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First published online March 30, 2006
Stem Cells Vol. 24 No. 7 July 2006, pp. 1769 -1778
doi:10.1634/stemcells.2005-0547; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Regeneration and Transdifferentiation Potential of Muscle-Derived Stem Cells Propagated as Myospheres

Rachel Sarig, Zadok Baruchi, Ora Fuchs, Uri Nudel, David Yaffe

Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, Israel

Key Words. Key Words • Leukemia inhibitory factor • Muscle regeneration • Myospheres • Transdifferentiation • Somatic stem cells • p27Kip1 • Myogenesis

Correspondence: Rachel Sarig, Ph.D., Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel. Telephone: 972-8-9342720;Fax: 972-8-9344125;email: rachel.sarig{at}weizmann.ac.il

Received November 7, 2005; accepted for publication March 17, 2006.
First published online in STEM CELLS EXPRESS   March 30, 2006.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
We have isolated from mouse skeletal muscle a subpopulation of slow adherent myogenic cells that can proliferate for at least several months as suspended clusters of cells (myospheres). In the appropriate conditions, the myospheres adhere to the plate, spread out, and form a monolayer of MyoD+ cells. Unlike previously described myogenic cell lines, most of the myosphere cells differentiate, without cell fusion, into thin mononucleated contractile fibers, which express myogenin and skeletal muscle myosin heavy chain. The presence of Pax-7 in a significant proportion of these cells suggests that they originate from satellite cells. The addition of leukemia inhibitory factor to the growth medium of the myospheres enhances proliferation and dramatically increases the proportion of cells expressing Sca-1, which is expressed by several types of stem cells. The capacity of myosphere cells to transdifferentiate to other mesodermal cell lineages was examined. Exposure of cloned myosphere cells to bone morphogenetic protein resulted in suppression of myogenic differentiation and induction of osteogenic markers such as alkaline phosphatase and osteocalcin. These cells also sporadically differentiated to adipocytes. Myosphere cells could not, so far, be induced to transdifferentiate to hematopoietic cells. When inoculated into injured muscle, myosphere-derived cells participated in regeneration, forming multinucleated cross-striated mature fibers. This suggests a potential medical application.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Therapeutic approaches for skeletal muscle degeneration by cell transplantation have been hindered by poor cellular survival rates and the limited spread of the injected cells (reviewed in [1–4]). Major efforts were made to identify the most suitable cells for transplantation.

Skeletal muscle myoblasts plated in cell culture adhere to the culture plates slower than fibroblasts. This offered a simple and very efficient method for a significant enrichment of muscle cell suspension for myogenic cells. In this procedure, the heterogeneous cell population obtained from trypsinized skeletal muscle is preplated in untreated cell culture plates. After the adherence of the "fibroblastic" cells, the unattached cells are collected and plated in gelatin-coated plates to enable the adherence of the myoblasts. The myoblasts proliferate as adherent cells and in the appropriate culture conditions fuse into multinucleated muscle fibers that express muscle-specific proteins and become contractile. Serial passaging of such cultures, using differential plating and cell cloning, resulted in the establishment of myogenic cell lines [5–7].

Following several reports on the possible existence of slower adherent stem cells in skeletal muscle cell cultures [8, 9], we re-examined the differential plating procedure. In this study, we assayed the content of the medium (which we previously used to discard) during the medium replacement after the myogenic cells adherence to the plate. Here, we report on the isolation and characterization of populations of cells that are able to proliferate for extended periods as floating clusters of rounded cells (myospheres). Under the appropriate conditions, the cells in the myospheres adhere to the culture plates and most of them elongate and, unlike previously described myogenic lines, differentiate without cell fusion into mononucleated contractile thin fibers (needles). In aged cultures, the differentiated needles often fuse and form multinucleated fibers.

Myosphere cells participate in the regeneration of injured muscle, forming large multinucleated cross-striated fibers.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 Acknowledgments
 References
 
Cell Culture

Myosphere Preparation.   Primary muscle cultures were prepared from 3- to 4-week-old mice using a modified version of the previously described preplating procedure [5–7, 10]. The hind-limb muscles of mice were isolated, and the fat and bones were discarded. The muscle was minced with scissors, enzymatically dissociated at 37°C with 0.05% trypsin-EDTA for 30 minutes, and then centrifuged at 2,500 rpm for 5 minutes. The cells were collected, and the trypsinization of the remaining undigested tissue was repeated three more times by adding fresh trypsin solution. On the fourth time, the cells were incubated for 30 minutes with 0.25% trypsin-EDTA. After centrifugation, the cells were suspended in the proliferation medium BIO-AMF-2 (Biological Industries Ltd., Kibbutz Beit Haemek, Israel, http://www.bioind.com), which contains fetal calf serum, steroids, basic fibroblast growth factor, insulin, glutamine, and antibiotics, either with or without leukemia inhibitory factor (LIF) (10 ng/ml; CytoLab Ltd., Rehovot, Israel, http://www.cytolab.com). As indicated in Results, different populations of myoblasts were isolated based on their adhesion characteristics.

Myospheres were serially passaged by allowing them to sediment by gravitation to the bottom of a test tube, and the old medium was removed by decantation followed by careful suspension of the myospheres in fresh medium and plating them in uncoated cell culture plates.

Adherent monolayer of myosphere-derived cells were grown in gelatin-coated plates in the proliferation medium. When the cultures approached confluence, the medium was changed to differentiation-enhancing medium, 10HI (Dulbecco’s modified Eagle’s medium [DMEM] containing 10% carefully selected horse serum, 0.04 units per ml insulin, 0.5% chick embryo extract, and penicillin-streptomycin).

Where indicated, the cells were isolated as described by Qu-Peterson et al. [9]. The trypsinized cells were serially passaged as nonadherent cells for 4 days. On the 5th day, the cells that attached to the plate either were collected as uncloned cell population or were sorted by fluorescence-activated cell sorting (FACS) to isolate single Sca-1+ cells for clonization as described below.

C2 Cells.   C2 cells [10] were kept frozen in –80°C and amplified in culture in DMEM medium containing 20% fetal calf serum and penicillin (100 units/ml)-streptomycin (0.1 mg/ml) in gelatin-coated plates. To avoid uncontrolled cell fusion, the cells were split before reaching confluence and not more than 2–3 days after plating. Intensive cell fusion was induced by changing the medium to 10HI.

FACS Analysis
The percentage of cells expressing the following antigenes was analyzed by FACScan: Sca-1 (phycoerythrin [PE]-conjugated; eBioscience, San Diego, http://www.ebioscience.com), CD45 (fluorescein isothiocyanate [FITC]-conjugated; eBioscience), and CD34 (FITC-conjugated; BD Biosciences PharMingen, San Diego, http://www.bdbiosciences.com/pharmingen). Cells were washed once with phosphate-buffered saline (PBS) and resuspended in 0.1 ml of ice-cold PBS. Mouse serum (1:10; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and Fc block (rat anti-mouse CD16/CD32; BD Biosciences PharMingen) were added, and the suspensions were incubated for 10 minutes on ice. Each sample was divided into two halves: one half received the antibody (diluted according to the manufacturer’s instructions), and the other half received the conjugated fluorescein only (PE/FITC). The tubes were incubated at 4°C for 30 minutes and then washed twice with ice-cold PBS. For the sterile sorting and collection of Sca-1+ cells, the cells were stained with Sca-1-PE as above, and the cells expressing Sca-1 were collected by FACSVantage.

Immunocytochemistry
Adherent cells were grown on gelatin- or fibronectin-coated glass coverslips. Intact myosphere cells were fixed on a glass slide using a CytoSpin centrifuge (Shandon Elliott, Sewickley, PA, http://www.shandon.com). The cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes and permeabilized with 0.2% Triton X-100 in PBS for 5 minutes. For blocking, the cells were incubated in PBS containing 0.1% Triton and 3% bovine serum albumin for 30 minutes at room temperature. For immunostaining, the cells were incubated for 1 hour with the following monoclonal antibodies diluted in the blocking solution: MyoD (1:100; Dako, Cytomation, Glostrup, Denmark, http://www.dakocytomation.com), myogenin (1:1; a kind gift from W.E. Wright, Department of Cell Biology, University of Texas Southwestern Medical Center at Dallas, Dallas), desmin (1:100, DE-U-10; Sigma-Aldrich), myosin heavy chain (MHC) (1:20, MF-20; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww), and Pax-7 (1:100; Developmental Studies Hybridoma Bank).

After three washes with PBS containing 0.1% Triton, the cells were stained for 30 minutes at room temperature with Alexa-488-labeled goat anti-mouse antibodies (1:150; Molecular Probes, Eugene, OR, http://probes.invitrogen.com) followed by 5 minutes of DAPI (4',6-diamidino-2-phenylindole dihydrochloride) staining (10 µg/ml). The cells were mounted with Elvanol and viewed under a Nikon fluorescence microscope (Tokyo, http://www.nikon.com) at a magnification of x200/x400. Pictures were taken with a 1310 digital camera (DVC, Austin, TX, http://www.dvcco.com).

Muscle Regeneration
To induce muscle injury, cardiotoxin (0.1 ml of 10 µM; Sigma-Aldrich) was injected into the gastrocnemius muscle of nude mice. Myosphere cells (~106 cells) were injected to the injured muscle the following day. All injections were done using a 27.5-gauge needle. The mice were sacrificed at the indicated time points, and the injected muscle was removed, together with a control noninjected gastrocnemius muscle, and subjected to X-gal staining as described below.

X-Gal Staining.   The muscles were fixed for 60–90 minutes (according to their size) in 4% paraformaldehyde, washed with PBS, and stained overnight with X-gal [11]. Where indicated, dehydrated muscles were cleared in benzyl alcohol/benzylbenzoate solution (1:2). For sectioning, stained muscles were postfixed overnight in 4% PFA, dehydrated, embedded in paraffin, and cut on a microtome (5–7 µM). The slices were deparaffinized with xylene (2 minutes), rehydrated (from 95% to 25% ethanol), counterstained with eosin, and mounted.

BMP Treatment and Osteogenic Markers
293T cells expressing BMP-4 were a kind gift from Dr. Eldad Tzahor (Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel). The cells were grown in culture for 4–5 days (until confluence), and the growth medium (DMEM with 10% fetal calf serum) was collected, filtered, and used as the source for BMP-4. The detection of alkaline phosphatase (AP) and osteocalcin was done according to Wada et al. [12].

Transplantation of Cells to Lethally Irradiated Mice
Mouse bone marrow (BM) cells were flushed from femurs using a 27.5-gauge needle attached to a 1-ml syringe and suspended in PBS. Myosphere cells that grew as a monolayer were trypsinized and suspended in PBS, and those that grew as nonadherent cells were collected, separated by pipetting, and suspended in PBS.

For the i.v. injections, recipient mice were exposed to two gamma irradiation doses of 600 rad from a cesium source (with a 3-hour separation between the two irradiations). Myosphere cells were injected into the tail veins of the mice 4–5 hours after irradiation. The amount of cells injected in each experiment is indicated in supplemental online Table 3. For the injections of cells into the BM, mice were irradiated with 950 rad 24 hours before transplantation. The mice were anesthetized prior to the injection, and their knee was flexed to 90°. Either myosphere cells (106 cells/mouse) or BM cells (5 x 105 cells/mouse) were injected into the BM cavity of the tibia using a 27.5-gauge needle.

Mice were kept in sterile conditions, and antibiotics (Cyproxine 100) were added to their water.

All animal studies were approved by the Institutional Animal Care and Use Committee of the Weizmann Institute of Science.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 Acknowledgments
 References
 
Most Myospheres Are Formed Mainly by Clonal Proliferation of Single Cells
Twenty-four hours after plating of freshly isolated adult mouse skeletal muscle cells, most living cells were attached to the bottom of the plate. The nutrient medium contained mostly cells debris. However, careful examination revealed the presence of very few small rounded cells suspended in the medium. Some of them were in clusters of two to four cells. One or 2 days later, many clusters containing up to 10–15 cells were very conspicuous. In the next few days, these myospheres continued to grow in size and number. These cells could be maintained by serial passages as suspended myospheres for at least several months without loosing their proliferation capacity (Fig. 1A).


Figure 1
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  		Figure 1. Cultures of myospheres. (A): Myospheres grown in suspension. (B): Outgrowth of myogenic cells from adherent myospheres. (C): Myosphere cells grown as an adherent monolayer. Myosphere cells adhere to the plate as rounded or spindle-shaped cells. (D): A monolayer of a clone R1#a1, derived from ROSA26 myosphere cells. Note the formation of a network of predominantly thin mononucleated fibers. (E): Magnification of (D). (F): After several passages of the adherent cells, foci of cells fusing into thick multinucleated fibers start to appear. The figure shows a fusing subline isolated from such a focus. (A–C): Phase-contrast. (D–F): Giemsa staining. Scale bars = 50 µm (A, C–E), 100 µm (B), and 250 µm (D).

 
To determine whether the myospheres were formed by cell aggregation or by clonal cell proliferation, we dissociated and cocultivated muscle cells isolated from wild-type mice of the strain 129 SVJ together with muscle cell populations derived from muscles of ROSA26 mice, which contain a transgene encoding a ubiquitously expressed bacterial ß-galactosidase [13]. After 4 days, the myospheres were collected by decantation of the medium and stained for ß-galactosidase activity. We found that the majority of myospheres consisted exclusively of either blue cells or unstained cells. Only 12% of the myospheres contained both stained and unstained cells, often clustered separately within the myosphere, suggesting fusion between myospheres (supplemental online Table 1). We therefore concluded that the myospheres were formed mainly by clonal proliferation of single cells that stayed together. However, there is also fusion between myospheres.

Cells from the Myospheres Spread Out and Form a Myogenic Monolayer
When myospheres grown in gelatin-coated plates are left for several days in the same plate, many of them adhere to the plate and start to spread out and to shade cells that adhere to the plate as single rounded cells or as spindle-shaped cells (Fig. 1B, 1C). Later, many of these cells elongate and form very thin myogenin-positive fibers (needles). Most of these fibers are mononucleated or containing two to three nuclei (Fig. 1D, 1E), and many of them are contractile. Growing these cells in the differentiation stimulating medium 10HI enhances the process of cell elongation as needles. Slow cell fusion and formation of a network of multinucleated fibers occur in aged cultures, indicating fusion between differentiated needles. In addition, serial passages of the cells for extended periods as adherent monolayers resulted in increased proportion of cells fusing into multinucleated fibers in response to 10HI medium. Clonal analysis indicated that this transition is stably inherited, suggesting an epigenetic change (Fig. 1F).

Isolation and Characterization of Myogenic Cell Lines Derived from Myospheres
Closer examination of freshly isolated uncloned myospheres revealed that, in many of them, some of the rounded cells twitched. Immunostaining revealed that most or all of the cells expressed MyoD and, in a small proportion of the cells within the myospheres, the nuclei were myogenin-positive, confirming their differentiated nature (Fig. 2). Most of the cells express Pax-7, suggesting their origin from muscle reserve cells (satellite cells) (Fig. 2).


Figure 2
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  		Figure 2. Myogenic markers expressed by myospheres. An uncloned myosphere population (cultured for 1 week) grown in the proliferation medium and immunostained with the indicated antibodies. Most of the cells (>90%) express MyoD and desmin, indicating that they belong to the skeletal muscle lineage. Few cells express myogenin (arrowhead), which may explain the contraction of single cells within the myosphere. Most of the cells (~70%) express the satellite cell marker Pax-7, suggesting that myosphere cells derive from satellite cells. Scale bars = 25 µm. Abbreviation: DAPI, 4,6-diamidino-2-phenylindole.

 
Serial passages and cloning of the proliferating attached cells described above or of dissociated myosphere cells resulted in the isolation of several myogenic clones. These clones proliferated as a mixture of rounded and spindle-shaped mononucleated cells (Fig. 1). Immunofluorescence staining with antibodies against MyoD, myogenin, desmin, and Pax-7 revealed intrinsic differences between clones. Most or all of the cells in all the clones expressed MyoD. Some clones did not express MyoD or expressed it in a low percentage of the cells when grown as myospheres. However, when these same clones were grown as a monolayer of adherent cells, most of the cells expressed MyoD. All clones tested were desmin-positive and expressed myogenin and skeletal muscle MHC after induction of myogenic differentiation. A small percentage of the cells in some clones retained the expression of Pax-7 but at much lower frequency than the uncloned myosphere cell population (not shown). When induced to differentiate, most of the clones formed contractile needles, as described above, whereas in some clones the cells fused and formed multinucleated fibers without prior differentiation into mononucleated needles. All the clones were CD45 and CD34–, indicating that they do not belong to the hematopoietic or endothelial lineage (supplemental online Table 2).

Thus, the pathway of differentiation of most of the myosphere-derived cells differs from that of the previously established myogenic cell lines. Whereas the common myogenic differentiation follows cell fusion, most of the myosphere-derived cells differentiate into contractile myogenin and myosin-expressing cells without cell fusion (Fig. 3A–3C).


Figure 3
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  		Figure 3. Single myosphere cells express myosin heavy chain (MHC) without cell fusion. When myospheres were grown in differentiation medium (10HI), most of the cells express MHC (green) either within the myospheres (A) or as single mononucleated adherent cells (B). This is in contrast to the pattern of differentiation in previously described myogenic cell lines, in which MHC appears after cell fusion, as shown here in the C2 cells (C). The myosphere clone R4#c that differentiates directly to multinucleated fibers expresses MHC only in the multinucleated fibers, similarly to C2 cells (D). Nuclei were stained with 4,6-diamidino-2-phenylindole (blue). Scale bars = 25 µm (A, B, D) and 50 µm (C).

 
Lin et al. [14] reported that in primary chick muscle cultures, myoblasts initiate synthesis of MHC prior to cell fusion. We have reinvestigated this question in rat and mouse primary myoblasts cultures isolated from muscle by differential plating. Using immunostaining, we have found that whereas in mouse cultures, a significant proportion of the mononucleated myoblasts express MHC at the onset of cell fusion, in rat myoblast cultures, the expression of MHC is confined mostly to the multinucleated fibers (not shown). Interestingly, in the mouse, myosphere-derived cell lines of which the spindle-shaped myoblasts fuse into multinucleated fibers without first forming needles (supplemental online Table 2), staining for MHC is confined almost exclusively to the multinucleated fibers (Fig. 3D). Thus, it seems that the differences in the sequence of events are determined by genetic and epigenetic factors. It should be noted that unlike the situation with primary mouse myoblasts, in which a fraction of the myoblast-shaped cells express MHC, in the myosphere derived cultures, almost the entire cell population differentiates into needle-shaped contractile mononucleated fibers.

It was recently shown that the expression of p27Kip1 controls the differentiation of C2 (C2C12) cells by upregulating MyoD levels in undifferentiated cells. Exogenous expression of p27Kip1 allowed low-density cultured C2 cells to differentiate and express MHC as single cells [15]. Our examination of endogenous p27kip1 expression revealed that C2 cells and myosphere-derived clones that differentiate directly to multinucleated fibers express p27kip1 only after their induction to differentiate by 10HI medium. In contrast, clones that differentiate as needles express p27kip1 already when cultured in the growth medium (supplemental online Fig. 1). These results indicate a difference in the sequence of signal transductions leading to terminal differentiation of muscle cells.

LIF Enhances Sca-1 Expression and Supports Cell Proliferation
Sca-1 is a stem cell marker of both hematopoietic and myogenic stem cells [16]. The percentage of Sca-1+ cells in uncloned myosphere cell populations 1 week after their isolation from the muscle ranged between 4% and 20% (Fig. 4). Using FACS, we have isolated myosphere clones, which originated from single Sca-1+ cells. Analysis of Sca-1 in these clones revealed that after amplification, the cells had a similar expression pattern of Sca-1 as the original unsorted cell population. Thus, using the regular growth medium and cloning, it was not possible to obtain Sca-1-enriched cell populations. LIF is known to inhibit the differentiation of mouse embryonic stem cells and of isolated multipotent adult progenitor cells [17, 18]. We have found that including LIF in the growth medium significantly enhanced the proliferation of Sca-1+-isolated clones. After propagation of these clones in medium containing LIF, they could continue to proliferate also without the addition of LIF. The addition of LIF to either the uncloned myosphere cell populations or the Sca-1+-isolated clones greatly increased the percentage of Sca-1+ cells (from 15%–80%) (Fig. 4). Moreover, the addition of LIF to Sca-1– cells for 7 days induced the expression of Sca-1 in 15% of the cells (not shown).


Figure 4
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  		Figure 4. LIF dramatically increases the proportion of cells expressing Sca-1. LIF was added to the proliferation medium of both uncloned and cloned myosphere cells populations. The effect of continuous exposure to LIF on Sca-1 expression was determined by fluorescence-activated cell sorting analysis 7 days later (top panel). Bottom panel: A graph summarizing three independent experiments. Abbreviation: LIF, leukemia inhibitory factor.

 
Interestingly, in some of the clones, the addition of LIF and enrichment of Sca-1+ cells reduced the percentage of cells expressing MyoD. However, changing to the differentiation-inducing medium 10HI enhanced MyoD expression and cell differentiation. Chulman et al. [19] showed that LIF reduced MyoD expression and cell fusion in the myogenic cell line C2. However, using primary muscle cultures, Vakakis et al. [20] showed that the LIF effect on myoblasts differentiation was reversible; thus, withdrawal of LIF from the medium and the replacement of the medium to 10HI resulted in myogenic differentiation. We have found that the addition of LIF to the primary uncloned population of myoblasts did not inhibit their differentiation; the cells fused and differentiated in the presence of LIF (not shown).

Myosphere Cells Fuse with C2 Cells
To reveal whether mononucleated myosphere cells that form needles can fuse with myoblasts that form multinucleated fibers, we mixed a myosphere clone (derived from ROSA26 mouse) that differentiates as mononucleated cells (R1#a1) together with C2 myoblasts, which form a network of large multinucleated fibers [10]. Exposure to 10HI medium resulted in the formation of many multinucleated fibers. Most of the fibers stained blue by X-gal, showing that the myosphere cells participated in the formation of the multinucleated fibers (supplemental online Fig. 2). This suggests that C2 cells provide a factor that accelerates cell fusion and that is deficient in pure populations of myosphere-derived cells. Interestingly, in these mixed cultures, almost all mononucleated cells were LacZ–, suggesting that the myosphere-derived cells were preferentially incorporated into the fibers.

Replacement of the C2 cells with a fibroblastic feeder layer also resulted in the acceleration of cell fusion of myosphere cells (not shown). However, using conditioned medium from either cultures of C2 or fibroblasts feeder layer did not affect the myosphere cell fusion, suggesting that the effect is mediated by cell contact.

Participation of Myosphere Cells in Regeneration of Damaged Muscle
To test the capacity of myosphere cells to participate in muscle regeneration, we injected cloned myosphere cells derived from ROSA26 mice into cardiotoxin-injured gastrocnemius muscle of nude mice (106 cells/muscle). Treated mice were sacrificed at various times after injection, followed by X-gal staining of the treated muscle. The regenerating, stained muscle was then sectioned. Microscopic examination of the sections revealed that the participation of myosphere cells in muscle regeneration was progressive. Sections of muscle taken 2–3 weeks after treatment showed small and local stained areas with little cross-striation. The stained area was much larger at 6 weeks after injection, whereas sections of muscle taken 2–3 months after injection contained large bundles of blue cross-striated fibers in the regenerating areas (Fig. 5). Some of the fibers were variegated, suggesting fusion between host and donor myogenic cells (Fig. 5B–5D; supplemental online Fig. 3). Transverse sections revealed the distribution of donor-derived fibers, organized both in clusters as well as dispersed single blue fibers (Fig. 5D).


Figure 5
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  		Figure 5. Participation of myosphere cells in muscle regeneration. (A): Gastrocnemius muscle of nude mice was injured by injection of cardiotoxin. One day later, the muscle was injected with 106 myosphere cells derived from a ROSA26 mouse, harboring a ß-gal transgene. Six weeks later, the muscle was fixed, X-gal-stained, and made transparent (by benzyl alcohol and benzyl benzoate). (B): The injured muscle was fixed and stained 2 months after injection, embedded in paraffin, sliced, and counterstained with eosin. The blue cross-striations indicate that myosphere cells are able to regenerate injured muscle. (C): Higher magnification of (B). (D): A transverse section showing the distribution of donor cells. The differences in the intensity of X-gal staining suggest variability in the proportion of host and donor nuclei in single fibers. Scale bars = 500 µm (A), 150 µm (B), 25 µm (C), and 60 µm (D).

 
Testing the Capacity of Myosphere Cells to Transdifferentiate into Osteogenic and Hematopoietic Cells
When left for a long period in differentiation medium, two clones (R21#4 and R21#8) spontaneously formed foci of adipocytes (Fig. 6A). Spontaneous transdifferentiation of satellite cells to adipocytes was recently described by Shefer et al. [21].


Figure 6
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  		Figure 6. Multipotential capacity of myosphere cells. (A): In the appropriate culture conditions (high density of differentiated muscle cells), cells in some clones spontaneously differentiated into adipocytes colonies detected by Oil Red O staining. In the myosphere-derived clones, exposure to bone morphogenic protein-4 induced the expression of the osteogenic markers alkaline phosphatase (B) and osteocalcin (C). Scale bars = 25 µm (A) and 50 µm (B).

 
To investigate the potential of myosphere cells to transdifferentiate to other mesodermal cell lineages, we exposed the myosphere cell clones to BMP-4. This resulted in acquisition of fibroblastic morphology, suppression of the expression of MyoD, inhibition of myogenic differentiation, and expression of osteogenic markers such as AP and osteocalcin (Fig. 6B, 6C) in all cells in all the tested clones (supplemental online Table 2). As reported for BMP-treated C2 cells [22], removal of the BMP resulted in regaining of the myogenic phenotype and disappearance of the osteogenic markers.

The growth of cloned myospheres in medium enriched with factors that support hematopoietic cell proliferation (Methocult GF; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) often resulted in enrichment of the cell population with cells expressing CD45 (Fig. 7A). However, these CD45+ cells enlarged in size, did not morphologically resemble hematopoietic cells, nor did they continue to proliferate (not shown). On the other hand, a great enrichment for CD45+ cells (up to 95%) was observed when we used a very early passage of uncloned myosphere cell populations (cultured for only 5 days prior to treatment) (Fig. 7A). This was probably due to CD45+ cells that exist in the early plating stage, as was shown by McKinney-Freeman et al. [23] and Polesskaya et al. [24]. Indeed, FACS analysis showed that myosphere populations harvested 5 days after the primary preparation contain 30%–40% CD45+ cells (Fig. 7B). The capability of the myosphere cells to express CD45 upon exposure to Methocult GF medium decreased with subsequent passaging (Fig. 7A). Moreover, separation by FACS of the cells that express CD45 from those that were CD45– and culturing of these populations separately in Methocult GF medium yielded hematopoietic colonies only in the culture of the CD45-expressing cells (data not shown). These results indicate that myosphere cells that do not express CD45 cannot transdifferentiate to hematopoietic cells in vitro and that the origin of the hematopoietic cells that were grown in Methocult GF is in the CD45-expressing cells that reside in the muscle.


Figure 7
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  		Figure 7. Myosphere cells did not transdifferentiate into hematopoietic cells. (A): Uncloned and cloned myosphere cells were cultured for 7 days in Methocult GF medium, and the percentage of cells expressing CD45 was analyzed by FACS. BM cells were used as a positive control. Freshly prepared uncloned population cultured only 5 days prior to treatment (5d) contained stem cells that gave rise to a high level of CD45+ cells. In contrast, uncloned population that was cultured for a long period prior to treatment (90 days [90d]) lost its ability to give rise to hematopoietic cells. The indicated clones gave rise to various levels of CD45+ cells; however, these cells did not morphologically resemble hematopoietic cells, nor did they continue to proliferate. (B): Freshly prepared myosphere cells (up to 7 days in culture) contain 30%–40% CD45+ cells. The graph represents FACS analysis of one such preparation. Right panel: Fluorescein isothiocyanate (FITC) only; left panel: anti-CD45-FITC. (C): Injection of myosphere cells directly into the BM did not rescue lethally irradiated mice. Injection of freshly prepared, uncloned myosphere cells (myospheres) into the BM (intra-bone marrow) of lethally irradiated mice did not improve their survival. Mice that were injected with BM cells as a control for the IBM injection procedure (BM) survived the irradiation. Abbreviations: BM, bone marrow; FACS, fluorescence-activated cell sorting; FS-CH, forward scatter-channel.

 
To test the capacity of myosphere cells to participate in hematopoiesis, we injected the cells into lethally irradiated mice. C3H and C57bl female mice were lethally irradiated, and 6–12 hours later the mice were injected intravenously with dissociated myospheres derived from C3H or C57bl male mice, respectively (supplemental online Table 3). In addition, because it was reported that muscle stem cells obtained from mix mice rescued lethally irradiated SJL mice [25], lethally irradiated SJL mice were injected with myospheres derived from mdx, SJL, or ROSA26 mice (that were inbred on the C57bl background, which is the genetic background of mdx). In 19 injection experiments that were made, no advantage of the injected mice was observed. In two experiments, there was a delay of 7–8 days in the mortality of some of the injected mice, compared to uninjected irradiated mice (supplemental online Table 3). Irradiated mice that were injected with BM cells served as a positive control that survived the irradiation (not shown).

In addition, C3H female mice were sublethally irradiated and injected with myospheres obtained from C3H male mice. Polymerase chain reaction (PCR) analysis of muscle, BM, peripheral blood, spleen, lungs, kidneys, and colon obtained from the injected mice did not detect the presence of the injected cells.

The first steps in the isolation of myogenic cells used by Cao et al. [25] were similar to our initial steps (differential plating). In recent experiments, we have attempted to follow the protocol of Cao et al. [25]; however, so far we have been unable to rescue the irradiated mice by injection of myogenic cells. The reason for the difference in the results is not yet clear.

To bypass a possible inefficient homing of the i.v. injected myosphere cells to the BM, we injected 106 cells of either uncloned myosphere population cultured for 5 days (and contain ~40% CD45+ cells) or myosphere clones (which do not express CD45) directly into the BM (intra-BM [IBM]) of isogenic lethally irradiated mice. We injected irradiated mice with 5 x 105 BM cells as a positive control for the procedure. Nevertheless, the mice injected with the myosphere cells (either uncloned early population or clones) died after 10–12 days, as did the uninjected irradiated mice. All mice that were injected IBM with BM cells survived the effect of irradiation (Fig. 7C).

Taken together, these experiments did not provide evidence for transdifferentiation of the myosphere cells to hematopoietic cells. Moreover, although primary muscle cultures contain hematopoietic stem cells, which can give rise to hematopoietic cells, both in vitro and in vivo ([23] and Fig. 7), they could not efficiently reconstitute the hematopoietic system of lethally irradiated mice.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 Acknowledgments
 References
 
The investigation reported here describes the reproducible isolation of myogenic cells derived from mouse skeletal muscle that can be propagated in suspension cultures as myospheres for an extended period. The myosphere cells retain their capacity to differentiate in vitro and to participate efficiently in muscle regeneration. The selection of those cells is based in principle on the old observation that skeletal muscle and cardiac myogenic cells adhere to the cell culture plate surface slower than most nonmyogenic cells that are obtained during the conventional preparation of muscle cell culture [5]. A partially similar approach was taken by Qu-Peterson et al. [9], who showed that cells that do not adhere to the plate during the first 5 days yield myogenic clones that differ from early adherent myoblasts in their capacity to repopulate degenerated muscle. Those cells were collected after 5 days and plated for further amplification as adherent cells. In the study reported here, we found that it is possible to propagate suspended cultures for at least several months without their losing the capacity to proliferate, differentiate, and participate in muscle regeneration.

Interestingly, unlike previously described rat and mouse myogenic cell lines, this procedure selected for cells that differentiate, mostly without cell fusion, into very thin elongated mononucleated myogenin and myosin-positive contractile cells. The formation of mononucleated contractile cells may indicate that these cells represent an earlier stage of myogenic differentiation, reminiscent of the mononucleated muscle cells of early somite [26]. It is possible that the continued proliferation of nonadherent cells selects for cell surface properties that lead to terminal differentiation without or prior to cell fusion. One possible candidate that enables terminal differentiation in single myogenic cells is p27kip1. This cyclin-dependent kinase inhibitor was shown to play a critical role in the N-cadherin-dependent signaling during myogenesis, and its forced expression in C2 cells resulted in their differentiation as single cells [15]. We show here that myosphere-derived cells express p27kip1 already when grown in the proliferation medium. It is possible that growing the cells as myospheres selects indirectly for cells expressing p27kip1 (e.g., perhaps by selecting for cells expressing altered levels of N-cadherin). Thus, conceivably, myosphere cells that express p27kip1 when cultured in the growth medium as single cells may bypass the requirement for cell-cell contact in order to differentiate.

As shown, the serial passages of uncloned mass cultures of myospheres resulted in a very efficient selection of myospheres consisting virtually of MyoD+ myogenic precursor cells. Cloning of single cells revealed mild heterogeneity between clones with regard to several markers, including expression of MyoD and the tendency of the cells to fuse and form multinucleated fibers or to differentiate first as mononucleated needles. Using the same experimental procedure with rat muscle cells, we obtained myosphere-like clusters of cells, most of which differentiated into a mixture of multinucleated muscle fibers and colonies of adipocytes (unpublished results) after adherence. As shown here, some cells of the mouse myosphere clones spontaneously transdifferentiated to adipocytes (Fig. 6A), suggesting that the rat adipocytes were derivatives of myogenic cells. Additional experiments are needed to determine the capacity of muscle cells of other species, including human cells, to yield myosphere cultures.

Several groups reported on the isolation of stem cells derived from various other tissues and their proliferation in suspension as spheres [27–29]. Neurosphere cells were shown to have pluripotent capability, giving rise to cells from all three germ layers, and participate in skeletal muscle and hematopoietic regeneration [30, 31]. Adult BM-derived cells have been shown to integrate into diverse adult tissues, including skeletal and cardiac muscle [32–35]. Several other stem cells were isolated and proliferated as adherent cells, which had either multipotent or pluripotent capability [18, 25, 36–38]. Despite these investigations, the issue of transdifferentiation of cells is still a matter of controversy, given that other findings indicated the inability of stem cells to transdifferentiate into other cell types [39–44].

In contrast to previously described pluripotent stem cells, which are immature and do not express proteins associated with specific differentiation (e.g., neurospheres, mesenchymal adult progenitor cells), the myosphere cells described here express the myogenic marker MyoD and are conceivably already more committed.

So far, we have not succeeded in rescuing irradiated mice by injection of myogenic cells (i.v. or IBM). This is in accordance with several reports on the incapability of CD45– cells derived from muscle to give rise to hematopoietic cells [45–49]. Moreover, although primary muscle cultures contain stem cells that can give rise to hematopoietic cells both in vitro and in vivo ([23] and Fig. 7), these cells could not rescue lethally irradiated mice (Fig. 7C). It is possible that hematopoietic stem cells from muscle do not give rise to certain essential hematopoietic cell types or that the short period during which the cells must replace a complete hematopoietic repertoire is not sufficient. Isolation of the hematopoietic stem cell population from muscle and their enrichment in vitro prior to transplantation may improve these results. However, this does not change the conclusion mentioned above that CD45– cells did not contribute to the regeneration of the hematopoietic system. It should be mentioned that Cao et al. [25] reported on the capacity of myogenic clones expressing MyoD (detected by reverse transcription-PCR) to repopulate the BM of lethally irradiated mice. The reason for the differences in the results needs further investigation. One possibility is that their clones contained a mixture of committed MyoD+ and uncommitted pluripotent cells.

C2 cells and primary satellite cells respond to BMP by expressing AP and osteocalcin and can also give rise to adipocytes [12, 22, 50–55]. BMP treatment induced the expression of AP and osteocalcin in all the cloned myosphere cell populations (Fig. 6B, 6C). These results suggest the potential of the myospheres to give rise to other mesodermal cell lineages. In accordance with the results obtained with C2 cells [22], the effect of BMP on myosphere cells was dependent on its continuous presence in the medium; withdrawal of BMP resulted in re-expression of MyoD and myogenic differentiation. Thus, the osteogenic nature of the response needs further investigation.

The capacity of myosphere-derived cells to participate in regeneration of injured muscle may be of potential clinical importance. It is therefore important to see whether this approach can be adapted for human myogenic cells.


   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
 
We thank Vivienne Laufer for editorial assistance and Drs. Esther Lustig and Alpha Peled for helping us with i.v. and IBM injections. The Pax-7 hybridoma (developed by A. Kawakami) and MF-20 hybridoma (developed by D.A. Fischman) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA. This research was supported by the Association Française contre les Myopathies (AFM), France.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 Acknowledgments
 References
 

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