IntroductionDuring the past decade, mesenchymal stem cells in adult bonemarrow, although forming a heterogeneous population, havebeen characterized by their ability to differentiate into varioustypes of tissue-specific cells (Pereira et al., 1995; Prockop,1997; Pittenger et al., 1999; Liechty et al., 2000) (reviewed byClark and Keating, 1995; Bianco and Cossu, 1999; Bianco andGehron-Robey, 2000). These cells produce colonies offibroblasts, osteogenic cells or adipocytes in vitro. In vivoexperiments have shown that they differentiate to form fibroustissue, bone or cartilage when transplanted into the appropriateanimal.

Wakitani et al. (Wakitani et al., 1995) were the first to showthat mesenchymal stem cells from rat bone marrow have theability to differentiate into myotubes when they are cultured inthe presence of 5-azacytidine. In vivo transplantation of suchcells into the muscles of X-chromosome-linked musculardystrophy (mdx) mice induces dystrophin-positive musclefibres (Saito et al., 1995). These results prompted investigatorsto speculate that the transplanted bone marrow cells can moveinto damaged muscle and grow into new muscle fibres in mice

(for a review, see Cossu and Mavilio, 2000). This also hasclinical implications, not only for muscular dystrophy but alsofor various tissue-specific hereditary disorders. Recently,several investigators have reported that transplanted bonemarrow cells participate in the muscle regeneration process inirradiated recipient mice (Ferrari et al., 1998; Gussoni et al.,1999; Bittner et al., 1999). In these studies, bone marrow cellswere distinguished from recipient cells by donor-specific geneexpression signals such as nuclear β-galactosidase or thepresence of a Y chromosome. However, muscle fibrescontaining donor-cell-derived chromosomes never exceeded1% of the total fibres in an average muscle.

It is widely accepted that postnatal growth and repair ofskeletal muscle are normally mediated by the satellite cells thatsurround muscle fibres. Satellite cells are mononucleateprecursor cells that are located beneath the basal lamina andsarcolemma of myofibres (Mauro, 1961) (for reviews, seeBischoff, 1994; Cullen, 1997). Satellite cells first appear in thelimbs of mouse embryos between embryonic day 16 and 18,and the cell number reaches a peak in neonatal mice. In adultmice, less than 5% of the total nuclei are satellite cells, and this

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The myogenic potential of bone marrow and fetal liver cellswas examined using donor cells from green fluorescentprotein (GFP)-gene transgenic mice transferred intochimeric mice. Lethally irradiated X-chromosome-linkedmuscular dystrophy (mdx) mice receiving bone marrowcells from the transgenic mice exhibited significantnumbers of fluorescence+ and dystrophin+ muscle fibres. Inorder to compare the generating capacity of fetal liver cellswith bone marrow cells in neonatal chimeras, these two celltypes from the transgenic mice were injected into busulfan-treated normal or mdx neonatal mice, and musculargeneration in the chimeras was examined. Cardiotoxin-induced (or -uninduced, for mdx recipients) muscleregeneration in chimeras also produced fluorescence+

muscle fibres. The muscle reconstitution efficiency of thebone marrow cells was almost equal to that of fetal livercells. However, the myogenic cell frequency was higher infetal livers than in bone marrow. Among the neonatal

chimeras of normal recipients, several fibres expressedthe fluorescence in the cardiotoxin-untreated muscle.Moreover, fluorescence+ mononuclear cells were observedbeneath the basal lamina of the cardiotoxin-untreatedmuscle of chimeras, a position where satellite cells arelocalizing. It was also found that mononuclearfluorescence+ and desmin+ cells were observed in theexplantation cultures of untreated muscles of neonatalchimeras. The fluorescence+ muscle fibres were generatedin the second recipient mice receiving muscle single cellsfrom the cardiotoxin-untreated neonatal chimeras. Theresults suggest that both bone marrow and fetal liver cellsmay have the potential to differentiate into muscle satellitecells and participate in muscle regeneration after muscledamage as well as in physiological muscle generation.

Key words: Muscle regeneration, Transplantation, Satellite cell

Summary

Muscle regeneration by reconstitution with bonemarrow or fetal liver cells from green fluorescentprotein-gene transgenic miceSo-ichiro Fukada 1, Yuko Miyagoe-Suzuki 2, Hiroshi Tsukihara 1, Katsutoshi Yuasa 2, Saito Higuchi 1,Shiro Ono 3, Kazutake Tsujikawa 1, Shin’ichi Takeda 2 and Hiroshi Yamamoto 1,*1Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan 2Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo 187-8502,Japan 3Division of Oncogenesis, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan*Author for correspondence (e-mail: hiroshiy@phs.osaka-u.ac.jp)

Accepted 19 December 2001Journal of Cell Science, 115, 1285-1293 (2002) The Company of Biologists Ltd

Research Article

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gradually declines with age (Cossu et al., 1983) (for reviews,see Campion, 1984; Mazanet and Franzini-Armstrong, 1986;Bischoff, 1994). In chicken, satellite cells appear during lateembryogenesis (Hartley et al., 1992). Adult bone marrowcontains mesenchymal stem cells from which muscle precursorcells can be supplied; however, there is no definitive evidenceas to whether or not satellite cells are recruited from bonemarrow to muscle under physiological or pathologicalconditions. Moreover, it has not yet been clarified whether thebone-marrow-derived cells exist as satellite cells in theradiation chimera experiments.

In this report we demonstrate that the injection of bonemarrow or fetal liver cells from EGFP-transgenic (GFP-Tg)mice into neonatal mice establishes tolerance to allogeneicdonor cells, and the muscle-reconstituting efficiencies of thesetwo cell populations were compared. Moreover, we provide thefirst report that neonatally injected cells are recruited to themuscle, where they probably reside as satellite cells, andparticipate in normal muscle development. The combinedprocedure – neonatal tolerance induction with cells from GFP-Tg mice – is a useful strategy for further studies of satellite cellrecruitment and muscle regeneration.

Materials and MethodsAnimals and cellsSpecific pathogen-free C57BL/6 mice aged between 6 to 8 weeks werepurchased from Charles River Japan (Yokohama, Japan). Specificpathogen-free mdxmice (of C57BL/10 background) were provided byCentral Laboratories of Experimental Animals (Kanagawa, Japan) andmaintained in our animal facility by brother-sister matings.Heterozygous EGFP transgenic (GFP-Tg) mice with a C57BL/6background (Okabe et al., 1997) were maintained in our animal facilityby mating with normal C57BL/6 mice. The C57BL/6 or GFP-Tg micewere mated at night, and females were examined the next morning.The day on which a vaginal plug was found was considered day 0 ofembryonic development. Gestational day 12 or 13 fetuses wereobtained from pregnant normal C57BL/6 mice time-mated with GFP-Tg heterozygous male mice. GFP-Tg fetuses were identified by briefexposure to 365 nm UV light as previously described (Okabe et al.,1997). Single cell suspensions of fetal liver cells from GFP-Tgembryos were prepared by gentle teasing of the tissue. Bone marrowcell suspensions were prepared by flushing the marrow from thefemora and tibiae of adult GFP-Tg mice, followed by gentle pipetting.mdx/scid mice were produced by mating mdx mice with scid mice(C57BL/6 background, purchased from Charles River Japan). The scidphenotype was determined by measuring serum immunoglobulin level.

Bone marrow or fetal liver cell reconstitutionBone marrow chimeras were established by the usual method forradiation chimeras. Adult (8 week old) mdx mice were lethallygamma-ray irradiated (10 Gy) using Gamma Cell (137Cs source,Nuclear Canada, Ontario, Canada). 40×106 bone marrow cells fromGFP-Tg mice were injected intravenously into the recipient micewithin 3 hours after radiation. The recipient mice had receivedampicillin (product of Sigma Chemicals Co., St. Louis, MO) in theirdrinking water (1 g/L) from 1 week before radiation to 2 weeks afterradiation and reconstitution. 4 weeks after reconstitution, peripheralblood was obtained and tested for chimerisms by measuring GFP+

cells with a flowcytometer. 12 weeks after reconstitution, the musclesand lymphoid organs of the mice were examined for muscleregeneration and chimerism, respectively.

Neonatal chimeras were established as follows (Fig. 1). Time-

mated pregnant C57BL/6 or mdx mice (at gestational day 17/18)received 20-40 mg/kg busulfan (Sigma Chemicals Co.)subcutaneously. Busulfan-treated neonatal mice were injectedintrahepatically with either 0.1-0.2×106 fetal liver cells or 1.0-10×106

bone marrow cells from GFP-Tg mice according to the method ofYoder et al. (Yoder et al., 1997). 4 weeks after reconstitution, bloodsamples were collected from the tail vein and used for chimerism tests.Chimeric mice with more than 5% donor-derived GFP+ cells amongtheir blood mononuclear cells were selected and used for furtherstudies of muscle regeneration. 4 to 13 weeks later (age 8-17 weeks),the muscles and lymphoid organs were examined.

Histological analysisChimeric mice were sacrificed by cervical dislocation. Tibialisanterior (TA) and vastus lateralis (VL) muscles, or diaphragms, wereisolated and frozen in liquid nitrogen-cooled isopentane. Cryosections(6 µm) were examined for GFP+ muscle fibres under a confocal laserscanning microscope (model MRC1024ES, Bio-Rad Laboratories,Hercules, CA). Excitation/emission wavelengths for GFP were 488nm/522 nm. Sections were then stained with hematoxylin/eosin (H-E).

ImmunohistochemistryFor immunohistochemical examinations, serial transversecryosections (6 µm) or cultured tissues were stained with variousantibodies. Alexa-568-conjugated monoclonal anti-dystrophin(MANDRA-1, Sigma) was a gift from M. Imamura (National Instituteof Neuroscience, Tokyo, Japan). Polyclonal rabbit anti-desmin (ICNPharmaceuticals, Inc.-Cappel Products, Aurora, OH) and anti-laminin(MEDAC GmbH, Wedel, Germany), together with rhodamine-conjugated goat anti-rabbit IgG (ICN Pharmaceuticals), were used tostain muscles. The signals were recorded photographically usingan Axiophot microscope (Carl Zeiss, Oberkochen, Germany) andlaser scanning confocal imaging system MRC1000 (Bio-RadLaboratories).

Test for blood chimerismBlood samples of 4-week-old busulfan-treated neonatal chimeraswere examined for chimerism. For flowcytometric analyses ofchimerism, mononuclear cells from the blood, spleen and thymus ofchimeras were stained with monoclonal antibodies, phycoerythrin-or biotin-conjugated anti-CD4 (RM4-5) and anti-CD8 (53-6.7)(PharMingen, San Diego, CA) for donor-derived T lymphocytes, andanalyzed together with GFP and phycoerythrin-Cy5-streptavidin(Cederlane, Westbury, NY) (Kawakami et al., 1999a; Kawakamiet al., 1999b). GFP fluorescence was measured at the same

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pregnant (E17-18) C57BL/6 mic e

neonata l mic e

Busu lfan injection (20-40mg/kg)

bone marrow or fetal liver cells (from GFP-Tg mic e)injection (intra- hepat ically)

Cardiotoxin injection (i.m.) (at 4wk old)

4-13 weeks

Exper imenta l protocol for neonata l chimera

test forchimerism

histolo gical analyses

test forchimerism

4 weeks

Fig. 1.Experimental protocols for chimeras. For more details see theMaterials and Methods section.

1287Muscular regeneration in neonatal chimeras

excitation/emission wavelength as FITC. Flow cytometric profileswere determined with a FACSCalibur analyzer and CELLQuestsoftware (Becton Dickinson Immunocytometry Systems, MountainView, CA).

Muscle regenerationSelected neonatal chimeras at 4 weeks of age, with >5% donor-derived GFP+ cells, were anesthetized with sodium pentobarbital (75mg/kg) (Abbott Laboratories, North Chicago, IL), and in someexperiments (Table 1), muscle regeneration was induced by injecting75 µl cardiotoxin (CTX; 10 µM, Latoxan, Rosans, France) into theTA muscle to necrotize the muscle according to the method of Daviset al. (Davis et al., 1993). Some mice were left CTX-untreated. Fourto 13 weeks later, muscle regeneration and chimerism were examined.

Muscle fibre explantationIn order to detect muscular satellite cells in the reconstituted chimeras,muscle fibres from extensor digitorium longus (EDL) muscles fromCTX-untreated neonatal chimeras were prepared and culturedessentially according to the methods of Bischoff (Bischoff, 1986) andRosenblatt et al. (Rosenblatt et al., 1995). Briefly, dissected musclewas incubated with 0.5% type I collagenase (WorthingtonBiochemical, Lakewood, NJ) in Dulbecco’s modified Eagle’s medium(Gibco BRL, Grand Island, NY) at 37°C for 90 minutes. The musclewas transferred to fresh growth medium, high-glucose DMEM(Gibco BRL) containing 10% FCS (Boehringer-Mannheim GmbH,

Mannheim, Germany) and penicillin (200 U/ml)/streptomycin (200µg/ml) (Gibco BRL), and incubated in a humidified incubator at 37°C,5% CO2 for 16 hours. The muscle mass was triturated with a fire-polished wide-mouth Pasteur pipette. Fibres were transferred to aMatrigel (Collaborative Biomedical, Bedford, MA)-coated Lab-Tekchamber (Nalge Nunc International, Naperville, IL) and cultured for4 days. The fibres and attached cells were fixed with 4%paraformaldehyde in phosphate-buffered saline at room temperaturefor 10 minutes. They were then permeabilized with 0.25% Triton X-100 (Nacalai Tesque, Kyoto, Japan) at room temperature for 20minutes, then non-specific binding was blocked by incubation with5% skim milk (in PBS) for 10 minutes. The fixed fibres and cells werestained with anti-desmin antibody at 4°C overnight and then withrhodamine-conjugated goat anti-rabbit IgG. Samples were examinedfor GFP+ and/or rhodamine+ cells under a confocal laser scanningmicroscope. Excitation/emission wavelengths for rhodamine are 554nm/573 nm.

Preparation of muscle-derived cellsFreshly isolated muscle-derived cells from neonatal chimeras wereprepared according to the method of Rando and Blau (Rando andBlau, 1994). CTX-untreated VL muscles from neonatal chimeras wereisolated, minced and digested with dispase II (2.4U/ml) (Boehringer-Mannheim) and 1% collagenase P (Boehringer-Mannheim)supplemented with CaCl2 to a final concentration of 2.5 mM. Theslurry, maintained at 37°C for 45 minutes, was triturated every 15minutes and passed through a 37µm nylon mesh. Single cell

Fig. 2.Flow cytometric analyses of leukocyte chimerisms. (A) Splenocyte chimerisms of radiation-bone marrow chimera. (B) GFP+CD4+ andGFP+CD8+ T cells of the gated fraction of (A). (C) GFP+ cells in the peripheral blood of neonatal-bone-marrow chimera 4 weeks afterreconstitution (4 weeks old). Splenic GFP+ cells (D) and splenic GFP+CD4+ and GFP+CD8+ T cells (E) of mouse C 9 weeks afterreconstitution. (F) GFP+ bone marrow cells of mouse (C). (G) GFP+ cells in the peripheral blood of a neonatal-fetal liver cell chimera 4 weeksafter reconstitution (4 weeks old). Thymic GFP+ cells (H), and CD4 and CD8 stainings of thymocytes (I) of mouse G at 10 weeks afterreconstitution. Splenic GFP+ cells (J) and its GFP+CD4+ and GFP+CD8+ T cells (K) of mouse G. (L) GFP+ bone marrow cells of mouse G.

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suspension was washed and injected into CTX-treated (24 hoursbefore) TA muscles of mdx/scidmice. 4 weeks later, muscle sectionswere histologically examined.

ResultsGFP+ muscle fibre generation in radiation-bone-marrowchimerasTwelve weeks after reconstitution, splenic lymphocytes fromradiation-bone-marrow chimeras were examined for GFPexpression. As shown in Fig. 2A, about 85-87% of splenocytesfrom chimeras expressed GFP (see also Table 1). A smallunidentified fraction (~13-15%), which expressed no GFP, mayrepresent either host-derived radio-resistant cells or rapidlydividing donor cells (Kawakami et al., 1999b). CD4+ and CD8+

T cells showed normal lymphocyte development in the mice(Fig. 2B). Thymic lymphocytes and splenic B cells were alsoreplaced by GFP+ cells at similar levels to total splenocytes(data not shown).

Several GFP+ fibres were readily identified in TA (Fig.3A,B), VL (Fig. 3C,D) muscles and diaphragm (Fig. 3E,F)sections and are clearly distinct from surrounding GFP-negative fibres. Because one of the GFP+ fibres expresseddystrophin, as shown in Fig. 3G-I, the fibre is thought to benewly generated from donor-derived precursor cells (Fig. 3J-L; this figure shows positive control sections of normalC57BL/6 muscle). Although the reason for the difference ofdystrophin expression in each fibre is not yet known, GFP+ butdystrophin-negative fibre was also seen.

GFP+ muscle fibre regeneration in neonatal chimerasLymphocyte chimerisms of neonatal chimeras were thenexamined. In order to enhance both tolerance and blood

chimerism, mice received busulfan, an anticancer drug forlympho-myeloid neoplasms, at embryonic day 17/18, thenreceived either adult bone marrow or fetal liver cells fromGFP-Tg mice within 16 hours of birth (Fig. 1). Typicalflowcytometry studies of the chimeras are shown in Fig. 2C-L.4 weeks after neonatal injection of donor bone marrow (Fig.2C) or fetal liver cells (Fig. 2G), the proportions of GFP+ bloodleukocyte were 26% or 18%, respectively. The frequency ofchimeras ranged from 1 to 64%, and mice having more than5% donor-derived GFP+ cells in their blood leukocytes wereselected. At the time of muscle examination, GFP+ splenocytes(Fig. 2D,E,J,K), thymocytes (Fig. 2H,I) and bone marrow cells(Fig. 2F,L) were also examined for chimerisms. Normalpatterns of splenic mature T cells or immature thymocyteswere observed. The donor-derived GFP+ cells were found tobe recruited to the recipient bone marrow. The chimerismsranged from 5 to 78% at the time when mice were killed (seeTable 1).

CTX-induced muscle regeneration of neonatal chimeras isshown in Fig. 4. The GFP+ muscle fibres of a neonatal bonemarrow chimera (Fig. 4B) or a fetal liver cell chimera (Fig. 4D,F) could be detected. The frequencies of GFP+ fibres aresummarized in Table 1. It is evident that the frequencies ofGFP+ fibres in radiation-chimeras do not exceed 2%. Theyshow better reconstitution efficiency in their lymphocytechimerisms than neonatal chimeras. It is interesting to note thatthe donor bone marrow or fetal liver cell number of theneonatal chimeras was only 1:4-1:40 (for bone marrow cells)or 1:200-1:400 (for fetal liver cells) of adult radiation-chimeraexperiments; however, the frequencies of GFP+ fibres in micewith lower lymphoid chimerisms are similar to those ofradiation chimeras. GFP+ fibres were also observed in theCTX-untreated neonatal chimeras (mdxas recipients) (Table 1;Experiment 6 and 7). This result suggested that the donor-

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Fig. 3.Detection of GFP+

muscle fibres in the radiationbone marrow chimeras.H-E staining (A,C,E) andGFP fluorescence (B,D,F) ofTA (A,B) and VL (C,D)muscles and diaphragm (E,F)of radiation-bone-marrowchimera. (G) GFP+ TAmuscle fibre; (H) dystrophinstaining (Texas-Red-conjugated anti-dystrophinantibody); (I) mixed color of(G) and (H); (J) GFPfluorescence; (K) dystrophin-staining; (L) the mixed colorof (J) and (K) from the TAmuscle of a normal C57BL/6mouse as a control. Bars,100µm (A-F) and 50 µm(G-I).

1289Muscular regeneration in neonatal chimeras

derived bone marrow (Experiment 6) or fetal liver (Experiment7) cells participate in the normal muscular regeneration in mdxmice. Muscle reconstituting efficiencies of adult bone marrow

and fetal liver cells were compared. As summarized in Fig. 5,when the efficiencies were calculated as GFP+ fibres/injectedcell numbers, fetal liver gave better results than bone marrow

Fig. 4.Detection of GFP+ muscle fibres and possible satellite cells in neonatal chimeras. H-E staining (A,C,E) and GFP fluorescence (B,D,F) ofCTX-treated TA muscles of neonatal chimeras. (A,B) Neonatal bone marrow chimera; (C-F) neonatal fetal liver cell chimera; (G,H) a singleGFP+ fibre with peripheral nucleus in a intact (CTX-untreated) neonatal bone marrow chimera; (I) detection of a GFP+ mononuclear cellbeneath the laminin layer (rabbit anti-laminin plus rhodamine-conjugated goat anti-rabbit IgG) (neonatal bone marrow chimera); (J-O) desmin-stained explanted fibres (J,L,M,O) were examined for GFP fluorescence (J,K,M,N). A GFP+ cell attaching to a single fibre was stained withdesmin (arrowheads of (L) and (O)). (P) Positive control of explanted muscle cells from GFP-Tg mouse (mixed color of desmin and GFP);(Q-T) are muscle sections of the secondary mdx/scidrecipient. Single cell suspension from intact (CTX-untreated) muscles of neonatal bonemarrow chimera was obtained and injected into CTX-treated TA muscle of the mdx/scidrecipient. GFP+ fibres were observed (arrowheads of(R) and (T)). Bar, 50 µm (A-H and Q-T).

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cells. Fetal livers may contain a higher frequency of muscleprogenitor cells than bone marrow.

GFP+ mononuclear cells in the intact muscles ofneonatal chimerasDuring the course of the study, we observed several GFP+

fibres in the CTX-untreated muscle of neonatal bone marrow(Fig. 4H) (C57BL/6 as recipient) as well as fetal liver cellchimeras (data not shown), but the frequency was low. It isevident from the H-E stained picture that the GFP+ fibre has aperipheral nucleus, suggesting that the fibre was generatedwithout damage by CTX. We speculate that these fibres werederived from GFP+ satellite cells in the normal muscle. Inaccordance with this observation, we detected GFP+

mononuclear cells residing beneath the laminin-positive basallamina of another CTX-untreated TA muscle (Fig. 4I). This isthe typical position in which muscle satellite cells reside. To

investigate which of the cells are myogenic progenitor cells,possibly satellite cells, and whether they contributed to musclegeneration, we performed a muscle explantation study. After 4days of explantation culture, desmin+ and GFP+ mononuclearcells were found attached to a single muscle fibre (Fig. 4J-L,M-O).

In order to confirm whether the GFP+ mononuclear cellsof neonatal chimeras are myogenic progenitors, single cell

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Table 1. Chimerisms of lymphocytes and muscle fibresExperiment number Mouse Donor Total GFP+ % %(spleen) Weeks after(strain/condition) number(1) cells Treatment fibre(2) fibre reconstitution(3) chimerism(4) reconstitution(5)

1 1 40×106 CTX(6) 1,670 25 1.50 85.8 12(mdx/radiation) 4 BM 1,947 25 1.28 87.1 12

5 1,405 15 1.07 86.9 12

2 3 1×106 CTX 2,498 12 0.48 7.5 8(B6/neonate) 9 BM 1,767 4 0.23 5.9 8

10 1,645 7 0.43 12.8 8

3 1 0.2×106 CTX 2,636 3 0.11 16.0 8(B6/neonate) 2 FL 2,144 15 0.70 31.7 9

3 1,313 16 1.22 37.9 104 820 4 0.49 27.4 145 1,606 1 0.06 5.0 106 2,163 16 0.74 21.2 12

4 3 4×106 CTX 1,144 12 1.05 13.0 12(B6/neonate) 5 BM 2,261 28 1.24 44.9 9

6 2,126 19 0.89 18.9 9

5 1 10×106 CTX 2,619 69 2.63 41.5 17(B6/neonate) 4 BM 1,934 26 1.34 62.7 17

5 1,854 20 1.08 65.4 17

6 1 6×106 None 1,771 19 1.07 39.8 13(mdx/neonate) 2 BM 1,850 20 1.08 34.2 8

3 1,282 21 1.64 36.8 84 1,485 24 1.62 28.2 13

7 1R 0.1×106 None 2,307 8 0.35 18.6 11(mdx/neonate) 1L FL 1,452 15 1.03 – –

3L 1,411 13 0.92 63.6 104L 1,683 21 1.24 68.4 86L 2,119 17 0.80 77.9 12

(1) Left VL (Experiment 1), right TA (Experiments 2-6) or left (L) or right (R) TA (Experiment 7) muscles were examined.(2) Total fibre numbers were counted.(3) GFP+ fibre×100/total fibre was calculated.(4) % chimerisms in the spleen (GFP+ lymphocytes×100/total splenic lymphocytes) were calculated.(5) Mice were killed and examined at the indicated weeks after the reconstitution.(6) Left VL (Experiment 1) or right TA (Experiments 2-5) muscles were treated with cardiotoxin (CTX) and muscles were examined.

(GF

P p

ositi

ve fi

bers

/ in

ject

ed c

ells

num

ber)

× 10

-6

BM FL BM FL

C57BL /6 mdx

0

50

100

150

200

250

0.00060.002

(CTX -treated) (untreated)

Fig. 5.Muscle-reconstituting efficiencies of bone marrow and fetalliver cells. Muscle-reconstituting efficiencies of neonatal bonemarrow and fetal liver cells were compared as a factor of GFP+

muscle fibres/injected cell numbers. From the data shown in Table 1,both CTX-treated normal C57BL/6 and CTX-untreated mdxrecipientmice were calculated.

1291Muscular regeneration in neonatal chimeras

suspension from CTX-untreated VL muscles was obtained andtransplanted into CTX-treated TA muscles of mdx/scid mice.As shown in Fig. 4R and T, muscles of the second recipientsshowed GFP+ fibres that were derived from intact muscles ofneonatal chimeras. Collectively, these results suggest that theGFP+ cells are located in the muscle as possible satellite cellsand may be participating in muscular generation upon muscledamage, as well as under physiological condition.

DiscussionThe chicken beta-actin promoter and cytomegarovirusenhancer-driven EGFP gene transgenic mouse strain is apowerful tool for studies tracing cell migration anddifferentiation in animals. The muscles of these mice expressubiquitous and strong fluorescence (Okabe et al., 1997), andthis prompted us to apply these mice to bone marrowtransplantation and muscle regeneration experiments. Incontrast to earlier bone marrow transplantation studies thatused nuclear β-galactosidase, the Y chromosome (Ferrari et al.,1998; Gussoni et al., 1999; Bittner et al., 1999) or GFP withthe aid of anti-GFP antibody (Orlic et al., 2001) as markers forthe detection of donor-derived skeletal or cardiac muscle fibres,GFP fluorescence can easily be detected by flow cytometry orfluorescence microscopy (Chalfie et al., 1994; Kawakami et al.,1999a) without any cofactor for light emission or any specificstaining procedures. As shown in this report, although thefrequency was not high enough, GFP+ fibres could be detectedin the muscles of chimeric mice.

It has long been known that the neonatal injection ofallogeneic cells induces tolerance to alloantigens of the donorcells and frequently establishes a chimeric state. Very recently,Liechty et al. (Liechty et al., 2000) described thetransplantation of human mesenchymal stem cells in uterointosheep at an early gestational period and the site-specificdifferentiation of these cells into to a variety of tissues, suchas chondrocytes, adipocytes, myocytes, cardiomyocytes, andthymic and bone marrow stromas, without any detectableimmune response against the human cells. Butler et al. (Butleret al., 2000) showed that neonatally induced tolerance toalloantigens in rats persisted, and skeletal tissue allograftssurvived, even though the rate of blood cell chimerism wasaround 3%. Thus transplantation to neonatal or embryonicanimals is a promising approach for stem cell therapy forhereditary diseases in animal models. The origin of satellitecells and the time at which they first migrate into muscle tissuesare not yet clear. Cossu et al. (Cossu et al., 1983) reported thatsatellite cells have a different sensitivity to phorbol ester thanmyoblasts. Taking advantage of this characteristic, the authorsshowed that satellite cells appear between day 16 and 18 ofembryonic development in mice. Muscle satellite cells accountfor about 30% of sublaminar muscle nuclei in neonatal mice,and this level decreases to less than 5% in adult mice (forreviews, see Campion, 1984; Mazanet and Franzini-Armstrong, 1986; Cossu and Molinaro, 1987; Bischoff, 1994).

These observations strongly suggested to us the idea ofintroducing muscle precursor cells at the earliest possible time,for example at birth. In the present study, neonatal injection ofallogeneic bone marrow or fetal liver cells combined withbusulfan treatment successfully induced a chimeric state inboth muscular precursor cells and hematopoietic cells.

Neonatal mice make it necessary to limit the cell dose andvolume of intravenous or intrahepatic injections; however,neonatal chimeras produced similar muscular reconstitution toadult radiation chimeras, although neonates have less efficientblood chimerisms. The immune response against proteins toviral vectors or to newly introduced gene products presents anew obstacle for both gene therapy and cell therapy. Once themice achieve radiation-induced or neonatally inducedimmunological tolerance, they can be challenged by anotherinjection of cell transplantation without any immune response.

In order to examine whether donor cells migrate intomuscles, we searched for GFP+ cells in the CTX-untreatedmuscles of neonatal chimeras. As shown in Fig. 4H,I, GFP+

fibre (H) as well as the GFP signal beneath the laminin-positivelayer (I) was present. We then explanted muscle fibres andcultured them for 4 days in vitro under non-differentiatingconditions. It was reported that naïve desmin+ single humanmuscle satellite cells in culture develop into two types of cells,one fuses into myotubes and the other persists for weeksamong the myotubes (Baroffio et al., 1996). The formerexpresses alpha sarcomeric actin, whereas the latter expressesdesmin. It is well documented that quiescent satellite cells inthe muscle express neither muscle-specific markers, such asdesmin and myosin, nor the MyoD family of muscle-specificregulatory molecules. However, after muscle injury (Helliwell,1988; Saito and Nonaka, 1994; Rantanen et al., 1995; Molnaret al., 1996) or short term culture in vitro (Allen et al., 1991;Kaufman et al., 1991; Creuzet et al., 1998), satellite cellsbecame desmin+ and MyoD+. This suggests that the desmin+

cell that attaches to a single fibre is probably a muscle satellitecell. Such cells were observed in the muscle fibre cultures inour present study (Fig. 4L,O), and they are GFP+. GFP+ donorbone marrow or fetal liver cells may be recruited as musclesatellite cells and may participate in muscle regeneration inmdxor CTX-treated muscle.

We then examined whether the fibre-attached cells expressM-cadherin (Donalies et al., 1991; Irintchev et al., 1994;Bornemann and Schmalbruch, 1994; Beauchamp et al., 2000),but they were negative in our experiments (data not shown).Beauchamp et al. (Beauchamp et al., 2000) reported that some(~20%) fibre-attached satellite cells in vitro do not express M-cadherin. We observed a few fibre-attached cells, so it ispossible that M-cadherin-negative fibre-attached satellite cellswere detected in these earlier experiments. To examine whetherthe GFP+ mononuclear cells become GFP+ fibres, wetransplanted the single cell suspension from muscles ofneonatal chimeras into immunodeficient mdx/scid secondaryrecipients. As shown in Fig. 4R,T, GFP+ fibres were generated.Muscle is a highly vascularised tissue and thereforecontamination from circulating blood cells can not beexcluded; however, the results suggest that the GFP+ cellsresiding in the CTX-untreated muscles are muscle precursorcells, probably satellite cells.

Recently, De Angelis et al. (De Angelis et al., 1999) reportedthat the large majority of mouse clones showing typicalsatellite cell morphology were derived from the dorsal aortanot from somites. Since the cells express several vascular cellmarkers, the postnatal satellite cells may be derived from avascular lineage. In the fetal liver, aorta-gonad-mesonephros(AGM)-area-derived hemangioblasts differentiate to bothendothelial cells for liver vessels and hematopoietic stem cells

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(Miyajima et al., 2000). Endothelial precursors also arise fromthe AGM (Ohneda et al., 1998). Because the AGM areaappears to be attached in clusters to the dorsal aorta, theprecursors of muscle satellite cells, vascular endothelial cellsand hematopoietic cells may derive from the same origin at anearly embryonic stage. They may move to fetal liver and thento bone marrow in adults. Thus, the bone marrow and fetal livermay have similar cell populations from which various types ofcells arise. As shown in Fig. 5, fetal liver cells exceeded bonemarrow cells in their myogenic potentials, and it suggeststhat fetal livers contained higher proportion of myogenicprogenitors than adult bone marrows.

The frequency of donor-derived muscle fibres is still too lowfor the treatment of muscular dystrophies. There are severalapproaches to overcome these problems. First, it is necessaryto enrich the muscular precursor cells from bone marrow orfetal liver cells by using various monoclonal antibodies againstcell-type-specific surface markers, such as c-Met, VEGF-receptor, Sca-1 and so on. Further trials to search for novelmarkers are also necessary. Second, it is not yet known whatkind of adhesion molecules are required for the migration ofprecursor cells into muscle. Neonatal or radiation-inducedtolerance, combined with injections of various monoclonalantibodies against adhesion molecules, will answer thisquestion. Lastly, developing or regenerating muscle mayproduce certain kinds of chemokines or cytokines that controlthe trafficking of precursor cells. These mediators and theirreceptors are likely to be critical for their migration. HGF andTGFβ are possible chemotactic factors for adult musclesatellite cells of the rat (Bischoff, 1997), but it is not yet knownwhat kind of factors are responsible for bone marrow- orfetal liver-derived precursors. Although the establishment oftolerance may not be immediately applicable to clinical trials,the experimental system, together with the above-mentionedstrategies for the proper reconstitution of muscle, will providevaluable information for future clinical application.

We thank M. Imamura and M. D. Ohto for the antibody used in thisstudy and for helpful advice, respectively. This work is supported bygrants-in-aid from the Ministry of Education, Culture, Sports, Scienceand Technology, and the Ministry of Health, Labour and Welfare ofJapan.

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