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Stem Cells 2005;23:610-618 www.StemCells.com
© 2005 AlphaMed Press

Two Different Roles of Purified CD45+c-Kit+Sca-1+Lin– Cells After Transplantation in Muscles

Momoko Yoshimotoa, Hsi Changa, Mitsutaka Shiotaa, Hirohiko Kobayashia, Katsutsugu Umedaa, Atsushi Kawakamib, Toshio Heikea, Tatsutoshi Nakahataa

a Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan;
b Department of Biological Science, Graduate School of Sciences, University of Tokyo, Tokyo, Japan

Key Words. Hematopoietic stem cells • Transplantation • c-Kit+Sca-1+Lin– • Muscle stem cells

Correspondence: Tatsutoshi Nakahata, M.D., Ph.D., Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: 81-75-751-3290; Fax: 81-75-752-2361; e-mail: tnakaha{at}kuhp.kyoto-u.ac.jp


   ABSTRACT
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent studies have indicated that bone marrow cells can regenerate damaged muscles and that they can adopt phenotypes of other cells by cell fusion. Our direct visualization system gave evidence of massive muscle regeneration by green fluorescent protein (GFP)–labeled CD45+c-Kit+Sca-1+Lin– cells (KSL cells), and we investigated the role of KSL cells in muscle regeneration after transplantation with or without lethal irradiation. In the early phase, GFP signals were clearly observed in all the muscles of only irradiated mice. Transverse cryostat sections showed GFP+ myosin+ muscle fibers, along with numerous GFP+ hematopoietic cells in damaged muscle. These phenomena were temporary, and GFP signals had dramatically reduced 30 days after transplantation. After 6 months, GFP+ fibers could hardly be detected, but GFP+ c-Met+ mononuclear cells were located beneath the basal lamina where satellite cells usually exist in both conditioned mice. Immunostaining of isolated single fibers revealed GFP+ PAX7+, GFP+ MyoD+, and GFP+ Myf5+ satellite-like cells on the fibers. Single-fiber cultures from these mice showed proliferation of GFP+ fibers. These results indicate two different roles of KSL cells: one leading to regeneration of damaged muscles in the early phase and the other to conversion into satellite cells in the late phase.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Various tissue-specific stem cells have been identified in epidermis [1], intestinal epithelium [2], testis [3], liver [4], brain [5], and muscle [6]. Until recently, it was thought that tissue-specific stem cells could only differentiate into their original tissue, but it has been demonstrated that they can also differentiate into other lineages. For example, cells of donor origin have been detected in liver, heart, vascular endothelium, skeletal muscles, and other organs after bone marrow (BM) transplantation [7–14]. Of special interest for our study is that BM-derived cells have been shown to participate in the regeneration of chemically damaged fibers in skeletal muscle [12]. Subsequent studies showed that dystrophin-positive myofibers were restored in mdx mice, an animal model of Duchenne’s muscular dystrophy, after transplantation of stem cells purified by fluorescence-activated cell sorting with Hoechst 33342 low-stained cells, also known as side population (SP) cells [15]. In short, stem cells in BM, comprising hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), have been found to be capable of regenerating damaged muscle fibers after BM transplantation.

It has also been demonstrated, however, that BM cells can adopt the phenotype of other cells by means of cell fusion [16, 17]. These investigators warned that in vivo "transdifferentiation" might result from cell fusion. The differentiation potential of BM cells beyond the lineage restriction of stem cells remains to be determined.

In addition to BM cells, SP cells have also been identified in muscle tissues [15, 18]. These muscle SP cells are reported to have hematopoietic as well as myogenic potential [19] and to express CD45 antigen, which is recognized as a hematopoietic cell marker. Another study gave evidence that CD45+ cells in skeletal muscle are of BM origin [20]. These reports thus indicate that hematopoietic cells of BM origin seem to be present in skeletal muscles. However, the correlations among HSCs, CD45+ cells in skeletal muscle, satellite cells, myogenic precursors, and muscle-derived stem cells have not yet been determined. It is important to clarify these relationships, both for scientific research and for the application of stem cell therapy.

To investigate the potential and the kinetics of HSCs in skeletal muscles, we transplanted c-Kit+Sca-1+Lin– (KSL) cells as enriched HSC fraction from green fluorescent protein (GFP) transgenic mice into lethally irradiated C57BL/6 mice or nonirradiated W/Wv neonates that can accept HSCs without myeloablation. We examined the time-course behavior of GFP+ cells in recipient muscles with a fluorescent stereo microscope and immunohistochemical staining during the early and late phases after transplantation. Our visualization system makes it possible to detect transplanted cells with GFP signals in intact organs without the need to make sections first and can easily trace their kinetics throughout the entire body [21]. With this system, we found that myeloablation enables KSL cells to migrate into damaged muscles and to regenerate muscle fibers in the whole body during the early phase following transplantation. In the late phase, progenies of KSL cells had remained in muscle tissues and gave rise to satellite cells with myogenic potential, regardless of whether the mice had been irradiated.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Mice
Pregnant female W/+ mice mated with Wv/+ mice were obtained from Shizuoka Laboratory Animal Center (Shizuoka, Japan). GFP transgenic mice were kindly provided by Dr. M. Okabe (Osaka University, Japan). The background of all these mice was C57BL/6. All cells except erythrocytes of transgenic mice expressed GFP protein. The mice were bred and maintained in a specific pathogen-free microisolator environment. Neomycin in acidic water was supplied to irradiated recipient adult mice during the first month after transplantation.

Cell Sorting
BM cells were labeled with a cocktail of biotinated primary antibodies for CD3 (145-2C11), B220/CD45R (RA3–6B2), Mac-1 (M1/70), Gr-1 (RB6-8C5), and TER119 (TR119). All antibodies were purchased from Pharmingen (San Diego, http://www.bdbiosciences.com/pharmingen). Lineage-negative (Lin–) cells were obtained by auto–magnetic cell sorting (MACS; Militenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com), according to the manufacturer’s instructions. Lin– cells were then stained with phycoerythrin (PE)–conjugated anti-Sca-1 antibody and allophycocyanin (APC)–conjugated anti-c-Kit antibody. KSL cells were collected by cell sorting on a FACS-Vantage (Becton, Dickinson, San Jose, CA, http://www.bd.com). To confirm that KSL cells are all hematopoietic cells based on the expression of CD45 antigen, we stained Lin– cells collected from wild-type C57BL/6 mice with fluorescein isothiocyanate (FITC)–conjugated anti-CD45 antibody, PE-conjugated anti-Sca-1 antibody, and APC-conjugated anti-c-Kit antibody.

Transplantation and Sequential Analysis
One to 5 x 103 of KSL cells with 2 x 105 Ly5.1 BM cells were injected into the tail vein of C57BL/6 adult mice that had received 9.0 Gy irradiation or into the orbital branch of the anterior facial vein of W/Wv neonates within 0–3 days after birth. On days 3, 10, 20, and 30 after transplantation, all muscles were observed under a fluorescent stereomicroscope (Leica, Heerbrugg, Switzerland, http://www.leica.com). At least five mice were analyzed in each time point.

Adherent Cell Culture
To see if KSL cells contain any mesenchymal cells, we cultured 1 to 5 x 103 per well of KSL cells with Mesen Cult Medium (Stem Cell Technologies, Vancouver, Canada, http://www.stemcell.com) in 24-well plates for 14 days.

Immunohistochemistry
Muscles were fixed with 4% paraformaldehyde, embedded in the optimal cutting temperature compound. Frozen sections of 7–3m thickness were mounted on silane-coated glass slides. For immunostaining of single-muscle fiber [22], fibers were fixed with 4% paraformaldehyde and were permeabilized with 0.5% (vol/vol) Triton X-100 phosphate-buffered solution (PBS). Rabbit or mouse anti-GFP (BD Biosciences Clontech, Palo Alto, CA, http://www.bdbiosciences.com/clontech/), rat anti-CD45 (Pharmingen), mouse anti-myosin (Zymed Laboratories, San Francisco, http://www.zymed.com), mouse anti-myogenin, mouse anti-MyoD1 (Dako, Carpinteria, CA, http://www.ump.com/dako.html), rabbit anti-c-Met, rabbit anti-Myf5 (Santa Cruz Biotechnology, Santa Cruz, CA, http://www.scbt.com), rabbit anti-laminin (Dako), and mouse-anti PAX7 (generated from mouse hybridoma [23]) were used as the primary antibodies. Either alkaline phosphatase (ALP)–conjugated anti-rabbit, Alexa488-conjugated anti-rabbit, Alexa350-conjugated anti-rabbit, streptavisin Alexa350 Cy3–conjugated anti-rat or anti-mouse, FITC-conjugated anti-rabbit or anti-mouse was used as the secondary antibody. Hematoxylin or Hoechst 33324 was used for nuclear staining. These samples were then examined under a fluorescent microscope (Olympus, Tokyo, http://www.olympus-global.com), an AS-MDW (Leica), or a confocal microscope (Olympus). Photographs were obtained with an Axio Cam (Carl Zeiss Vision GmbH, Hallbergmoos, Germany, http://www.zeiss.com) or an AS-MDW (Leica).

FISH Analysis
For the detection of GFP DNA in myonuclei of the tissues at 30 days and 6 months after transplantation, fluorescence in situ hybridization (FISH) analysis was done. GFP detection was made using a polymerase chain reaction (PCR) digoxigenin (DIG) probe synthesis kit (Roche, Basel, Switzerland, http://www.roche.com). Hybridization was done according to the instructions of in situ hybridization kit (Nippon Gene, Toyama, Japan, http://www.nippongene.jp). In brief, frozen sections were fixed in 100% ethanol, followed by incubation in 90%, 80%, 70%, and 50% ethanol. Sections were incubated in 50 µg/ml of proteinase K for 15 minutes at 37°C. The GFP probe was denatured at 95°C and added to each slide following incubation on a 95°C hotplate. The sections were hybridized overnight at 42°C in a humidified chamber. Sections were washed three times in 50% formamide in 2x sodium chloride/sodium citrate (SSC) pre-warmed to 42°C, then in 0.1x SSC at 42°C. Sections were washed in PBS, and a hybridized GFP probe was detected using peroxidase-conjugated anti-DIG antibody (Dako), following tyramide signal amplification (Perkin Elmer Life and Analytical Science, Inc., Boston, http://las.perkinelmer.com).

Single-Fiber Culture
Single muscle fibers were explanted in matrigel-coated plates, as described previously [24]. Briefly, the bilateral soleus muscles were removed from the animal and incubated in collagenase for 90 minutes at 37°C. Digested fibers were then carefully explanted onto matrigel-coated 24-well plates. A plating medium, consisting of 10% horse serum (HS) and 0.5% chick embryo extract (Gibco, Carlsbad, CA, http://www.invitrogen.com) in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Chemical Corp., St. Louis, http://www.sigma-aldrich.com) was added. The medium was replaced on day 4 with a growth medium consisting of 20% fetal calf serum (FCS), 10% HS, and 1% chick embryo extract in DMEM, and on day 8 with a differentiation medium, consisting of 2% FCS, 10% HS, and 0.5% chick embryo extract in DMEM.

PCR Analysis
Total DNA was extracted with a Dneasy Tissue Kit (Qiagen, Valencia, CA, http://www1.qiagen.com) from the 24-well plates used for culturing single fibers from hind limb muscles 6 months after transplantation of KSL cells. The primers for GFP were (5'–3') CTG GTC GAG CTG GAC GGC GAC G and CAC GAA CTC CAG CAG GAC CAT G. For nested GFP they were ACA AGT TCA GCG TGT CCG GCG A and CTT CTC GTT GGG GTC TTT GCT C. The PCR cycle using AmpliTaq Gold (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) for the GFP or nested GFP primer comprised 8 minutes at 95°C, 35 cycles at 94°C for 30 seconds, at 64°C for 30 seconds, and at 72°C for 40 seconds, followed by 7 minutes at 72°C.


   RESULTS
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
KSL Cells Can Repair Muscle Damage in Early Phase After Transplantation
To investigate the process of settlement of donor hematopoietic cells into skeletal muscles, HSC fraction, KSL cells [25], derived from BM of GFP-transgenic mice, were transplanted into lethally irradiated adult mice by tail vein injection. The entire KSL cell fraction was hematopoietic and expressed CD45 (Fig. 1Go). The purity of KSL cells after sorting was 98%. To exclude the possibility of contamination of mesenchymal cells, we cultured 1 to 5 x 103 per well of KSL cells with Mesen Cult medium for 14 days. No adherent cells were observed (data not shown).



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  		Figure 1. Purification of KSL cells. After collecting Lin– cells by auto-MACS, KSL cells were sorted by FACS Vantage. (A): Lin– gating. (B): Sorting gate for KSL cells. (C): All KSL gated cells express CD45. (D): The purity of KSL cells after sorting was 98%. Abbreviation: FSC, forward scatter.

 
After transplantation, the muscles in the entire body were examined, first with a fluorescent stereomicroscope and then with immunohistochemical staining, to detect the GFP signals. On day 3 after transplantation, no GFP signals could be detected in any muscles. On days 10 and 20, powerful GFP signals in the muscle fibers throughout the body were observed under a fluorescent stereomicroscope such as in the intercostal, thigh, abdominal, greater pectoral, and external ocular muscles (Fig. 2AGo). Transverse sections showed muscle fibers of variable sizes with centrally localized nuclei, which represents the regenerative status (Fig. 2BGo). Among these muscle fibers, GFP+ region–like muscle fibers were detected (Fig. 2B, bGo, arrowheads; faintly red region). There were mainly two types of GFP+ regions. One region consisted of GFP+ fibers (Fig. 3BGo), which were confirmed by typical cross-striations (Fig. 3AGo) and by immunostaining with anti-myosin antibody (Fig. 3C, DGo). And the other consisted of GFP+ mononuclear cells (Fig. 3EGo), most of which were not stained with anti-myosin (Fig. 3G, HGo) but stained with anti-CD45 antibody (Fig. 3F, HGo). In the region of GFP+ fibers, myogenin+ myoblasts or myotubes were detected around the fibers (Fig.4D, EGo, arrowheads). Interestingly, CD45low myogenin+ GFP+ cells were also detected (Fig. 4B–EGo, arrows) but only very few in number. This replacement by GFP+ fibers could be observed only from day 10 to day 20 and had dramatically decreased on day 30 (Fig. 2A, g–iGo). FISH analysis revealed a small number of GFP-DNAs in myonuclei (data not shown). These results indicated that the irradiation for myeloablation evoked muscle injury and that KSL cells engrafted in damaged muscles, fusing the host’s muscle fibers, and participated in muscle regeneration in the early phase of transplantation.



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  		Figure 2. (A): Visualization of transplanted GFP+ cells in muscles on days 10, 20, and 30. Appearances of various muscles under a fluorescent stereomicroscope are shown. Muscle tissues were removed from recipient mice and observed under a fluorescent stereomicroscope on days 10, 20, and 30 after transplantation. a, d, g: Intercostal muscle (the white lines indicate the shape of ribs). b: Abdominal muscle. c: Thigh muscle. e: Greater pectoral muscle. f: External ocular muscle (the white line indicates the shape of the eyeball). h: Dorsal muscle. Bars: a–c, e, 2 mm; d, 500 µm; f–g, i, 1 mm; h, 200 µm. (B): Immunohistostaining of the section of muscle tissues on day 10. a: Negative control. b: The section was stained with anti-GFP antibodies (alkaline phosphatase, faintly red region; arrowheads), and hematoxylin was used for nuclear staining.

 


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  		Figure 3. GFP+ regions in intercostal muscle on day 20. Intercostal muscles were removed from recipient mice and fixed with 4% paraformaldehyde and made into frozen sections, as described in Materials and Methods. There were two types of GFP regions: (A–D) GFP+ fibers and (E–H) GFP+ mononuclear cells. Each section was stained with anti-GFP antibody (B, D, E, H, green), anti-myosin antibody (C, D, Cy3 red; G, H, Alexa350 blue), and anti-CD45 antibody (F, H, Cy3 red). (A): Phase contrast (D, H): Merge. Bars: D, 10 µm; H, 20 µm.

 


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  		Figure 4. Myogenic phenotype of a CD45+ cell in muscle tissues on day 20. (A, B): GFP+ muscle structure showed cross striations. These sections were stained with anti-CD45 antibody (C, Cy3 red) and anti-myogenin antibody (D, blue). (E): Merge. Myogenin+ myoblast- or myotube-like cells were detected (D, E, arrowhead), and an elongated GFP+ cell coexpressed myogenin and CD45 (B–E, arrow).

 
KSL Cells Settle in Muscle Tissue Like a Satellite Cell
Sections of the muscle tissues obtained 30 days and 6 months after transplantation were stained with anti-GFP and anti-laminin (a marker of the basal lamina) antibodies, showing that several GFP+ cells were located inside the basal lamina with laminin expression (Fig. 5AGo), which is where satellite cells are usually found. GFP+ cells under the basal lamina were coexpressed with c-Met antigen (a marker of satellite cells) (Fig. 5EGo, arrows). Furthermore, we isolated single fibers from soleus muscles 2 months after transplantation and stained them with anti-PAX7, MyoD, or Myf5, which are specific satellite cell markers. We detected Myf5+ GFP+ satellite cells on the fibers (Fig. 6A–DGo), but neither PAX7+ GFP+ cells nor MyoD+ GFP+ cells (data not shown). These results suggested that GFP+ KSL cells migrate into muscle tissues, with some of them localizing beneath the basal lamina expressing satellite cell markers.



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  		Figure 5. GFP+ cells localized like satellite cells long term after transplantation. Sections from 6 months after transplantation were stained with laminin (A, Cy3 red; D, E, Alexa350 blue) and c-Met (C, E, Cy3 red) GFP+ cells localized under the basal lamina (A, E, arrows) and were costained with c-Met (E, arrows). Bar: 20 µm.

 


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  		Figure 6. Immunostaining of single fibers. Single fibers were isolated from (A–D) irradiated mice 2 months after transplantation and from (E–P) W/Wv mice 1 or 2 months after transplantation and were stained with satellite cell–specific markers. (A, E, I, M): Anti-GFP antibody (FITC green, arrowheads). (B, N): Anti-Myf5 antibody (Cy3 red, arrowheads). (F): Anti-MyoD1 antibody (Cy3 red, arrowhead). (J): Anti-PAX7 antibody (Cy3 red, arrowhead). (D, H, L, P): Merge. Bars: D, P, 50 µm; H, L, 100 µm.

 
KSL Cells Can Settle in Muscle Tissues Without Muscle Damage
Tissue-specific stem cells occupy niches—microenvironments that maintain self-renewal activity and multipotency of stem cells. Since it is known that irradiation depletes endogenous satellite cells and that injected muscle precursors can replace them [26], we used lethal irradiation for the transplantation assay. However, lethal irradiation also evokes various responses in the body. To exclude the influence of irradiation damage, we next transplanted KSL cells into W/Wv neonates. W/Wv mice possess a c-Kit gene mutation and can accept transplanted HSCs in a BM niche without irradiation [27]. Moreover, transplantation into W/Wv neonates results in a higher chimeric ratio than does transplantation into adults [28]. Our experiments using transplantation into W/Wv neonates showed no evidence of GFP+ muscle fibers at any time, whereas BM cells were almost entirely replaced. However, small GFP+ mononuclear cells could be detected between muscle fibers as early as 30 days after transplantation (Fig. 7AGo). Some GFP+ cells were also detected beneath the laminin-positive basement membrane (Fig. 7BGo), and some of them were also stained with anti-c-Met antibody (Fig. 7CGo). We also examined isolated single fibers 1 month after transplantation and detected MyoD+ GFP+ cells and PAX7+ GFP+ cells on the fibers (Fig. 6H, LGo). We could also detect Myf5+ GFP+ cells on the fibers 2 months after transplantation (Fig. 6PGo). These results showed that KSL cells and/or their progenies could migrate into undamaged muscle tissues expressing satellite cell–specific markers also, and that a suitable microenvironment for them might exist in skeletal muscles.



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  		Figure 7. Analysis of the potential of GFP+ satellite-like cells in muscle 6 months after transplantation. Thirty days and 6 months after nonirradiated transplantation into W/Wv neonates, GFP+ mononuclear cells were detected between muscle fibers under fluorescent stereomicroscope (A, arrows). In the sections, immunostaining for laminin and c-Met was performed (B, C). (A): Appearance of rib and rib muscle under fluorescent stereomicroscope on day 30. Some GFP+ mononuclear cells were detected (arrows). The white lines indicate the shape of ribs. (B): Immunohistochemistry with anti-laminin (Cy3 red) and anti-GFP antibodies. A confocal microscope was used to determine the precise location of GFP+ cells. GFP+ cells beneath basal lamina (arrow) were detected on day 30 and 6 months later. (C): GFP+ cell under laminin-positive basal lamina (Alexa350 blue) was also stained with anti c-Met antibody (Cy3 red, arrow). (D–F): Single-fiber culture was performed with single fibers isolated from recipient mice 6 months after transplantation, followed by immunostaining. C, negative control; D, anti-myosin (Cy3 red) staining; E, anti-GFP-ALP (red) staining. D and E were same position. GFP-ALP+ fibers were confirmed to be muscle fibers using myosin Cy3 staining. (G): PCR analysis of extracted DNA from single-fiber culture. DNA was extracted from the single-fiber culture followed by PCR for GFP DNA, as described under Materials and Methods. 1: negative control; 2: peripheral blood of GFP transgenic mouse; 3: single fiber culture. Bars: A, 1 mm; B, C, 20 µm; D, 200 µm; E, F, 50 µm.

 
KSL-Derived Cells in Muscle Can Generate Muscle Fibers In Vitro in Long Term After Transplantation
So far it has been reported that damaged muscles by chemical agent or stress were regenerated by donor cells after BM transplantation [12, 15]. However, these muscle-regeneration assays in vivo cannot clarify whether donor cells participate in regenerating muscles directly from the settled muscle or indirectly from settled BM. To determine whether GFP+ mononuclear cells in muscle tissues can differentiate into muscle fibers, a single-fiber culture was performed. Six months after transplantation with KSL cells, muscle fibers were isolated from the hind limb muscles of W/Wv mice or lethally irradiated mice transplanted, and single fibers were cultured onto matrigel-coated plates. After 14 days, most of the satellite cells had migrated from the fibers, proliferated, and formed new muscle fibers, which were confirmed to be myosin-positive by immunohistochemical staining (Fig. 7D, EGo). Since the GFP signal in vitro was too weak to be detected by fluorescent microscope, we determined the presence of GFP+ cells by means of immunohistochemical staining using the anti-GFP antibody or by GFP DNA amplification with the aid of PCR. Staining with the anti-GFP antibody proved the presence of GFP+ myofibers (Fig. 7FGo), which were also myosin-positive. In addition, GFP DNA was also confirmed by PCR to be present in samples extracted from culture dishes of single-muscle fiber (Fig. 7GGo). No GFP+ fibers were generated in the single-fiber culture in the early phase of transplantation in irradiated mice (data not shown). In brief, GFP+ KSL cell that engrafted muscle tissues could repair damaged muscle but not produce muscle fibers in vitro in the early phase. In the long term, however, they acquired the potential to differentiate into muscle fibers like satellite cells also in vitro. Since the single-fiber culture is a functional assay of the presence of satellite cells, it can be said that GFP+ KSL cells can give rise to satellite cells with myogenic potential in the long term after transplantation.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this study, we showed early repair of damaged muscles by KSL cells and first demonstrated that KSL cells could differentiate into satellite-like cells in skeletal muscle in the long term after transplantation (Fig. 8Go).



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  		Figure 8. Schematic representation of hypothesized behavior of transplanted KSL cells in muscle tissues. See Discussion for a detailed explanation. Abbreviation: HSC, hematopoietic stem cell.

 
The thoroughly studied and identified characteristics of surface markers for HSCs showed that our KSL cells contained a dominant stem cell population. A single CD34–/low c-Kit+Sca-1+Lin– cell (CD34– KSL cell) has been demonstrated to be capable of reconstituting long-term hematopoiesis in about 30% of lethally irradiated adult mice [25]. Our 1 x 103 of KSL cells contained about 5%–10% of CD34– cells, which was enough to reconstitute BM of the irradiated mice, and hardly any CD45– cells (less than 0.1%; Fig. 1Go). And we cultured 1 to 5 x 103 of KSL cells for 14 days and obtained no adherent cells. It can therefore be safely assumed that the 1 x 103 of KSL cells were originally all hematopoietic and did not contain any mescenchymal cells, which do not express CD45 antigen, demonstrating that CD45+ KSL cells can differentiate into muscle fibers. So far, unfractionated BM cells have been used to demonstrate transdifferentiation to muscle fibers [12, 29]. LaBarge and Blau [30] reported recently on the biological progression from adult BM cells to muscle stem cells to muscle fiber, and their findings support our hypothesis. Their study used unfractionated BM cells, which contained both HSCs and MSCs. In contrast, we used CD45+ KSL cells as the enriched HSC fraction, and we showed that KSL cells could contribute to muscle regeneration in both the early and late phases after transplantation. Very recently, it has been reported that a single HSC generated skeletal muscle [31, 32]. These reports support our results. BM SP cells are the same as HSCs and are reported to be involved in muscle regeneration after transplantation, but no satellite cells derived from BM SP cells have been documented [15, 31]. Our study, by analyzing single fibers, is the first to demonstrate that CD45+ KSL cells can give rise to satellite cells both in vivo and in vitro. Since BM SP cells and KSL cells are still heterogeneous populations, further classification of HSCs may lead to the identification of a subpopulation specific to muscle regeneration.

It has been reported that mononuclear cells harvested from murine skeletal muscle are capable of hematopoietic reconstitution in lethally irradiated mice and that these muscle-derived hematopoietic progenitor cells are originally derived from BM [20, 33]. However, determination of the time when these hematopoietic cells settle in muscles after transplantation, as well as the histological identification of their location in muscle tissues, has not yet been accomplished. Our observations demonstrate that GFP+ mononuclear cells gather in damaged muscles before BM has been fully replaced. Nearly all of these GFP+ mononuclear cells consist of CD45+ in muscle fibers. Interestingly, a few CD45low myogenin+ GFP+ cells were also detected, and this implies that transplanted KSL cells might have an important and direct role in regeneration in the very early phase after transplantation.

Thirty days after transplantation, single fibers from irradiated mice were not stained by anti-PAX7, anti-MyoD, or anti-Myf5 antibody, and they did not grow in vitro in culture, whereas myofibers could develop in vitro in single-fiber culture from age-matched nontreated mice. Because it is said that irradiation eliminates satellite cells [26, 34], these phenomena might mean that satellite cells had been damaged by irradiation at this time point. We speculated that, after 30 days, host satellite cells recovered from radiation damage, producing new muscle fibers, which replaced GFP+ fibers gradually. We assume that GFP+ fibers were diluted, fusing with host-derived muscle fibers. Simultaneously, GFP+ cells remain beneath the basal lamina as satellite cells in muscle tissue and acquire the potential to differentiate into muscle fibers in the long term after transplantation. In this late phase, muscle regeneration can be obtained from GFP+ satellite-like cells, as well as from endogenous satellite cells, as was confirmed by single-fiber culture. We therefore propose two different roles of KSL cells in muscle regeneration: repair of muscles damaged by irradiation in the early phase, and generating satellite cells in the late phase.

In the case of transplantation into W/Wv neonates, GFP+ cells engrafted into muscle tissues as early as 30 days after transplantation without irradiation. Interestingly, we could detect satellite cell markers MyoD, PAX7, and Myf5 on GFP+ cells on muscle fibers as early as 1 or 2 months after transplantation. GFP+ single fibers can develop in vitro at the same time (data not shown). This means that KSL cells can migrate into muscle fibers and become satellite-like cells without muscle damage. We therefore speculate that KSL cells might be part of a physiological satellite cell source. Neonatal environment might give some effect to KSL cells. And it is not yet clear why hematopoietic cells engraft into undamaged muscles. The fact that CD45+ hematopoietic cells engraft into skeletal muscles suggests the existence of some muscle-specific molecular mechanism for HSC engraftment. During the processes of HSC homing and engraftment into BM, the contribution of some adhesion molecules such as VCAM-1, which is a ligand for {alpha}4 integrins, has been reported [35, 36]. HSCs are reported to have {alpha}4 integrins [37]. Furthermore, laminin, which is a ligand for {alpha}4 integrin, which in turn is a component of muscle basement membrane, may be important for the maintenance of hematopoietic cells in skeletal muscles.

About 1,000 KSL cells, which proved to be enough to reconstitute BM of irradiated mice, are also sufficient to distribute throughout all the muscles in the body and regenerate all damaged muscles such as intercostals, diaphragm, and limb muscles. This is an important finding in terms of potential for clinical application. Patients with muscular dystrophy suffer from respiratory distress due to respiratory muscle weakness. Our findings of powerful engraftment of transplanted KSL cells in the entire body, including respiratory muscles, may be the first step on the way to the establishment of therapeutic strategies using HSCs. Further studies are needed, however, to establish efficient engraftment of HSCs to muscle tissues and to identify responsible molecules.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study was supported by Health and Labour Science Research Grant, Research on Human Genome, Tissue Engineering Food Biotechnology, Ministry of Health, Labour and Welfare, Tokyo; by Grant-in-Aid for Science Research on Priority Areas no. 122150-67; and by Grant-in-Aid for Creative Scientific Research no. 13GS-0009. This study was also supported by Research Grant no. 13B-1 for Nervous and Mental Disorders and no. H13-iyaku-043, both from the Ministry of Health, Labour and Welfare.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 2, 2004; accepted for publication January 13, 2005.



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S. Baba, T. Heike, M. Yoshimoto, K. Umeda, H. Doi, T. Iwasa, X. Lin, S. Matsuoka, M. Komeda, and T. Nakahata
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