Correspondence to: Johnny Huard, Director: Growth and Development Laboratory, Department of Orthopaedic Surgery and Molecular Genetics and Biochemistry, Children's Hospital of Pittsburgh and University of Pittsburgh, Pittsburgh, PA 15261.
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Abstract |
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Several recent studies suggest the isolation of stem cells in skeletal muscle, but the functional properties of these muscle-derived stem cells is still unclear. In the present study, we report the purification of muscle-derived stem cells from the mdx mouse, an animal model for Duchenne muscular dystrophy. We show that enrichment of desmin+ cells using the preplate technique from mouse primary muscle cell culture also enriches a cell population expressing CD34 and Bcl-2. The CD34+ cells and Bcl-2+ cells were found to reside within the basal lamina, where satellite cells are normally found. Clonal isolation and characterization from this CD34+Bcl-2+ enriched population yielded a putative muscle-derived stem cell, mc13, that is capable of differentiating into both myogenic and osteogenic lineage in vitro and in vivo. The mc13 cells are c-kit and CD45 negative and express: desmin, c-met and MNF, three markers expressed in early myogenic progenitors; Flk-1, a mouse homologue of KDR recently identified in humans as a key marker in hematopoietic cells with stem cell-like characteristics; and Sca-1, a marker for both skeletal muscle and hematopoietic stem cells. Intramuscular, and more importantly, intravenous injection of mc13 cells result in muscle regeneration and partial restoration of dystrophin in mdx mice. Transplantation of mc13 cells engineered to secrete osteogenic protein differentiate in osteogenic lineage and accelerate healing of a skull defect in SCID mice. Taken together, these results suggest the isolation of a population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing.
Key Words: dystrophin, gene transfer, BMP-2, stem cells, bone formation
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Introduction |
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The development of muscle stem cells for transplantation or gene transfer (ex vivo approach) as a new method for treatment of patients with muscle disorders has become more attractive and challenging in the past few years (Barroffio et al., 1996;
The efficiency of cell transfer might be improved by using muscle stem cells, which potentially display unique features, including: self-renewing cells that produce progeny; arise early in development and persist throughout life; and long-term proliferation and multipotency. In fact, muscle satellite cells have long been considered the myogenic cells responsible for postnatal muscle growth, regeneration and repair for the maintenance of skeletal muscle (
It has been established that mesenchymal stem cells derived from bone marrow (
The satellite cells, a subpopulation of muscle-derived cells, may have stem cell-like characteristics (
A recent report has suggested that only a discrete minority of myoblasts can survive after implantation and thus may represent a population of myogenic stem cells (
Taken together, these results suggest that satellite cells are highly heterogeneous in nature. This prompted our attempt to investigate whether our highly purified muscle-derived cells (preplate technique) will express markers of stem cells and differentiate into osteogenic lineage. In this report, we show that the highly purified myogenic cells derived by the preplate technique express markers indicative of stem cells. Isolation and characterization of a clonal population from these highly purified myogenic cells revealed that a clonal cell population (mc13) express both stem cell and satellite cell-specific markers. More importantly, the mc13 cells have the capacity to differentiate into both myogenic and osteogenic lineage in vitro and in vivo. Thus, our results suggest that a subpopulation of muscle-derived cells possess stem cell-like characteristics and can differentiate into multiple lineages.
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Materials and Methods |
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Isolation of Muscle-derived Cells
Primary muscle cells were isolated from 3-wk-old mdx mice (C57BL/10ScSn mdx/mdx, The Jackson Laboratory) using a technique previously described (1 h, the supernatant was withdrawn from the flask and replated in a fresh collagen-coated flask. The cells that adhered rapidly within this 1-h incubation were mostly fibroblasts (
56 serial platings, the culture was enriched with small, round cells (pp6;
Clonal Isolation of Purified Muscle-derived Cells
To isolate clones from pp6, the slow adhering primary muscle cells were transfected with a plasmid encoding for the ß-galactosidase, minidystrophin (
We have recently investigated two additional clones of muscle-derived cells from the highly purified muscle-derived cells (pp6). These clones of muscle-derived cells (MD1 and MD2) were not transfected by the plasmid that encodes for the minidystrophin, lacZ, and neomycin resistance genes. These cells were cloned using a limiting dilution derived technique by which the cells from the preplate #6 (pp6) population were: seeded at a low density in culture flasks; cultivated for 1 wk until colonies appeared; and single colonies were then trypsinized and the detached cells from the individual colonies picked up and seeded in a culture flask. The expansion of these new clonal cells in large quantity was performed using a similar protocol as described for mc13.
Immunohistochemistry on Muscle Cells In Vitro
Cells were plated in a 6-well culture dish and fixed with cold methanol for 1 min. After rinsing with PBS, cells were blocked with 5% horse serum at room temperature for 1 h. The primary antibodies were diluted in PBS as follows: antidesmin (1:100; Sigma-Aldrich), biotinylated anti-mouse CD34 (1:200; BD PharMingen), rabbit antimouse Bcl-2 (1:1,000; BD PharMingen); rabbit antimouse m-cadherin (1:50); mouse antimouse MyoD (1:100; BD PharMingen); mouse antirat myogenin (1:100; BD PharMingen); rabbit antimouse Flk-1 (1:100; Research Diagnostics); and biotinylated anti-Sca-1 (1:200; BD PharMingen). The primary antibodies were applied overnight at room temperature. Appropriate biotinylated secondary antibodies for nonbiotinylated primary antibodies were applied for 1 h at room temperature. The cells were rinsed with PBS then incubated at room temperature with 1/300 streptavidine conjugated with Cy3 fluorochrome for 1 h. The cells were visualized by fluoroscopy, and the percentage of positive cells was calculated by counting positively stained cells in 10 randomly chosen 20x fields.
RT-PCR Analysis of Muscle-derived Cells
Total RNA was isolated using TRIzol reagent (Life Technologies). Reverse transcription (RT) was carried out using SuperScriptTM preamplification system for first strand cDNA synthesis (Life Technologies) according to the instructions of the manufacturer. PCR amplification of the targets was performed in 50 µl reaction mixture containing 2 µl of RT material, 5 U/100 µl Taq DNA polymerase (Life Technologies), and 1.5 mM MgCl2. CD34 primers were designed by using Oligo software. The sequences of the other primers are from references as follows: myogenin (
Cell Characterization by Flow-cytometry Analysis
Cultures of muscle-derived cells were harvested before analysis with a 1:2 dilution of trypsin/EDTA solution diluted in HBSS (0.5% trypsin/5.3 mM EDTA initial concentration; Life Technologies). Cells were then spun, washed, counted, and divided into two groups (experimental and control). A 1:10 mouse serum (Sigma-Aldrich) in PBS solution (0.5% BSA, 0.1% sodium azide) and Fc Block (rat antimouse CD16/CD32; BD PharMingen) were added to each cell pellet for 10 min on ice. Optimal amounts of rat antimouse mAbs were predetermined and added directly to each tube for 30 min. Each experimental tube received FITC-conjugated CD45, R-PEconjugated CD117(c-kit) and biotin-conjugated Sca-1 (all from BD PharMingen). A control tube for each cell type received equivalent amounts of FITC-conjugated, biotin-conjugated, and R-PE-conjugated isotype standards (BD PharMingen). After several rinses, streptavidin allophycocyanin conjugate (APC; BD PharMingen) was added to each tube, including controls, and incubated on ice for 20 min. Just before analysis, 7-amino-actinomycin D (7-AAD, Via-Probe, BD PharMingen) was added to each tube for dead cell exclusion. A minimum of 10,000 live cell events were analyzed on a FACSCalibur (Becton Dickinson) flow cytometer.
Immunohistochemistry on Muscle Tissue In Vivo
The cryosections of muscle samples from a 4-wk-old normal mouse (C-57 BL/6J, The Jackson Laboratory) was fixed with cold acetone for 2 min and preincubated in 5% horse serum diluted in PBS for 1 h. For CD34, Bcl-2, and laminin/collagen type IV, the following primary antibodies were used: biotin anti-mouse CD34 (1:200 in PBS; BD PharMingen); rabbit antimouse Bcl-2 (1:1,000; BD PharMingen); and rabbit antilaminin (1:100 in PBS; Sigma-Aldrich) or antimouse collagen type IV (1:100 in PBS; Chemicon). The CD34 and Bcl-2 was also colocalized with Hoechst 33258 (bis-Benzimide, 1/100 in PBS; Sigma-Aldrich) to stain the nuclei. For dystrophin staining, sheep antihuman D-10 antibody (1:250 dilution in PBS) was used as the primary antibody. After several rinses in PBS, a biotin-conjugated anti-sheep was subsequently used (1:250 dilution in PBS). Streptavidin-FITC (bone) and streptavidin-Cy3 (muscle) at a dilution of 1:250 dilution in PBS were used; immunoreaction was observed by fluorescence microscopy (Nikon, Eclipse E-800). Finally, the colocalization of ß-galactosidase, osteocalcin, and 4',6-diaminido-2-phenylindole (DAPI; Sigma-Aldrich) stain nuclei was performed using the following protocol. The muscle sections were incubated with DAPI at a dilution of 1/100 in PBS to stain the nuclei; a biotinylated antiß-galactosidase antibody (1/100 in PBS; Sigma-Aldrich), followed by streptavidin conjugated to fluorescein (Gal-13, 1/300 in PBS; Sigma-Aldrich) to stain the ß-galactosidase expressing nuclei; and a goat antimouse osteocalcin (1:100 in PBS; Chemicon Co), followed by an incubation with a Cy3-conjugated anti-goat antibody (1/100 in PBS; Sigma-Aldrich) to label the osteocalcin expressing cells. The colocalization of the cells expressing ß-galactosidase, osteocalcin with the nuclear labeling was visualized by fluorescence microscopy using an E-800 Nikon microscope.
Stimulation with rhBMP-2, Osteocalcin Staining, and Alkaline Phosphatase Assay
The mc13 and nonpurified muscle-derived cells (npmc) were plated in triplicate at a density of 12 x 104 per well in 12-well collagen-coated flasks. The cells were stimulated by addition of 200 ng/ml recombinant human BMP-2 (rhBMP-2) to the media. The media was changed on days 1, 3, and 5 after initial plating. The control group also had media changed on these days, without rhBMP-2. After 6 d of rhBMP-2 stimulation, cells were counted using a hemacytometer, and cell lysates were harvested by repeated freeze-thaw cycles. The alkaline phosphatase activity in the cell lysate was measured using a commercially available kit (Sigma-Aldrich) that utilizes color change in the reagent due to the hydrolysis of inorganic phosphate from p-nitrophenyl phosphate. The color change was analyzed on a spectrophotometer, and the data was expressed in international units: ALP activity per liter normalized to 106 cells (U/L/mil cells). Statistical difference among the different groups was analyzed using t test (*P < 0.05). The mc13 cells with or without rhBMP-2 stimulation were also analyzed on day 6 for expression of desmin. The desmin immunoreactivity was determined with a mouse antidesmin (1:100; Sigma-Aldrich), followed by a biotinylated anti-mouse (1/100; Sigma-Aldrich), and finally a streptavidin-conjugated Cy3 (1/300; Sigma-Aldrich). The immunofluorescence was visualized by an inverted miscropscope (Diaphot, Nikon); the number of desmin expressing cells were monitored and compared between the rhBMP-2 stimulated and nonstimulated cells.
In Vivo Differentiation of Muscle-derived Cells in Myogenic and Osteogenic Lineages
Myogenic.
The mc13 cells were injected (5 x 105 cells) intramuscularly in the hind limb muscle of mdx mice. The mdx mice were killed at 7 d after injection, and the injected muscles were frozen, cryostat sectioned, and assayed for dystrophin (see above) and LacZ expression (
Osteogenic.
The mc13 and npmc were transduced with an adenovirus encoding for rhBMP-2 and injected intramuscularly in the hind limb muscles of SCID mice. Genetics Institute, Cambridge, MA, generously provided the BMP-2-125 plasmid that contains the rhBMP-2 cDNA. A replication defective, E1 and E3 gene-deleted adenoviral vector was engineered to encode rhBMP-2 under the human cytomegalovirus promoter (
The mc13 and the npmc were transduced with the adenoviral vector (MOI = 50). After 4 h of incubation with the adenovirus at 37°C, equal volume of serum containing media was added to the cell culture for 24 h. The transduced cells were then trypsinized, centrifuged, washed twice with HBSS, and 0.51.0 x 106 cells were injected into exposed triceps surae of SCID mice (The Jackson Laboratory) using a 30-gauge needle on a gas-tight syringe. At 1415 d, animals were killed with cervical dislocation. The injected hind limbs were analyzed by radiography, and triceps surae were isolated and flash frozen in 2-methylbutane buffered in PBS, precooled in liquid nitrogen. The frozen samples were cut into 510-µm sections using a cryostat (Microm, HM 505 E, Fisher Scientific) and stored at -20°C until further study.
Skull Defect Assay
3 68-wk-old female SCID mice (The Jackson Laboratory) were used in both the control and experimental groups. The mice were anesthetized with methoxyflurane and placed prone on the operating table. Using a number 10 blade, the scalp was dissected to the skull, and the periosteum was stripped. A 5-mm full-thickness circular skull defect was created using a dental burr, with minimal penetration of the dura. A collagen sponge matrix (HelistatTM, Colla-Tec, Inc) seeded with 0.51.0 x 106 mc13 cells, either with or without adBMP-2 transduction, was placed to fill the skull defect. The scalp was closed using a 4-0 nylon suture, and the animals were allowed to ad lib food and activity. After 14 d, the mice were killed, and the skull specimens were analyzed both grossly and microscopically. For von Kossa staining, slides were fixed in 4% formaldehyde and were then soaked in 0.1 M AgNo3 solution for 15 min. After exposure to light for at least 15 min, the slides were washed with PBS and stained with hematoxylin and eosin for histological evaluation. The identification of the mc13 within the newly formed bone in the skull defect was performed using the LacZ staining. The number of LacZ positive mc13 cells within and outside the osteoid was consequently monitored.
Fluorescent In Situ Hybridization (FISH) Using Y-probes
The FISH technique was used to follow the fate of the injected male npmc genetically engineered to express BMP-2 into the skeletal muscle of female mice (
Standard Cytogenetic Method for Metaphase Preparations of mc13
The standard cytogenetic method for metaphase preparation of mc13 was performed using a previously described protocol (
Soft Agar Technique
The technique of cell growth on soft agar was performed as previously described (
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Results |
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Characterization of the pp6 Cells In Vitro
Cells isolated from primary muscle tissue contain a mixture of fibroblasts, myoblasts, adipocytes, and hematopoietic cells. However, the muscle-derived cells can be enriched using the preplate technique based on their differential adherence characteristics of primary muscle cells to collagen-coated flasks (
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As shown in Table 1, cells in pp6 fractions were expressing myogenic markers, including desmin+, MyoD+/-, myogenin-/+. The pp6 cells were also c-met and MNF positive (RT-PCR), two genes which are expressed at an early stage of myogenesis (
Marker Analysis of the Clonal Muscle-derived Cells Isolated from pp6
The biochemical markers expressed by mc13 cells were analyzed using RT-PCR, immunohistochemistry, and flow cytometry. The markers expressed by mc13 clone were compared with those of pp6 and fibroblasts. As summarized in Table 1, mc13 cells were positive for the expression of desmin, c-met, MNF, myogenin (+/-), and MyoD (RT-PCR). These results suggest that this clonal population of mc13 contained cells at different stages of differentiation. The mc13 cells were positive for m-cadherin (+/-) and Bcl-2 (+/-), but negative for CD34 expression. They were highly positive for the expression of Flk-1 and Sca-1. Similar to that observed with the pp6 cells, the mc13 cells were negative for CD45 and c-kit (see Table 1).
Two additional clones (MD1/MD2) were also investigated. These clonal cells have been isolated from the pp6, but were not transfected with the plasmid to express ß-galactosidase, minidystrophin, and the neomycin resistance gene. These two clones share similarities with mc13 and pp6 since they express desmin, MyoD+, myogenin+, c-met+, MNF+, and Flk-1+ (see Table 1). The MD1 and MD2 cells are also CD45- (see Table 1); in contrast to the mc13, they are positive for CD34 and negative for m-cadherin and Bcl-2 (see Table 1). The MD1 and MD2 cells were also compared with the pp6 and mc13 for their expression of Sca-1 and c-kit. Similar to that observed with pp6 and mc13, the MD1 and MD2 cells are Sca-1+ and c-kit- (see Table 1).
In Vivo Localization of CD34+ and Bcl-2+ Cells
To identify the location of CD34+ and Bcl-2+ cells in vivo, muscle tissue sections from gastrocnemius of normal mice were stained using anti-CD34 and antiBcl-2 antibodies. The CD34 positive cells constituted a small population of muscle-derived cells (Fig 1 A). Colocalization of CD34 expressing cells (Fig 1 A) with laminin, which stained the basal lamina (Fig 1 B), revealed the location of these CD34+ cells within the basal lamina (Fig 1C and Fig D). The colocalization of the CD34+ cells with a nuclear staining (Hoescht 33258) indicated the presence of cells instead of small blood vessels that are also positive for CD34 (Fig 1C and Fig D). In fact, the expression of CD34 by vascular endothelial cells has been shown in previous studies (
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In Vitro Differentiation of Clonal Muscle Stem Cells into Osteogenic Lineage
To further characterize the subpopulation of muscle-derived cells that may have stem cell-like capabilities, the mc13 clone isolated from the pp6 population was further subjected to in-depth analysis. We investigated whether mc13 cells have the potential to differentiate into different lineages by examining their response to rhBMP-2 stimulation. The cells were plated on a 6-well culture dish in identical density and allowed to become confluent with and without exposure to 200 ng/ml rhBMP-2. Within 34 d, there was a striking morphologic difference between mc13 cells exposed to rhBMP-2 and control cells. Without stimulation of rhBMP-2, mc13 cells started to fuse into multinucleated myotubes (Fig 2 A). When exposed to 200 ng/ml rhBMP-2, however, cells remained mononucleated and did not fuse (Fig 2 B). When cell density reached >90% confluency, the untreated culture fused to form multiple myotubes (Fig 2 C), whereas the treated cells became circular and hypertrophic (Fig 2 D, see arrows). Using immunohistochemistry, these hypertrophic cells were analyzed for the expression of osteocalcin. Osteocalcin is a matrix protein that is deposited on bone, specifically expressed by osteoblasts. These hypertrophic cells in the rhBMP-2treated mc13 were found highly positive for expression of osteocalcin (Fig 2 E, see arrows). This suggests that the rhBMP-2stimulated mc13 cells can no longer fuse into myotubes and differentiate into osteoblasts.
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We monitored the level of desmin as mc13 cells undergo morphologic differentiation with rhBMP-2 stimulation as described above. Freshly isolated mc13 cells were uniformly positive for desmin (Fig 3A and Fig B). However, within six days of exposure to rhBMP-2, the percentage of desmin positive cells significantly decreased to 3040% (*P < 0.05), whereas the control cells not exposed to rhBMP-2 remained 90100% desmin positive (Fig 3 C). This result indicates that a large number of mc13 cells lose their desmin expression upon stimulation with rhBMP-2 (Fig 3 C). It is unclear whether this decrease in percentage of desmin positive cells was due to increased proliferation of a small number of cells that lose desmin expression or due to a large number of cells responding to rhBMP-2. However, in light of the complete absence of multinucleated myotubes in flasks containing rhBMP-2, it seems more likely the decreased percentage of desmin positive cells is due to the loss of myogenic characteristics of mc13 cells.
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We also tested for alkaline phosphatase activity in rhBMP-2stimulated mc13 cells. The alkaline phosphatase activity has been used as a biochemical marker for cells differentiating in osteoblastic lineage (
In Vivo Differentiation of mc13 Cells into Myogenic and Osteogenic Lineages
To assess the in vivo characteristics of mc13 cells, the cells were intramuscularly injected into hind limb musculature of mdx mice to determine whether these clones were capable of differentiating through the myogenic lineage in vivo. The ß-galactosidase and dystrophin expression were followed to confirm the ability of mc13 cells to enhance muscle regeneration and partially restore dystrophin. Before injection, LacZ staining of the stably transfected mc13 revealed that 90100% of cells were expressing ß-galactosidase (data not shown). 5 x 105 mc13 cells were injected intramuscularly in the hind limb muscles of mdx mice; the animals were killed at 7 d after transplantation. The hind limbs of the injected animals were harvested for histology and immunohistochemical analysis. Multiple LacZ (Fig 4 A) and dystrophin positive myofibers (Fig 4 B) were readily identified at the injection site (Fig 4A and Fig B). We have monitored the number of myofibers that coexpress LacZ/dystrophin and found 379 ± 256 positive myofibers at 7 d after injection (Fig 4 E). This demonstrates that mc13 cells, when injected into the dystrophin deficient mdx mice, can differentiate through the myogenic lineage in vivo and consequently enhance muscle regeneration and partially restore dystrophin expression in the dystrophic muscle.
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More importantly, we have tested whether mc13 cells can be systemically delivered to dystrophic muscles. The mc13 cells (5 x 105) were intravenously injected in the tail vein of mdx mice, and the animals were killed at 7 d after injection. We observed a small number of LacZ positive myofibers (Fig 4 C) coexpressing dystrophin in the hind limb of the injected animals (Fig 4C and Fig D, asterisk). A lower number of LacZ and dystrophin positive myofibers were observed after the systemic delivery of the mc13 cells when compared with the i.m. injection (Fig 4 E). This result suggests that mc13 cells can be delivered systemically to the target tissue for partial restoration of dystrophin expression. We have also investigated whether the mc13 cells were also disseminated in other tissues by testing the presence of dystrophin positive cells in nonmuscle tissues of the injected animals. We have been unable to detect any dystrophin positive cells in the lung, spleen, liver, kidney, and brain of the injected animals at 7 d after injection (data not shown).
The colocalization of dystrophin and ß-galactosidase expression in the membrane of various myofibers after i.m. and i.v. injection of mc13 is unclear. In fact, some myofibers display a different level of ß-galactosidase expression in the membrane versus the cytoplasm, whereas other myofibers are ß-galactosidase negative in the cytoplasm and positive in the membrane. The potential fusion of ß-galactosidase with dystrophin and their transport to the membrane may explain this predominance of ß-galactosidase expression in the membrane.
To test the pluripotent characteristics of mc13 in vivo, the cells were transduced with an adenoviral vector encoding rhBMP-2 (adBMP-2). The mc13 cells were then injected into hind limbs of SCID mice, and bone formation was monitored radiographically and histologically at 1415 d after injection. The LacZ and dystrophin were assessed in vivo to follow the fate of the injected cells. Previous experiments have shown that 7090% of these cells are typically successfully transduced with our adenoviral vectors (our unpublished data). ELISA of mc13 cells transduced with adBMP-2 showed that infected cells are capable of producing a significantly higher amount of rhBMP-2 (*P < 0.05) when compared with control cells that were not transduced by the vector (Fig 5 A). The BMP-2 detected in the nontreated cells with the ELISA technique is attributed to nonspecific background detection. Radiographic analysis of hind limbs of injected SCID mice revealed robust ectopic bone formation within 14 d of injection (Fig 5 B, see arrow). Histologic analysis using LacZ staining of the ectopic bone showed that LacZ positive mc13 cells were uniformly located within the mineralized matrix or lacunae, a typical location where osteoblasts and osteocytes are found (Fig 5 C). To further confirm the role of mc13 in formation of the ectopic bone, the muscle sections were also stained for the expression of dystrophin. As shown in Fig 5 D, the ectopic bone contained cells highly positive for dystrophin, further implicating that mc13 cells are intimately participating in bone formation.
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To determine whether the genetically engineered mc13 expressing BMP-2 can express bone protein, we have colocalized ß-galactosidase expressing nuclei, osteocalcin expression, and nuclei staining (DAPI) by immunohistochemistry (Fig 5, EH). We have identified nuclei expressing ß-galactosidase (Fig 5 F, FITC/green, see arrow), which express osteocalcin (Fig 5 G, cy3/red, see arrow), and colocalized with nuclei staining (Fig 5 E, DAPI/blue, see arrow). The triple colocalization of DAPI/osteocalcin and ß-galactosidase (Fig 5 H, see arrow) suggest that the genetically engineered mc13 can differentiate in bone lineage and consequently express bone protein (osteocalcin). We were also capable of detecting ß-galactosidase expressing cells (Fig 5 F, arrowhead) that were not colocalized with osteocalcin positive cells (Fig 5 G, arrowhead), suggesting that some of the injected mc13 were not expressing osteocalcin (Fig 5, EH, arrowheads).
As a control, similar experiments were carried out with male npmc, which are highly fibroblastic in nature. We have observed that npmc genetically engineered to express BMP-2 also supported robust ectopic bone formation in skeletal muscle (Fig 6 A). The FISH technique was used to identify the Y chromosome positive cells and revealed that the injected npmc cells were located, in contrast to mc13, outside of the osteoid (Fig 6 B, arrow); a complete absence of Y chromosome positive cells was found within the newly formed osteoid (Fig 6 C, arrowheads). This suggests, as previously described by our group (
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Enhancement of Bone Healing by Genetically Engineered mc13 Cells
To test the clinical applicability of our findings and to further support the functional properties of mc13, we investigated whether the mc13 cells engineered to express BMP-2 can be used to enhance healing of a bone defect. We created 5-mm skull defects in skeletally mature (68-wk-old) female SCID mice using a dental burr. A 5-mm size skull defect has been shown to be a nonhealing defect in previous mouse models (
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Standard Cytogenetic and Tumorigenicity Assay on mc13
We have also investigated the karyotype of these cells and tested whether they can be grown in soft agar as an indicator of their tumorigenicity (not illustrated). The mc13 cells analyzed had an unknown passage history, but likely over 20 passages. The normal diploid number of chromosomes (2n) seen in mice (Mus musulus) is 40 (
The soft agar test (
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Discussion |
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We have observed that the preplate technique enriches for a population of muscle-derived cells that express both early myogenic markers, including desmin, c-met, MNF, and Bcl-2, and stem cell markers (Sca-1, Flk-1, CD34). In vivo staining showed that these cells were localized within the basal lamina, a location normally occupied by satellite cells. Clonal analysis from these highly purified muscle-derived cells (pp6) showed that, as observed with the pp6 cells (
An immortalized clonal cell line from mouse myoblast, C2C12, has been shown to decrease expression of myogenin and MyoD mRNA, and increase expression of alkaline phosphatase, osteocalcin, and parathyroid-dependent 3', 5'-cAMP in response to rhBMP-2 in vitro (
A recent in vivo study has demonstrated that in human hematopoietic cells, CD34+KDR+ population had the highest pluripotent characteristics (
Interestingly, the pp6 cells are CD34+, whereas mc13 cells are CD34-. However, we cannot rule out that mc13 initially expressed CD34, but the extensive selection with G418 may have differentiated the mc13 clone, resulting consequently in their loss of CD34 and the gain of m-cadherin expression. In fact, we have recently investigated two new clones (MD1 and MD2) from the pp6 to determine whether the marker expression by the mc13 were unique or also expressed by other clonal cell populations originated from the same pp6 population. These clonal cells have been isolated from the pp6, but were not genetically engineered with the plasmid to express ß-galactosidase and dystrophin. These two clones were similar to mc13 for most of the myogenic and stem cell marker expression in vitro, but in contrast to mc13, they are positive for CD34 and negative for m-cadherin, as well as Bcl-2. These results suggest that mc13 might be isolated from a small population of CD34- cells within the pp6 population or the mc13 were originally CD34+, but during the selection, they differentiated into CD34- cells.
Although the expression of CD34 can account for a difference between the mc13 and pp6/MD1/MD2, recently it has been published (
The relationship between mc13 and other populations of muscle-derived stem cells, such as the side population (SP) of muscle-derived cells described by
The relationship of the clonal mc13 cells with satellite cells is still unclear, but various features of these cells suggest a close relationship with satellite cells. These features include: the expression of myogenic markers including m-cadherin, which is known as a specific marker for satellite cells by the mc13 cells (
Although mc13 can efficiently regenerate skeletal muscle after i.m. injection, their ability to migrate to skeletal muscle after i.v. injection remains an important feature of these cells. Whereas a lower number of LacZ and dystrophin positive myofibers were found after i.v. injection of mc13 when compared with the i.m. injection, the mechanism by which these cells can be disseminated by the bloodstream and return to the skeletal muscle is important and requires further investigation. The development of approaches to improve the systemic delivery of these cells will be investigated. The absence of dystrophin positive cells at 7 d after injection within the lung, liver, spleen, kidney, and brain after i.v. injection of mc13 is surprising. These results suggest that the injected cells get specifically disseminated in the skeletal muscle, although we cannot rule out that the injected cells were present on these nonmuscle tissues at an early time point after injection, but died in the nonmuscle tissue.
The number of positive myofibers found in the injected animals after i.m. and i.v. injection of mc13 was also compared with that reported by
We have also compared our results with a recent publication from
The presence of stem cells in skeletal muscle makes this tissue an attractive source of cells for cell-transplantation therapy to enhance the healing of various tissues of the musculoskeletal system. We have in fact investigated in this study whether the genetic engineering of mc13 cells to express rhBMP-2 can be used to enhance closure of a nonhealing skull defect. Our results suggest that the mc13 was capable of inducing and, more importantly, participating in ectopic and orthopic bone formation when genetically engineered to express BMP-2. It is likely that the transplanted muscle cells are acting as a delivery vehicle for rhBMP-2, as well as a source of cells that differentiate into osteoblasts. The observation that 95% of the transplanted mc13 genetically engineered to express BMP-2 are located within the newly formed bone suggests that the vast majority of these cells are differentiating into osteogenic lineage and participating in bone formation. Based on these results, the stimulation with BMP-2 is required to differentiate these mc13 cells in osteogenic lineage to consequently improve bone healing. Our data suggest that the mc13 will differentiate into skeletal muscle, but the addition of an extra stimuli, such as the BMP-2 used in this experiment, will push them to differentiate in osteogenic lineage. These results suggest that muscle tissue is a valuable resource for osteoprogenitor cells to be used in clinical setting to improve bone healing.
Finally, we have investigated the karyotype of these cells and tested whether they can be grown in soft agar as an indicator of their tumorigenicity. Our results suggest that the number of chromosomes within the mc13 cells are normal and more importantly, they are totally incapable of growing on soft agar. This result, in addition to the lack of any adverse effect(s), at least histologically, such as tumor development, after their injection (mc13) in either skeletal muscle or other tissues in immunodeficient SCID mice, suggest that the mc13 are not tumorigenic.
In summary, the isolation and purification of muscle-derived stem cells offers an opportunity to elucidate cellular and molecular mechanisms of organogenesis. Taken together, our results suggest the isolation of a clonal population of muscle-derived stem cells capable of improving both muscle regeneration and bone healing. These new results will shed more light on the functional properties of the muscle-derived stem cells and further support that muscle tissue may become a valuable resource for the isolation of osteoprogenitor cells capable of improving bone healing. Further characterization of these muscle-derived stem cells will open an array of possibilities for advancement of tissue engineering and tissue transplantation techniques.
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Footnotes |
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Joon Yung Lee and Zhuqing Qu-Petersen contributed equally to the work.
1 Abbreviations used in this paper: adBMP-2, adenovirus bone morphogenetic protein-2 construct; ALP, alkaline phosphatase; DAPI, 4',6-diaminido-2-phenylindole; DMD, Duchenne muscular dystrophy; MNF, myocyte nuclear factor; npmc, nonpurified muscle-derived cells; pp, preplate; rhBMP-2, recombinant human BMP-2; RT, reverse transcription.
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The authors wish to thank Marcelle Pellerin and Ryan Pruchnic for their technical assistance, and Dr. Lilian Hsu and Dana Och for their assistance with the manuscript. The authors also wish to thank the Genetics Institute (Cambridge, MA) for the human recombinant BMP-2 and the antibody against BMP-2, Stephen Hardy for graciously providing us with the Cre-lox adenoviral system, and Dr. Miranda Grounds for providing us with the Y-probes.
This work was supported in part by grants to Dr. Johnny Huard from the National Institutes of Health (1 P60 AR44811-01, 1PO1 AR45925-01), the Pittsburgh Tissue Engineering Initiative (PTEI), and the William F. and Jean W. Donaldson Chair at Children's Hospital of Pittsburgh.
Submitted: 23 December 1999
Revised: 19 June 2000
Accepted: 30 June 2000
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