; Smn^F7/+) were transplanted using the same protocol. Hematopoietic chimerism was examined from blood samples collected 1 and 3 months after BMT. Fluorescence-activated cell sorting (FACS) analysis of GFP-positive (GFP⁺) cells, hematopoietic CD45⁺ cells, or Gr-1⁺ granulocytes was performed on transplanted mutant (n = 39) and control (n = 10) mice. One month after BMT, a mean of 55 ± 2.4% and 55.3 ± 5% of CD45⁺ cells were GFP⁺ in mutant and control mice, respectively. FACS analysis demonstrated that 3 months after BMT, long-term hematopoietic reconstitution was similar in mutant and control mice (61 ± 3% and 64 ± 6.5% of GFP⁺ CD45⁺ cells, respectively). Similar results were obtained for GFP⁺ Gr-1⁺ cells (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 1. Green fluorescent protein (GFP) muscle fibers derived from bone marrow are present in transplanted mutant mice. Double immunofluorescent staining of GFP (red) and basal lamina (laminin, green) on transverse sections of gastrocnemius of 8-month-old control (A) and mutant (B) mice transplanted with wild-type bone marrow (wtBM) revealed GFP+ myofibers (arrow). Nontransplanted mutant mouse was used as negative control (D). GFP+ muscle fibers are also present in mutant mice transplanted with mutant bone marrow (mutBM) (C) (details in Results). Scale bar = 100 µm.

View larger version (70K): [in this window] [in a new window]   Figure 2. Skeletal muscle regeneration is improved in mutant mice transplanted with wild-type bone marrow. At low magnification (A–D), soleus muscle from 8-month-old control mice (A), nontransplanted mutant mice (B), and mutant mice transplanted with wild-type bone marrow (wtBM) (C) is framed by a dotted line. Note the larger size of the entire soleus of wtBM transplanted (C) compared with nontransplanted mutant mouse (B). At higher magnification (A'–D'), note the increased proportion of regenerating myofibers (with central nuclei, arrow) in mutant mouse transplanted with wtBM (C') compared with nontransplanted (B'). In mutant mouse transplanted with mutant bone marrow (mutBM) (D, D'), note the reduction in size of the entire soleus (D) and the lower proportion of regenerating myofibers (D') compared with mouse transplanted with wtBM (C'). Scale bar = 300 µm (A–D), 50 µm (A'–D').

View larger version (32K): [in this window] [in a new window]   Figure 3. Transplantation of wild-type but not mutant bone marrow cells improves the myopathic phenotype of Smn mutant mice. (A): Total number of regenerating myofibers in the entire soleus. Asterisk indicates statistically significant higher number of regenerating myofibers in mutant mice transplanted with wtBM (n = 7) compared with nontransplanted mutants (n = 5; p = .01). (B): Total number of mature myofibers in the entire soleus. Asterisk indicates statistically significant higher number of mature myofibers in control mice (p < .0001) compared with mutant mice transplanted with wtBM (n = 7) or nontransplanted mutant (n = 5). (C): Percentage of CD34 or Pax7 satellite cells on extensor digitorum longus isolated myofibers. Asterisks indicate statistically significant higher proportion of CD34 or Pax7 satellite cells in mutant mice transplanted with wtBM (n = 5) compared with nontransplanted mutants (n = 5). When mutant mice were transplanted with mutBM, no more benefit was observed in either the number of regenerating myofibers (n = 5) (A) or the proportion of CD34 or Pax7 satellite cells (n = 5) (C) compared with mutant mice transplanted with wtBM (p < .01 for each comparison). (D): Rotarod test. Motor capacity was similar in all groups until 5 months of age, then a marked and statistically significant decline (indicated by asterisk) was observed in untreated mutants (n = 10) compared with mutant (n = 15) or control (n = 4) mice transplanted with wtBM. Abbreviations: mutBM, mutant bone marrow; wtBM, wild-type bone marrow; m, month(s).

View larger version (18K): [in this window] [in a new window]   Figure 4. Increased proportions of Pax7 and CD34 satellite cells on freshly isolated muscle fibers of mutant mice transplanted with wild-type but not mutant bone marrow. Immunolabeling experiments of CD34 (A–C) and Pax7 (D–F) were performed on isolated muscle fibers from extensor digitorum longus of nontransplanted mutant mice (A, A', D, D'), mutant mice transplanted with wild-type bone marrow (wtBM) (B, B', E, E'), and mutant mice transplanted with mutant bone marrow (mutBM) (C, C', F, F'). An increased proportion of CD34 or Pax7+ satellite cells (arrows) was observed in mutant transplanted with wtBM compared with nontransplanted mutant mice or mutant mice transplanted with mutBM (see Fig. 3 for quantification). Satellite cell nuclei and myonuclei were stained with 4,6-diamidino-2-phenylindole (A', B', C', D', E', F'). Scale bar = 50 µm.

Skeletal muscle improvement of transplanted mutant mice was not caused by a change in Cre-mediated deletion of Smn in skeletal muscle. Indeed, the quantification of transcripts of the Cre recombinase transgene in skeletal muscle did not reveal any differences among transplanted, nontransplanted, and untreated mutant mice. These data indicate that the improvement of the muscular phenotype cannot be ascribed to changes in Cre-mediated deletion of Smn (supplemental material).

Activation of Skeletal Muscle Regeneration Is Mediated by a Biological Activity of Bone Marrow-Derived Cells
Surprisingly, wild-type bone marrow transplantation of mutant mice revealed a low proportion of GFP⁺ muscle fibers (2%), which contrasted with a significant increase in the number of regenerating myofibers (1.6-fold increase). These data indicate that BM cells had a very limited capacity to replace or fuse to mutant myofibers (2%), in agreement with previous reports. Nevertheless, ^wtBM transplantation was able to attenuate the myopathic phenotype of mutant recipient mice through an improvement of skeletal muscle regeneration. These results suggested an activation of muscle regeneration mediated by a biological activity of the BMDCs.

To test this hypothesis, a myoblast proliferation assay was performed. Proteins were extracted from quadriceps muscle of 8-month-old mutant mice transplanted with ^wtBM (six mice) and nontransplanted mutant mice (four mice) and added to the medium of primary cultures of wild-type myoblasts (Smn^+/+) 2 days after purification. Myoblast proliferation was evaluated by comparing the number of myoblasts before (day 2) and after a 1-day incubation with protein extracts (day 3). We found a 5.3-fold increase in the number of myoblasts in the presence of 10 µg of protein extracts from mutant mice transplanted with ^wtBM compared with a 3.5- or 2.7-fold change using nontransplanted muscle protein extracts or mock, respectively (p < .0001; Fig. 5). These data strongly suggest that activation of myoblast proliferation was mediated by a biological activity derived from skeletal muscle of mutant mice transplanted with wild-type bone marrow.

View larger version (17K): [in this window] [in a new window]   Figure 5. Evidence for a biological activity of bone marrow-derived cells on myoblast proliferation. Myoblast proliferation was evaluated as the ratio of wild-type myoblast number at day 3 (1 day after incubation with the protein extracts or mock) to that at day 2 (before incubation). The same fields were examined at both stages. Zero corresponds to the mock (phosphate-buffered saline without protein extract). Student's t test revealed statistically significant higher myoblast proliferation (indicated by *) in the presence of muscle extracts from mutant mice transplanted with wtBM (six mice) compared with nontransplanted (four mice) or mutant mice transplanted with mutBM (three mice). Note the dose effect of protein extracts from mutant mice transplanted with wtBM (0–10 µg) on myoblast proliferation ratio. Asterisks indicate p value <.05. Abbreviations: mutBM, mutant bone marrow; wtBM, wild-type bone marrow.

To evaluate the role of BM-derived muscle cells in the muscle improvement of recipient mutant mice, mutant mice were transplanted with mutant bone marrow. Mutant bone marrow cells carry the (HSA-Cre, Smn^F7/F7) genotype, and Cre-mediated deletion of Smn exon 7 will only occur in differentiated muscle fibers. Therefore, in BM-derived circulating blood cells, the Smn locus (Smn^F7/F7) remains intact since the Cre recombinase is not expressed. In contrast, in BM-derived muscle cells, the Cre recombinase is expressed, leading to the deletion of Smn (Smn^7/7; Table 1). The GFP transgene was transferred into a (HSA-Cre, Smn^F7/F7) background through successive backcrosses, and (GFP, HSA-Cre, Smn^F7/F7) mice were selected as donors. Transplantation was performed on irradiated 2-month-old non-GFP mutant mice (HSA-Cre, Smn^F7/F7; n = 11) using the same protocol. Hematopoietic reconstitution of the mutant mice transplanted with ^mutBM (65 ± 8% and 61 ± 6% of GFP⁺CD45⁺ cells, 1 and 3 months after transplantation, respectively) was similar to mutant mice transplanted with ^wtBM. Immunofluorescent labeling of GFP on transverse muscle sections revealed 1 ± 0.1% of GFP⁺ muscle fibers in 8-month-old mutant mice transplanted with ^mutBM (47 GFP⁺ of 4,579; five mice; Fig. 1). Importantly, the total number of regenerating muscle fibers in the entire soleus (246 ± 27) as well as the proportion of CD34⁺ (2.4 ± 0.1%) and Pax7⁺ (2.6 ± 0.2%) satellite cells per 100 myofiber nuclei were markedly reduced in 8-month-old mutant mice transplanted with ^mutBM (five mice) compared with mutant mice transplanted with ^wtBM (p < .009 for each test; Figs. 2–4). The results of ^mutBM transplantation were strikingly similar to those found in nontransplanted mutant mice. Taken together, these data indicate that ^wtBM-derived muscle cells are more likely to be the cells able to activate muscle regeneration of recipient mutant mice transplanted with wild-type bone marrow.

Moreover, myoblast proliferation assay was performed and did not reveal any significant increased myoblast proliferation after a 1-day incubation with 10 µg of protein extracts from mutant mice transplanted with ^mutBM (3.5-fold change) compared with nontransplanted mutant mice or mock (3.4- and 2.7-fold change, respectively; Fig. 5).

These data strongly suggest that activation of myoblast proliferation was mediated by a biological activity of skeletal muscle cells from mutant mice transplanted with wild-type but not mutant bone marrow. Several candidate pathways could account for this activation and were tested using real-time PCR analysis of transcripts normalized to aldolase. The Notch signaling pathway has been shown previously to mediate activation of muscle regeneration [28], but no differences in transcript levels of Notch targets, including HES-1, HEY-1, and HEY-L, were observed in our mice (Fig. 6C) [29]. In addition, no evidence for increased angiogenesis was found in skeletal muscle of mice transplanted with ^wtBM since no change in VEGF expression was observed in comparison with nontransplanted mutant mice (Fig. 6C). In contrast, expression analysis of the gene encoding the HGF, a known growth factor that activates quiescent satellite cells in skeletal muscle [30–32], revealed dramatic changes. A tremendously increased expression of HGF was observed in skeletal muscle of 8-month-old mutant mice transplanted with ^wtBM (1.5 ± 0.08; n = 5) compared with ^mutBM (0.45 ± 0.09; n = 5) or nontransplanted mutants (0.045 ± 0.007; n = 5, p < .0001 for each comparison; Fig. 6A). These results were further supported by Western blot analysis of HGF in skeletal muscle of mutant mice transplanted with ^wtBM (n = 5) compared with nontransplanted mutants (n = 5; Fig. 6B).

View larger version (23K): [in this window] [in a new window]   Figure 6. Expression analysis of HGF, Notch targets, and vascular endothelial growth factor a (VEGFa). (A): Real-time polymerase chain reaction (PCR) amplification analysis of transcripts encoding HGF in skeletal muscle of nontransplanted mutant mice (n = 5), mutant mice transplanted with wild-type bone marrow (wtBM) (n = 5), mutant mice transplanted with mutant bone marrow (mutBM) (n = 5), and untreated mutant (n = 5). Asterisks indicate statistically significant changes (p < .0002). (B): Western blot analysis of HGF (active form) in skeletal muscle from nontransplanted mutant mice (lanes 1–3) and mutant mice transplanted with wtBM (lanes 4–6). A marked increased amount of HGF is observed in mutant mice transplanted with wtBM compared with nontransplanted mutant mice and actin expression. (C): Real-time PCR amplification analysis of transcripts encoding HES-1, HEY-1, HEY-L, and VEGFa transcripts in skeletal muscle of nontransplanted mutant mice (n = 3) and mutant mice transplanted with wtBM (n = 3). For transcript analysis, data are presented as the mean ratio ± SE values of a given gene to aldolase expression. Abbreviation: HGF, hepatocyte growth factor.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

The biological activity of BM-derived cells was further suggested by the in vitro myoblast proliferation assay showing an enhanced activation of myoblast proliferation in the presence of proteins extracts from skeletal muscle of mutant mice transplanted with ^wtBM but not with ^mutBM. Importantly, a tremendous upregulation of the HGF gene was found in skeletal muscle of mutant mice transplanted with ^wtBM compared with mice transplanted with ^mutBM or nontransplanted mutant mice. HGF, a ligand for the c-met protooncogene product, has emerged as an important candidate molecule in muscle regeneration and has been shown to act in a paracrine manner. The c-met receptor is present on quiescent satellite cells in skeletal muscle, and HGF has the ability to activate satellite cells [30–32], suggesting that HGF plays an important role in regulating the early phases of muscle regeneration. Activation of satellite cells attracted by a factor released by bone marrow-derived cells could account for muscle regeneration improvement in our model. Although our experiments do not provide mechanistic evidence that HGF is in fact responsible for the beneficial effects, they do show that bone marrow transplantation upregulates the expression of a candidate soluble molecule able to activate satellite cells. Interestingly, the induction of the HGF signaling pathway following BMT has recently been shown to promote pancreatic regeneration in diabetic rats, suggesting a similar mechanism [34]. In addition to its role in the skeletal muscle and pancreatic regeneration, HGF is one of the most potent survival-promoting factors for motor neurons [35]. SMA being mainly characterized by motor neuron degeneration, the neurotrophic effect of HGF upregulation mediated by BMT should be explored in the neuronal mouse model of SMA [9, 10].

Thanks to the Cre-loxP system, we were able to exclude the BM-derived circulating blood cells as candidate to harbor this biological activity. Therefore, no difference exists at the Smn locus in circulating blood cells of wild-type or (HSA-Cre, Smn^F7/F7) mice. In contrast, the transplantation of BM cells derived from wild-type (Smn^+/+) but not mutant (HSA-Cre, Smn^F7/F7) donors provides wild-type Smn alleles to deficient pre-existing myofibers or is able to form new wild-type myofibers. Even at a low percentage, ^wtBM-derived myofibers can be regarded as candidate cells able to activate muscle regeneration through a biological activity. We cannot exclude other candidates, such as cells of muscle lineage, homing in skeletal muscle and coming from bone marrow. Additional experiments should contribute to determining the fraction of bone marrow-derived cells homing in skeletal muscle and participating in the upregulation of HGF. Although the nature of this biological activity still remains to be characterized, these results provide strong evidence that this activity may attenuate a myopathic phenotype by delaying or preventing myofiber loss.

It has been shown that the proportion of muscle fibers incorporating adult BMDCs depends on the type of skeletal muscles or physiological stress applied to muscles [22, 23]. In this study, 2-month-old mutant mice were used as recipients. Since mutant mice older than 6 months of age show dramatic loss of myofibers associated with progressive motor defects, BM transplantation on these mutant mice or in the severe muscular model of SMA [8] should be investigated to determine whether the recruitment of BMDCs into skeletal muscle may be correlated with the severity of myofiber loss of recipient mice. Higher contribution of BMDCs to myofibers using physiological stress might allow stronger beneficial effects. Elucidating the nature of BMDCs able to fuse to or to differentiate into myofibers on one hand and the pathway underlying HGF gene upregulation, which may represent an attractive paracrine mechanism to support this biological activity, on the other hand should be helpful in identifying new means to improve skeletal muscle regeneration.

	DISCLOSURES

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

Top Abstract Introduction Materials and Methods Results Discussion Disclosures Acknowledgments References

 
This work was supported by Institut National de la Santé et de la Recherche Médicale, the Association Française contre les Myopathies, Families of Spinal Muscular Atrophy (USA), the Conseil Régional d'Ile de France, and the Fondation Bettencourt Schueller. We are very grateful to C. Collins for helpful suggestions in isolating myofibers, A. Joutel for advice on investigation of the Notch pathway, and E. Tournier-Lasserve and A. Fischer for fruitful comments. We thank P. Ardouin and A. Rouches (Institut Gustave Roussy) for assistance in the irradiation process and M. Okabe for providing us with GFP transgenic mice.

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