A Population of Myogenic Stem Cells That Survives Skeletal Muscle Aging - 《stemcells》干细胞学杂志干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 26 下一页

返回列表

查看: 455275\|回复: 254	go A Population of Myogenic Stem Cells That Survives Skeletal Muscle Aging [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 00:56 |只看该作者 |倒序浏览 |打印

作者：Charlotte A. Collinsa, Peter S. Zammitb, Ana Prez Ruizb, Jennifer E. Morgana, Terence A. Partridgec作者单位：aThe Dubowitz Neuromuscular Unit, Department of Paediatrics, Imperial College London, Hammersmith Hospital, London, United Kingdom;bRandall Division of Cell and Molecular Biophysics, King ) s, N& }% O6 |' {8 j7 h& I- M

   * l/ Y* R' S1 W+ p3 o8 z
      9 Y5 e9 n1 _; ?6 \! Y$ e5 l, B
                     + q: }9 i* V7 _8 c9 D
         $ b2 N* E  x& F/ R: X6 |, g
         ) `! ?' @5 l; T  o( n
         ; J8 [/ @+ [' n& I- y



      ( F# y$ P, \0 `- J2 n2 }
      - [- \1 x  I. e6 h3 `+ m( `! w
      【摘要】2 t% A( ]5 S* P0 A4 w
   Correspondence: Jennifer E. Morgan, Ph.D., The Dubowitz Neuromuscular Unit, Department of Paediatrics, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K. Telephone:  44 (0)20 8383 2125; Fax:  44 (0)20 8383 2187; e-mail: jennifer.morgan@imperial.ac.uk; or Peter S. Zammit, Ph.D., Randall Division of Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, U.K. Telephone:  44 (0)20 7848 8217; Fax:  44 (0)20 7848 6435; e-mail: peter.zammit@kcl.ac.uk1 d+ B' T/ w9 Q' N

Age-related decline in integrity and function of differentiated adult tissues is widely attributed to reduction in number or regenerative potential of resident stem cells. The satellite cell, resident beneath the basal lamina of skeletal muscle myofibers, is the principal myogenic stem cell. Here we have explored the capacity of satellite cells within aged mouse muscle to regenerate skeletal muscle and to self-renew using isolated myofibers in tissue culture and in vivo. Satellite cells expressing Pax7 were depleted from aged muscles, and when aged myofibers were placed in culture, satellite cell myogenic progression resulted in apoptosis and fewer total differentiated progeny. However, a minority of cultured aged satellite cells generated large clusters of progeny containing both differentiated cells and new cells of a quiescent satellite-cell-like phenotype characteristic of self-renewal. Parallel in vivo engraftment assays showed that, despite the reduction in Pax7  cells, the satellite cell population associated with individual aged myofibers could regenerate muscle and self-renew as effectively as the larger population of satellite cells associated with young myofibers. We conclude that a minority of satellite cells is responsible for adult muscle regeneration, and that these stem cells survive the effects of aging to retain their intrinsic potential throughout life. Thus, the effectiveness of stem-cell-mediated muscle regeneration is determined by both extrinsic environmental influences and diversity in intrinsic potential of the stem cells themselves.
) F' I. m" R8 c2 A( v( S- t( [
Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Stem cell Satellite cell Aging Skeletal muscle Self-renewal Regeneration1 }& c, P* s. u, b0 {7 j" W# L
   INTRODUCTION9 }& g+ U  V' r4 F

Skeletal muscle consists predominantly of a single differentiated cell type, the contractile myofiber. It is supplied with new nuclei during growth and regeneration by the satellite cell, a quiescent stem cell located beneath the basal lamina surrounding each myofiber , but the age-related loss of muscle bulk and regenerative ability may involve an impairment of this mechanism.( \* Z( b) ^7 ?% X% n

There is debate as to the extent to which ineffective muscle regeneration in aged mice is determined by changes in the extrinsic environment that inhibit the regenerative ability of otherwise competent satellite cells .3 y. z7 ~4 l7 e  T: _, _  F
" K3 P8 _  E( `, [
There is no agreement as to whether satellite cell numbers decrease with age. Reports of age-related decline in the numbers of satellite cells in the rat tibialis anterior (TA) muscle . This study, however, provides no information on satellite cell behavior in vivo.
) \9 J4 \( X* i: Q1 D' w
Here, we show that aging affects myofiber-associated satellite cells differentially. Although the number of satellite cells expressing the paired-box transcription factor Pax7 declines with age, this is partly counterbalanced by cells lacking myogenic markers that become associated with the satellite cell niche. In culture, the nonmyogenic cells undergo apoptosis, and the total yield of progeny from satellite cells associated with aged compared with young myofibers is reduced. Notably, however, a few satellite cells associated with aged myofibers do generate large clusters of progeny that comprise both cells undergoing myogenic differentiation and cells of a quiescent satellite cell phenotype characteristic of self-renewal. Moreover, this smaller population of satellite cells associated with individual aged myofibers can, when grafted, regenerate and self-renew as effectively as the larger population of satellite cells associated with young myofibers. These findings lend support to the idea that adult muscle regeneration is largely mediated by a minor subset of stem-like satellite cells that survive the effects of aging.: d$ `! j& i; E8 j/ ?5 p* q
4 P6 t) Y, f3 M/ `  ?$ h! F' m
MATERIALS AND METHODS

Single Myofiber Isolation
& h+ G" C6 g! G8 I$ W( u
Single myofibers were prepared from the EDL muscles of young and aged 3F-nLacZ-2E . "Aged" mice were used between 22 and 30 months of age, and "young" mice were used between 1 and 2 months of age.
9 i3 f: ~: N4 A; m8 p
Isolation of Satellite Cells from Single Fibers

Single myofibers were prepared from the EDL muscles of young and aged 3F-nLacZ-2E .7 b8 N6 a+ J* G# q

Culture of Myofibers in Suspension

Batches of 20¨C30 myofibers were incubated in plating medium  at 37¡ãC and 5% CO2.) b9 b1 i6 a3 @
' f& N1 c4 s! \& S! }
Culture of Myofibers on Matrigel

We coated 8-well Lab-Tek Chamber Slides (Nunc, Rochester, NY, http://www.nuncbrand.com) with Matrigel (BD Biosciences, San Diego, http://www.bdbiosciences.com) (1 mg/ml in Dulbecco's modified Eagle's medium). One myofiber with 250 µl of plating medium was placed in each well, and cultures were maintained at 37¡ãC/5% CO2. A 50% medium change was performed every 48 hours from 72 hours after plating.
1 M. H" `2 d7 T& [5 k1 ?6 h
Immunocytochemistry of Isolated Myofibers and Satellite Cells& w; n' |, `9 O4 u. x. S
/ w  Z2 R" d: ^. s5 a6 L4 t
We fixed 3F-nLacZ-2E myofibers or adherent cultures with 4% paraformaldehyde and incubated in 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) solution or immunostained as previously described . Primary antibodies used were mouse monoclonal anti-Pax7 (Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/dshbwww), mouse monoclonal anti-MyoD1 (clone 5.8A; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com), rabbit polyclonal anti-MyoD (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), rabbit polyclonal anti-myogenin (Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-ß-gal (Molecular Probes, Carlsbad, CA, http://probes.invitrogen.com), and rabbit polyclonal anti-laminin (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). For secondary detection, AlexaFluor 488-conjugated or AlexaFluor 594-conjugated goat anti-mouse Ig (Molecular Probes), tetramethylrhodamine isothiocyanate-conjugated swine anti-rabbit Ig (DakoCytomation), and AlexaFluor 488-conjugated goat anti-rabbit Ig were used. We used 4',6-diamidino-2-phenylindole (DAPI) (10 µg/ml) to counterstain nuclei. Myofibers isolated from Myf5nLacZ/  myofiber-engrafted mdx-nude TA muscles were fixed as above and then incubated overnight in X-gal solution or permeabilized in 0.5% Triton X-100 and incubated overnight with antibodies to Pax7 and ß-galactosidase (ß-gal). For TdT-mediated dUTP-biotin nick end labeling (TUNEL) assays, an APO-BrdU TUNEL Assay Kit (Molecular Probes) was used according to the manufacturer's protocol, with minor modifications. We detected bromodeoxyuridine (BrdU) incorporation at DNA break sites using an AlexaFluor 488-labeled mouse anti-BrdU antibody.

Grafting into Mouse Muscles/ n  G! Q7 s. P7 A$ `
& `9 ~) H) p8 K& k2 s4 D' S
Individual donor myofibers were grafted into the preirradiated TA muscles of mdx-nude host mice as previously described . In one experiment, 200 satellite cells sorted by flow cytometry on the basis of size and granularity were grafted into preirradiated mdx-nude host mouse muscles.2 ]& ~) _, K. H) m  n' v

Model of Muscle Injury

To investigate the ability of donor cells to regenerate tissue after acute injury, 4 weeks after grafting mice were reanesthetized, and 10 µl of Notechis scutatus scutatus notexin (Latoxan, Valence, France, http://www.latoxan.com) (10 µg/ml) was injected into each engrafted TA muscle . Vetergesic (buprenorphine; Alstoe Ltd Animal Health, York, U.K., http://www.alstoe.co.uk) 0.05 mg/kg was administered for postoperative analgesia.
8 D1 R/ `* ?9 L* h7 c, i
Immunohistochemistry of Tissue Sections
' N. f* @" ]% @9 w; \* j- ^8 b4 L
Engrafted muscles were frozen in isopentane cooled in liquid nitrogen. Three or four serial 7-µm cryosections were collected at 100-µm intervals throughout the entire muscle. Sections were immunostained with P7 rabbit polyclonal anti-dystrophin and BF34 mouse monoclonal anti-neonatal myosin antibodies and counterstained with DAPI .1 I, P$ @5 O5 @  q' O! b; j2 |

Microscopy4 i  I  ~: V3 b0 E7 s' I
$ \/ E- h4 R/ ]
Fluorescent and bright-field microscopy and image capture were performed using an epifluorescence microscope (model Axiophot; Carl Zeiss, Jena, Germany, http://www.zeiss.com). Digital images were acquired with a Charge-Coupled device (model RTE/CCD-1300-Y; Princeton Instruments Inc., Trenton, NJ, http://www.piacton.com) at ¨C10¡ãC using MetaMorph version 4.5r5 software (Molecular Devices Corporation, Sunnyvale, CA, http://www.moleculardevices.com). Images were optimized globally for brightness and contrast and assembled into figures using Adobe Photoshop CS (Adobe Systems Incorporated, San Jose, CA, http://www.adobe.com).

Quantification+ F) q; c+ H; Q% b2 u

In myofiber suspension and adherent cultures, counts were made of cells that contained the blue reaction product of X-gal staining (which quenches DAPI fluorescence) and of DAPI  cells that had been categorized by immunostaining. Data from multiple myofibers or myofiber cultures were pooled to give a population mean ¡À SEM for cells in each category.0 j: Z3 G" D! [* v# q4 r- J
. h3 a) U$ U) E
To analyze the numbers of new myofibers generated from single myofiber grafts, counts were made of the maximum number of donor dystrophin  myofibers in immunostained sections and ß-gal  myofibers or nuclei in adjacent X-gal-stained sections of host muscles. Data in each plot were pooled from multiple donor animals as indicated in figure legends.
* @2 k$ w+ Y" N) C$ Q+ \1 A% b
RESULTS& W+ m" s# m: a

Aging Results in Reduced Numbers of Pax7  Satellite Cells. }5 E* L  H2 w

The number and phenotype of satellite cells were compared on single myofibers from EDL muscles of six aged (mean age 735 ¡À 42 days) (n = 107) and seven young (mean age 43 ¡À 5 days) (n = 77) 3F-nLacZ-2E mice, in which ß-gal reports expression of the myosin light chain MLC3F gene in fast myofibers , and the basal lamina component laminin identified sublaminal satellite cells, whereas any other associated cells appeared as DAPI  nuclei beneath or outside the basal lamina (Fig. 1A¨C1D).0 j1 N$ T3 w' k6 o2 e. g6 x0 C& H
" a# ~5 W- G, G! n
Figure 1. Myogenic progression of aged satellite cells in myofiber suspension culture. (A, B): Myofibers isolated from the extensor digitorum longus (EDL) muscles of aged 3F-nLacZ-2E mice, stained in 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) solution and coimmunostained for Pax7 and laminin. Myonuclei contain the blue reaction product of X-gal staining, which quenches DAPI immunofluorescence. (A): Example of a Pax7  satellite cell beneath the basal lamina of a myofiber. (B): Example of a Pax7¨C cell that appears to be encapsulated by the basal lamina. Scale bar, 10 µm. (C): Bar graph depicting the mean numbers of 3F(ß-gal)  myonuclei in myofibers from seven young and six aged mice. (D): Bar graph depicting the mean numbers of Pax7  satellite cells and Pax7¨C basal lamina-associated cells in myofibers from young and aged mice. A total of 107 aged and 77 young myofibers were analyzed. Each error bar represents a standard error between the mean counts obtained from six or seven animals. (E¨CG): Myofibers isolated from the EDL muscles of an aged 3F-nLacZ-2E mouse and cultured in suspension for 96 hours, stained in X-gal, and then immunostained. The blue reaction product of X-gal staining marks expression of 3F(ß-gal) in both myonuclei and differentiated satellite cell progeny. (E): Myofiber immunostained for Pax7 and MyoD. A rare cluster of cells that contains a Pax7  MyoD¨C cell of a quiescent satellite-cell-like phenotype (arrow) as well as differentiating 3F(ß-gal)  and MyoD  progeny. (F): Myofiber stained for MyoD and myogenin. A typically small cluster of cells contains two myogenin  MyoD  cells in an early-stage of differentiation (arrows) as well as DAPI  cells, which do not express either myogenin or MyoD. (G): Myofiber immunostained for Pax7 and myogenin. An unusually large cluster contains 3F(ß-gal)  cells, differentiation-committed myogenin  cells, and undifferentiated Pax7  cells. Scale bar, 10 µm. Abbreviations: ß-gal, ß-galactosidase; DAPI, 4,6-diamidino-2-phenylindole.

Counts of 3F(ß-gal)  myonuclei in young and aged myofibers were not significantly different (Mann-Whitney, p > .05) (Fig. 1C). Counts of Pax7  cells, seen in an exclusively sublaminal satellite cell position (Fig. 1 A), revealed means of 4.6 ¡À 0.6 per young myofiber and 2.4 ¡À 0.3 per aged myofiber (Fig. 1 D), a significant difference (Mann-Whitney, p / f. y8 A  l7 D
) U4 [: g' P3 z) F+ I- b0 z5 N
Aging Perturbs Myogenic Progression of Activated Satellite Cells5 ?" \" J/ n* H' r7 K3 N
, X8 r+ W$ `/ A5 @$ r3 q. v$ F
To investigate the functional potential of aged satellite cells, we cultured isolated myofibers in suspension, preserving a near-normal relationship between satellite cell and myofiber .9 S& F3 [7 n6 U8 \7 R- X
& Z/ y" x0 @- h3 }6 g8 g
Total numbers of progeny associated with aged and young myofibers at 0-, 24-, and 48-hour time points were not significantly different (Kolmogorov-Smirnov two sample test, p > .05 in each case), but in cultures from aged mice a proportion of the total DAPI  cells expressed no myogenic markers. Whereas at 0 hours most Pax7  cells were MyoD¨C, when sampled at 24 hours and 48 hours the majority of stained cells in cultures from both young and aged mice coexpressed Pax7 and MyoD, and by 48 hours occasional couplets of dividing Pax7  MyoD  cells were observed. Thus, a proportion of aged satellite cells retained the ability to initiate an active myogenic program, remaining responsive to the same mitogenic environment as young satellite cells.
3 n+ X5 C" h% e* M
From 48 hours onward, satellite cells proliferated to generate clusters of progeny. At both 72 hours and 96 hours, myofibers from aged mice had fewer associated large clusters of cells and significantly fewer total cells than myofibers from young mice (Kolmogorov-Smirnov two sample test, p
' y% D5 _0 `. M/ G/ M/ B
Figure 2. Quantitative and phenotypic analysis of satellite cell progeny in myofiber suspension cultures. (A, B): Bar charts depicting phenotypic characterization of satellite cell progeny in myofiber suspension cultures. Myofibers were fixed and analyzed after 0, 24, 48, 72, or 96 hours in suspension culture. Batches of myofibers from the same mice were stained in 5-bromo-4-chloro-3-indolyl-ß-D-galactoside solution to reveal ß-gal activity and then coimmunostained for Pax7 and MyoD, MyoD and myogenin, or Pax7 and myogenin. Values are population means of the number of satellite cells in each category (¡À SEM) per myofiber isolated from two young mice (A) (n = 15¨C21 per category) or three aged mice (B) (n = 30 per category). (C, D): Bar charts depicting individual counts of the total number of cells associated with each cultured young or aged myofiber. Counts are ranked in order of the total number of cells associated with each myofiber. Abbreviations: ß-gal, ß-galactosidase; DAPI, 4,6-diamidino-2-phenylindole; h, hours; myog., myogenin.- c; D2 ]+ Z7 P# s/ }

Phenotypically, many of the progeny of young satellite cells were in various stages of myogenic differentiation by 72 hours, expressing either myogenin or 3F(ß-gal), but in aged fiber cultures, few satellite cell progeny reached this stage until 96 hours (Figs. 1E¨C1G, 2). Concomitant with the appearance of differentiated myogenin  and 3F(ß-gal)  cells, a subset of progeny downregulated MyoD but maintained robust Pax7 expression, thus opting out of immediate differentiation and acquiring the phenotype characteristic of quiescent satellite cells (Fig. 1E¨C1G). In cultures from young muscles, Pax7  MyoD¨C cells formed on average 13.5% ¡À 1.7% of the total population at 72 hours and 13.1% ¡À 1.9% of the total population at 96 hours, significantly more than in cultures from aged muscles (1.3% ¡À 0.6% of the total progeny at 72 hours and 2.7% ¡À 0.7% at 96 hours; Mann-Whitney, p
" J' b, S6 U5 o5 ?% T, O7 @! b
Apoptosis of Nonmyogenic Cells During Satellite Cell Activation and Proliferation
: Q1 f( T4 X8 s6 Q+ l; F7 L
We compared the rate of apoptosis between the progeny of cells associated with young and aged myofibers using the TUNEL assay, which specifically labels DNA breaks in apoptotic nuclei ), suggesting apoptosis of previously proliferating myogenic cells. Thus, the nonmyogenic cells associated with aged myofibers appear highly vulnerable to apoptosis, and it also seems likely that, at least in vitro, a significant proportion of the progeny of aged satellite cells lose their myogenic potential and succumb to apoptosis.4 X2 k2 }2 T$ F% }& C6 Q+ V) W
7 m; }* `! a  K+ @: f3 d( ?
Figure 3. Apoptosis of nonmyogenic cells during myogenic progression of aged satellite cell progeny. (A): TUNEL assay to identify apoptotic nuclei on extensor digitorum longus myofibers from young and aged 3F-nLacZ-2E mice after culture in suspension. (i, ii): 72-h aged muscle-derived myofiber with two TUNEL  cells (green, one arrowed). (iii, iv): 72-h aged muscle-derived myofiber bearing a cluster of cells in which five nuclei are TUNEL  (green, arrows mark two examples). Scale bars, 25 µm. (B): Bar chart showing the numbers of TUNEL  and TUNEL  MyoD  cells associated with myofibers isolated from three young or three aged mice after 0 h, 24 h, 48 h, and 72 h in suspension culture. Values are population means of the number of satellite cells in each category (¡À SEM) per myofiber. A total of 30 myofibers was analyzed per time point per age group. Each error bar represents a standard error. (C, D): Progeny of young and aged satellite cells in long-term adherent myofiber cultures. Single myofibers were isolated from three young and three aged 3F-nLacZ-2E mice and cultured on matrigel for 96 h or 216 h. Fixed cultures were stained in 5-bromo-4-chloro-3-indolyl-ß-D-galactoside and then immunostained for Pax7 and MyoD. (C): Culture from aged mouse analyzed at 96 h. (D): Culture from aged mouse analyzed at 216 h. Scale bars, 10 µm. (E): Bar chart depicting mean counts of phenotypically characterized nuclei per freshly isolated myofiber at 0 h (n = 20¨C22) or myofiber culture at 96 h (n = 23¨C43) or 216 h (n = 20¨C27) in cultures from young or aged mice. Each error bar represents a standard error. (F): Bar charts depicting individual counts of the total numbers of nuclei generated in each myofiber culture. Data are ranked in order of the total number of nuclei per culture. Abbreviations: ß-gal, ß-galactosidase; DAPI, 4,6-diamidino-2-phenylindole; h, hours; TUNEL, TdT-mediated dUTP-biotin nick end labeling.3 H* Z: z' Q5 O% E5 `' C' m$ S

Myogenic Potential of Aged Satellite Cells in Long-Term Culture! `  _$ w3 a1 i+ ^9 L! b! E4 h$ ?

To examine longer time points, we maintained myofibers isolated from three young and three aged 3F-nLacZ-2E mice as individual adherent cultures on Matrigel, which permits cells to migrate from the myofiber and proliferate extensively. By 96 hours, the majority of satellite cell progeny in both young and aged cultures coexpressed Pax7 and MyoD, a phenotype characteristic of proliferating myogenic cells (Fig. 3C). By 216 hours, most nuclei had undergone myogenic differentiation, expressing 3F(ß-gal) and/or incorporation into multinucleate myotubes (Fig. 3D). The percentage of total differentiated nuclei was higher in cultures derived from young mice (82.2% ¡À 4.0%) than in cultures derived from aged mice (66.1% ¡À 4.2%). In cultures from both young and aged mice, most of the remaining mononucleate cells expressed Pax7 and/or MyoD, indicating their myogenic character. Aged myofiber cultures contained significantly fewer total progeny than young myofiber cultures both at the 96-hour (Mann-Whitney, p

Satellite Cells Associated with Aged Myofibers Mediate Extensive Regeneration After Engraftment# [0 |2 J9 x  y' M; @- {9 P
1 }+ U$ T" M4 N" ^% i
To assay the myogenic potential of aged satellite cells in vivo, we isolated myofibers from the EDL muscles of three aged 3F-nLacZ-2E mice (mean age 735 ¡À 48 days) and grafted a single myofiber, with its associated satellite cells, into each of the irradiated TA muscles (n = 32) of 16 24-day-old mdx-nude mice. Examination of muscles 4 weeks after grafting revealed that 40.1% (13/32) of them contained substantial clusters of donor-derived myofibers identifiable by both 3F(ß-gal) and dystrophin expression (Fig. 4). In two cases, more than 100 new dystrophin  myofibers were formed, requiring extensive proliferation of donor cells to give rise to an estimated 10,000¨C20,000 differentiated donor myonuclei  despite the fact that the aged myofibers had significantly fewer associated Pax7  satellite cells.

Figure 4. Myogenic potential of aged satellite cells in single myofiber grafts. (A): Sections of an irradiated mdx-nude tibialis anterior (TA) muscle that had been engrafted with a single myofiber isolated from the extensor digitorum longus (EDL) muscle of an aged 3F-nLacZ-2E mouse 4 weeks previously. The satellite cells (average 2.4) present in the graft proliferated and gave rise to a large cluster of donor muscle identified by 5-bromo-4-chloro-3-indolyl-ß-D-galactoside staining, locating expression of 3F(ß-gal) (i) and, in a serial section, immunohistochemistry for dystrophin protein (ii), which is not expressed by the host. Asterisks mark the same myofiber in each section. Scale bar, 100 µm. (B): Bar chart depicting the numbers of donor ß-gal  and dystrophin  myofibers generated from 32 individual grafts of single myofibers derived from the EDL muscles of three aged 3F-nLacZ-2E mice and grafted into the irradiated TA muscles of 16 host mdx-nude mice. Four-week time point. Ranked in order of the number of dystrophin  myofibers. Abbreviation: ß-gal, ß-galactosidase.

To compare the regenerative potential of equal numbers of satellite cells from young and aged muscles, we sorted 3F-nLacZ-2E EDL myofiber-derived satellite cells on the basis of size and granularity . We grafted 200 sng satellite cells into the preirradiated right TA muscle and 200 cells from the residual satellite cell population into the irradiated left TA of host mice. Five mice were grafted with satellite cells from aged donors and five mice with satellite cells from young donors. Muscles were removed for analysis 4 weeks after grafting.( w6 k; L+ r! L9 t1 @

Preparations of sng cells from young muscle were 82% Pax7 , comparable to the 93% Pax7  in our previous preparations from diaphragm , whereas only 40% of the sng cells from aged muscle were Pax7  . Interestingly, 43% of the residual satellite cell population from young and 44% of the residual satellite cell population from aged EDL muscles were Pax7 .
7 b4 v) n- v4 K* ?( U% `$ n( Z
Donor muscle regeneration at graft sites of sng and residual satellite cell populations showed no significant age-related difference in performance (p > .1 in all cases). Donor muscle was found in four out of five muscles grafted with aged sng cells (41 ¡À 17.7  dystrophin  fibers; 8 ¡À 3.99 ß-gal  fibers) and in all five muscles grafted with young sng cells (42.4 ¡À 16.8 dystrophin  fibers; 8 ¡À 2.66 ß-gal  fibers). Residual cells formed donor muscle in only one out of five muscles grafted with cells from each age group, probably reflecting contamination of this population with a few stem-like satellite cells, as flow cytometry does not separate cell populations with 100% accuracy.

The apparent better performance of sng cells over the remainder of the population was significantly different for the young (p = .015 for both ß-gal and dystrophin) but not the aged (p > .1 in all cases) group, the latter probably because of the small sample size, and was highly significant when the two age groups were combined (p = .005 for ß-gal; p = .002 for dystrophin). Thus, satellite cells from young and aged donors show equivalent capacity to regenerate skeletal muscle, and this property is associated with the sng rather than the Pax7  phenotype./ w: _- t% K6 Y4 p$ v
0 Y) l% e3 A2 k" t7 Q  h* U
Satellite Cells Associated with Aged Myofibers Generate New Satellite Cells In Vivo

To investigate the ability of aged satellite cells to self-renew and generate new satellite cells in vivo, we grafted single myofibers derived from the EDL muscles of Myf5nLacZ/  mice, in which ß-gal reports expression of Myf5 in the nuclei of satellite cells and also in newly-formed myofibers .& S' a3 a0 X/ m  W1 d& Y5 I
0 s& M: Q  a) q( `
Figure 5. The reduced population of satellite cells on each aged myofiber contains equivalent in vivo myogenic and self-renewal potential to the larger population of satellite cells on each young myofiber. (A): Myofibers isolated from an irradiated mdx-nude (tibialis anterior) TA muscle 5 weeks after grafting with a single myofiber, with its associated satellite cells, derived from the extensor digitorum longus (EDL) muscle of an aged Myf5nLacZ/  mouse. (i): Staining with 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) reveals Myf5(ß-gal)  nuclei in a satellite cell position (arrowed). (ii¨Civ): Immunostaining with antibodies to Pax7 and ß-gal (counterstained with DAPI) shows a donor-derived satellite cell (arrowed). (B, C): Transverse sections of irradiated mdx-nude TA muscles 5 weeks after engraftment with a single myofiber, and its associated satellite cells, derived from the EDL muscle of an aged Myf5nLacZ/  mouse. Serial sections stained in X-gal (i), immunostained for dystrophin and neoMyHC (ii), or stained with H&E (iii). (B) is a noninjured control, and (C) was injured by injection of notexin 1 week before analysis. (B): The inset shows a Myf5(ß-gal)  nucleus in a peripheral satellite cell position (i). Arrows mark the same myofiber in each series. Scale bars, 200 µm (B) and 100 µm (C). (D¨CG): Bar charts depicting numbers of dystrophin  myofibers, dystrophin  neoMyHC  myofibers, and ß-gal  nuclei generated from single Myf5nLacZ/  myofibers 5 weeks after grafting. Ranked in order of the number of dystrophin  myofibers. (D, F): The left TA muscles of host mice were engrafted with single myofibers derived from four young donor mice. (E, G): The contralateral right TA muscles were engrafted with single myofibers derived from four aged donor mice. (D, E): The engrafted muscles of 12 host mice functioned as noninjured controls. (F, G): The engrafted muscles of 14 host mice were injected with notexin 4 weeks after grafting and analyzed 1 week later. Abbreviations: ß-gal, ß-galactosidase; DAPI, 4,6-diamidino-2-phenylindole.% J; y7 H' @) M: d3 t1 O: J4 Z
8 b: v% b) |. d% @/ |% ^
Table 1. Counts of satellite cells generated from individual grafts of Myf5nLacZ/  extensor digitorum longus single myofibers

Myogenic and Self-Renewal Potential of Aged and Young Satellite Cells Are Equivalent# b1 @. V" T( M3 c% X' T

To directly compare the myogenic potential of satellite cells from young and aged muscles and their daughter satellite cell progeny generated from these transplanted satellite cells in vivo, we grafted single myofibers derived from the EDL muscles of four young (mean age 51 ¡À 5 days) and four aged (mean age 703 ¡À 11 days) donor Myf5nLacZ/  mice into the irradiated TA muscles of 26 host mdx-nude mice. Each left TA was grafted with a myofiber from a young donor and each contralateral TA with a myofiber derived from an aged donor. Four weeks later, both TA muscles of 14 host mice were injured by injection of the notexin, the remaining 12 host mice functioning as noninjured controls. Notexin destroys 75%¨C80% of myofibers in injected muscle within 24 hours, sparing blood vessels and satellite cells . Muscles were removed 1 week later (5 weeks after grafting) to assess the regenerative response.

In noninjured muscles, similar numbers of donor dystrophin  myofibers were generated from young and aged myofiber grafts. Most dystrophin  myofibers did not express neoMyHC or myonuclear Myf5(ß-gal) and were large and mainly peripherally nucleated, a phenotype characteristic of mature nonregenerating muscle (Fig. 5B). Occasional Myf5(ß-gal)  nuclei were located predominantly at the periphery of myofibers, consistent with a satellite cell phenotype (Fig. 5B, inset). Several engrafted muscles that had been injected with notexin 1 week before removal contained compact clusters of donor dystrophin  myofibers, which were small and centrally nucleated and contained cytoplasmic neoMyHC and myonuclear Myf5(ß-gal), showing them to have been newly regenerated by donor-derived myogenic progenitors during the week following injury (Fig. 5C). Grafts derived from young muscles and from aged muscles were very similar with regard to both the proportion of engrafted muscles that contained donor muscle and the total numbers of newly-regenerated donor myofibers formed (Fig. 5D¨C5G). Thus, in vivo, aged satellite cells underwent self-renewal to generate new satellite cells, which, after experimental injury of the engrafted muscle, exhibited equivalent regenerative potential to satellite cells formed by self-renewal of young satellite cells.
6 F6 Q; E" f0 S- Z
DISCUSSION0 F0 P9 B% D6 T! j
. Z( b8 V) Y# K( t6 n( M
Skeletal muscle in the aging individual manifests a progressive failure to maintain itself, leading to a loss of tissue mass and function. Satellite cells are reduced in number in aged muscle and exhibit a range of functional impairments in tissue culture . This apparent paradox led us to investigate directly the function of aged satellite cells themselves, using parallel culture and engraftment assays as measures of regenerative potential.

We show that the number of Pax7  myofiber-adherent satellite cells is reduced in preparations from aged muscles, in agreement with the findings of another recent tissue culture study . We extend these observations by showing that this decline is associated with the presence of cells that do not express either Pax7 or other myogenic markers. Our tissue culture data show that such nonmyogenic cells are highly vulnerable to apoptosis and that the total yield of progeny from satellite cells derived from aged muscle is significantly lower than that of satellite cells derived from young muscle. Importantly, however, a subpopulation of satellite cells on aged myofibers remains capable of generating large clusters of progeny that contained both differentiating cells and other cells with a quiescent satellite cell phenotype (Pax7  and MyoD¨C and myogenin¨C), signifying the retention of both regenerative and self-renewal potential.

As a direct assay of regenerative potential in vivo, we grafted single myofibers with their associated satellite cells into irradiated mdx-nude mouse muscles. This model provides an optimized environment for assessing the contribution of stem cell populations to muscle regeneration .' u, t3 Z3 t- g" q
1 |: r$ T8 v- ]( M2 C- e
We found that the smaller population of satellite cells associated with each aged myofiber (mean 2.4) had equivalent regenerative and self-renewal potential to the larger population of satellite cells associated with each young myofiber (mean 4.6). As shown previously , injury of the recipient muscle provokes an increase in the frequency of donor-derived muscle regeneration. On the basis of this, we speculate that, in a proportion of grafts, the donor satellite cells are viable but not recruited into an active myogenic program until later stimulation by notexin-induced local muscle damage. Overall, our data show that, in vivo, a reduction in aged satellite cell population size does not result in any significant functional deficit. These findings support the idea that a stem cell subset of adult satellite cells is primarily responsible for muscle regeneration and, moreover, suggest that such cells are selectively retained within the aged satellite cell niche.

In grafts of EDL satellite cells sorted on the basis of size and granularity , the amount of muscle formed by sng cells from the two age groups was similar, despite the number of Pax7  satellite cells in this fraction prepared from aged muscle being far smaller. In young muscles, the stem-cell-like cells must be Pax7 , since the frequency of Pax7¨C cells on young myofibers is far below the frequency of young myofiber grafts that successfully regenerate muscle. However, a high proportion of aged myofibers have both Pax7  and Pax7¨C cells associated with them, and we cannot directly demonstrate any relationship between Pax7 expression and stem cell properties. Notably, although the young and aged residual cell populations contained a similar percentage of Pax7  cells to the aged sng fraction, they formed less muscle. This suggests that size and granularity is the best predictor of high regenerative capacity, leaving Pax7 positivity as perhaps essential but not sufficient.7 n/ V2 c4 ]/ v6 ^/ D" ]2 }

We and others  have shown that total satellite cell number declines with aging, and data from our culture experiments suggest that failure of effective self-renewal is a likely explanation for this. Although a subset of satellite cells in myofiber preparations from aged muscles remains capable of generating large clusters of progeny, containing both differentiated cells and new quiescent satellite-cell-like cells, the majority of the population generates few progeny, of which very few reacquire the satellite-cell-like phenotype associated with self-renewal.
0 ?' \) q& [% |' h; P, ~: {2 b
The Pax7¨C population of aged myofiber-associated cells may either be the product of the aberrant myogenic progression of aged satellite cells or interlopers from outside the myogenic lineage that have penetrated the basement membrane and, thus, acquired the same anatomical situation as satellite cells ." g4 D1 X5 k1 Z6 L( Y. }
  d+ J% K, E$ v) y$ j* x% Z6 V
Phenotypically homogeneous populations of hematopoietic stem cells are known to exhibit significant functional heterogeneity, the basis of which remains incompletely understood  implied that muscle stem cells can be instructed to adapt their function. Our engraftment experiments support this view by showing that satellite cells from young and aged muscles behave similarly in response to the same optimized in vivo environment. However, our data show that only a minority of the satellite cell population possesses this stem cell function. If all satellite cells responded similarly to the same environment, then the greater numbers of satellite cells associated with young myofibers would generate new muscle in greater quantity and with greater frequency than the smaller numbers of satellite cells associated with aged myofibers, whereas in fact there was no such difference.0 A" J9 ?2 l3 Z5 A
8 Y0 ^: H% L' s" f- ~5 [/ R' K
CONCLUSIONS

We demonstrate that a subset of functional myogenic stem cells persists in aged skeletal muscle and provide evidence that these stem cells are present with similar frequency in the satellite cell compartments of young and aged muscles. This indicates that muscle stem cell potential is as much a product of the intrinsic properties of the cell as of the extrinsic influences of the muscle environment. The stem cell subpopulation of muscle satellite cells represents a prominent and specific target for any therapeutic regime that might be applied to regeneration-defective aging, sarcopenic, or dystrophic muscle.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
8 X- p3 |% ^* n8 r
The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS
: i( m6 k* r  J- i- z
C.A.C. was funded by the Muscular Dystrophy Campaign (RA1/685 and RA3/711) and MYORES Network of Excellence, contract 511978, from the European Commission 6th Framework Programme. P.S.Z. is funded by the Medical Research Council (MRC). A.P.R. is funded by a Fundaci¨®n Ram¨®n Areces postdoctoral fellowship. J.E.M. is funded by an MRC Joint Collaborative Career Development Award in Stem Cell Research. T.A.P. was funded by the MRC and a Blaise Pascal chair awarded by the Ecole Normale Sup¨¦rieure. The Pax7 antibody developed by A. Kawakami was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA. The authors are grateful to Dr. Margaret Buckingham for providing the 3F-nLacZ-2E and Myf5nLacZ/  mouse strains and to Dr. Didier Montarras and Dr. Margaret Buckingham for advice on fluorescence-activated cell sorting and providing the Pax3 (green fluorescent protein) reference cells.* m3 V# \  a( Z5 b; v$ c7 U7 A4 y0 t

C.A.C. is currently affiliated with the Wellcome Trust Centre for Stem Cell Research, University of Cambridge, U.K.
      【参考文献】
9 A- J: K2 x2 ]9 v; H

Mauro A. Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 1961;9:493¨C495." F( U3 s! m/ h6 l& S
4 p& k- f& U3 v
Moss FP, Leblond CP. Satellite cells as the source of nuclei in muscles of growing rats. Anat Rec 1971;170:421¨C435.
# u8 w3 J% I& l1 S. }% Y
Snow MH. An autoradiographic study of satellite cell differentiation into regenerating myotubes following transplantation of muscles in young rats. Cell Tissue Res 1978;186:535¨C540.$ J" u  C. {$ k

Zammit PS, Golding JP, Nagata Y et al. Muscle satellite cells adopt divergent fates: A mechanism for self-renewal? J Cell Biol 2004;166:347¨C357.

Collins CA, Olsen I, Zammit PS et al. Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 2005;122:289¨C301.
* A5 L. y" J1 W& [: k" E/ H+ b. t
Collins CA. Satellite cell self-renewal. Curr Opin Pharmacol 2006;6:301¨C306.( Z; P0 z; F* |# P& ^5 Z4 J

Sadeh M. Effects of aging on skeletal muscle regeneration. J Neurol Sci 1988;87:67¨C74.) `5 {1 t# N; e4 t: l

Grounds MD. Age-associated changes in the response of skeletal muscle cells to exercise and regeneration. Ann N Y Acad Sci 1998;854:78¨C91.! M) w+ z1 f: B( a; c2 }

Conboy I, Rando T. Aging, stem cells and tissue regeneration: Lessons from muscle. Cell Cycle 2005;4:407¨C410.. Q5 [! x6 h' c- u7 [

Carlson BM, Faulkner JA. Muscle transplantation between young and old rats: Age of host determines recovery. Am J Physiol 1989;256:C1262¨CC1266.! J/ d5 r/ E2 h

Conboy IM, Conboy MJ, Wagers AJ et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 2005;433:760¨C764.3 T3 ]) |/ O4 b* C
; P# B4 d  Y2 }- \8 v, f9 @
Dedkov EI, Borisov AB, Wernig A et al. Aging of skeletal muscle does not affect the response of satellite cells to denervation. J Histochem Cytochem 2003;51:853¨C863.1 k9 i0 w8 D' x+ n' I
' X' W4 L; H$ x) Z8 ?
Brack AS, Bildsoe H, Hughes SM. Evidence that satellite cell decrement contributes to preferential decline in nuclear number from large fibres during murine age-related muscle atrophy. J Cell Sci 2005;118:4813¨C4821.

Gibson MC, Schultz E. Age-related differences in absolute numbers of skeletal muscle satellite cells. Muscle Nerve 1983;6:574¨C580.% M9 t: B6 Q& \! y$ n$ `- ~
1 V+ `/ m2 z: J( S
Shefer G, Van de Mark DP, Richardson JB et al. Satellite-cell pool size does matter: Defining the myogenic potency of aging skeletal muscle. Dev Biol 2006;294:50¨C66.* h& D+ J+ j7 N; J
/ [, m5 R6 Y3 Z  i1 x* L( z
Snow MH. The effects of aging on satellite cells in skeletal muscles of mice and rats. Cell Tissue Res 1977;185:399¨C408.2 a. P" ~4 N1 S# a! R
# S- p1 S; `! ?
Schafer R, Zweyer M, Knauf U et al. The ontogeny of soleus muscles in mdx and wild type mice. Neuromuscul Disord 2005;15:57¨C64.
1 U( }/ P6 x2 [5 p
Nnodim JO. Satellite cell numbers in senile rat levator ani muscle. Mech Ageing Dev 2000;112:99¨C111.. j' T$ j$ B& s! ?7 O/ {' N
  i; d6 y: I1 e. t$ C! X
Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528¨C1530.3 E7 q! U: O% L0 l- y) V9 e

Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123¨C134.. d, f8 G) K8 J, Y, b  S0 n

Tamaki T, Akatsuka A, Ando K et al. Identification of myogenic-endothelial progenitor cells in the interstitial spaces of skeletal muscle. J Cell Biol 2002;157:571¨C577.
1 c% J! J0 p4 C
Allen RE, McAllister PK, Masak KC. Myogenic potential of satellite cells in skeletal muscle of old rats. A brief note. Mech Ageing Dev 1980;13:105¨C109.
0 }& C- I! d& T6 X+ Z
Tajbakhsh S, Rocancourt D, Buckingham M. Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice. Nature 1996;384:266¨C270.
% k3 t/ ]5 U  e% R( d" d
Partridge TA, Morgan JE, Coulton GR et al. Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature 1989;337:176¨C179.
3 Q' z% o  j1 N1 i( O+ M
Rosenblatt JD, Lunt AI, Parry DJ et al. Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 1995;31:773¨C779.
- e6 L5 l& x: E6 w" g( e
Montarras D, Morgan J, Collins C et al. Direct isolation of satellite cells for skeletal muscle regeneration. Science 2005;309:2064¨C2067.

Beauchamp JR, Heslop L, Yu DS et al. Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells. J Cell Biol 2000;151:1221¨C1234.
2 E* R/ l5 S' c" ~' k  M
Gross JG, Morgan JE. Muscle precursor cells injected into irradiated mdx mouse muscle persist after serial injury. Muscle Nerve 1999;22:174¨C185.2 Q8 @. c/ d. ^5 B/ R0 d

Kelly R, Alonso S, Tajbakhsh S et al. Myosin light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice. J Cell Biol 1995;129:383¨C396.9 e( Y# {" U' ~) J8 \
( }. H* r6 p' l# S# a' ]
Seale P, Sabourin LA, Girgis-Gabardo A et al. Pax7 is required for the specification of myogenic satellite cells. Cell 2000;102:777¨C786.
+ j# Y6 v* ^$ v, \& z4 T: U
Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 1992;119:493¨C501.' N4 y/ I( M: Y9 i! H, {* J
' @6 L- y2 f% U: N
Tajbakhsh S, Bober E, Babinet C et al. Gene targeting the myf-5 locus with nlacZ reveals expression of this myogenic factor in mature skeletal muscle fibres as well as early embryonic muscle. Dev Dyn 1996;206:291¨C300.

Harris JB. Myotoxic phospholipases A2 and the regeneration of skeletal muscles. Toxicon 2003;42:933¨C945.

Morgan JE, Hoffman EP, Partridge TA. Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse. J Cell Biol 1990;111:2437¨C2449.
  Y5 E! B' j4 D; R0 Q
Lemischka IR, Raulet DH, Mulligan RC. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell 1986;45:917¨C927.
2 E, n& x8 l7 ]% d2 C! O5 i
Sherwood RI, Christensen JL, Conboy IM et al. Isolation of adult mouse myogenic progenitors: Functional heterogeneity of cells within and engrafting skeletal muscle. Cell 2004;119:543¨C554.
" x- ]  B* e: ^+ h6 R) |( n6 w
Trupin GL, Hsu L. The identification of myogenic cells in regenerating skeletal muscle. II. Early mammalian regenerates. Dev Biol 1979;68:72¨C81.
( T* E7 b- Q$ E8 Y) v  z; B
Cossu G, Bianco P. Mesoangioblasts¡ªvascular progenitors for extravascular mesodermal tissues. Curr Opin Genet Dev 2003;13:537¨C542.
2 }  A0 I2 X* |1 f& F5 N
Oustanina S, Hause G, Braun T. Pax7 directs postnatal renewal and propagation of myogenic satellite cells but not their specification. Embo J 2004;23:3430¨C3439.6 u# l* h2 O3 Y  n" q, ], j
4 r  ]; e; e3 M& S
Muller-Sieburg CE, Sieburg HB. Clonal diversity of the stem cell compartment. Curr Opin Hematol 2006;13:243¨C248.
5 i  I* v2 \0 @( x
Beauchamp JR, Morgan JE, Pagel CN et al. Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source. J Cell Biol 1999;144:1113¨C1122.0 E8 |. L: ^' v. u( |7 @+ y3 G; u. B0 N

Rantanen J, Hurme T, Lukka R et al. Satellite cell proliferation and the expression of myogenin and desmin in regenerating skeletal muscle: evidence for two different populations of satellite cells. Lab Invest 1995;72:341¨C347.
- V2 u/ N4 i6 }' D- }. a
Schultz E. Satellite cell proliferative compartments in growing skeletal muscles. Dev Biol 1996;175:84¨C94.

Partridge T. Reenthronement of the muscle satellite cell. Cell 2004;119:447¨C448.) @( v  i: [. |7 J- X6 O* N

Rouger K, Brault M, Daval N et al. Muscle satellite cell heterogeneity: In vitro and in vivo evidences for populations that fuse differently. Cell Tissue Res 2004;317:319¨C326.: y& S' O/ m  F# w

Shinin V, Gayraud-Morel B, Gomes D et al. Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells. Nat Cell Biol 2006;8:677¨C687.

收藏0 分享0 顶0 踩0