Evaluation of Sca-1 and c-Kit As Selective Markers for Muscle Remodelling by Non - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:54 |只看该作者 |倒序浏览 |打印

作者：Sharon H.A. Wonga,b,c, Kym N. Lowesa,b,c,j, Ivan Bertoncellod,e,f, Anita F. Quigleya,b,c,h,j, Paul J. Simmonsd,g, Mark J. Cookc,h, Andrew J. Kornberga,b,i, Robert M.I. Kapsaa,b,c,h,j作者单位：aNational Muscular Dystrophy Research Centre, Fitzroy, Victoria, Australia;bHoward Florey Institute, Parkville, Victoria, Australia;cDepartment of Medicine, The University of Melbourne, Fitzroy, Victoria, Australia;dStem Cell Laboratory, Peter McCallum Cancer Research Institute, East Melbourne, Vict
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      【摘要】
   Bone marrow (BM)-derived cells (BMCs) have demonstrated a myogenic tissue remodeling capacity. However, because the myoremodeling is limited to approximately 1%¨C3% of recipient muscle fibers in vivo, there is disagreement regarding the clinical relevance of BM for therapeutic application in myodegenerative conditions. This study sought to determine whether rare selectable cell surface markers (in particular, c-Kit) could be used to identify a BMC population with enhanced myoremodeling capacity. Dystrophic mdx muscle remodeling has been achieved using BMCs sorted by expression of stem cell antigen-1 (Sca-1). The inference that Sca-1 is also a selectable marker associated with myoremodeling capacity by muscle-derived cells prompted this study of relative myoremodeling contributions from BMCs (compared with muscle cells) on the basis of expression or absence of Sca-1. We show that myoremodeling activity does not differ in cells sorted solely on the basis of Sca-1 from either muscle or BM. In addition, further fractionation of BM to a more mesenchymal-like cell population with lineage markers and CD45 subsequently revealed a stronger selectability of myoremodeling capacity with c-Kit/Sca-1 (p < .005) than with Sca-1 alone. These results suggest that c-Kit may provide a useful selectable marker that facilitates selection of cells with an augmented myoremodeling capacity derived from BM and possibly from other nonmuscle tissues. In turn, this may provide a new methodology for rapid isolation of myoremodeling capacities from muscle and nonmuscle tissues.4 `5 M! |: F9 I3 @0 ]
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Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Bone marrow Stem cells Stem cell antigen- c-Kit mdx Muscular dystrophy Muscle regeneration Mice6 H% z& G) _* d( S% X6 x) a( K6 g- k
   INTRODUCTION8 K7 `& y4 s# m. q  r; }

Duchenne's Muscular Dystrophy (DMD) is an X-linked recessive (Xp21.1) disorder characterized by a lack of dystrophin protein in the membrane-associated cytoskeleton of muscle fibers .

Several factors hinder the success of myoblast transplant therapy in DMD, particularly host immune rejection of donor myoblasts . An alternative cell source is, therefore, required for autologous remodeling of dystrophic muscle.

Bone marrow (BM)-derived cells (BMCs) have a demonstrated capacity to differentiate into mature cells of the heart, liver, kidney, lungs, gastrointestinal tract, skin, bone, skeletal muscle, cartilage, and brain in vivo . We propose that a rare subpopulation(s) of cells capable of remodeling muscle, but highly diluted when WBM is transplanted, exists in BM. Ideally, if the specific BMC population(s) with myoremodeling ability can be isolated and enriched, then SC transplantation may provide a clinically relevant level of muscle engraftment.; `: w& }$ T$ J- }& Y
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Presently, it is still not known what characteristics of muscle-derived progenitors define a cell that can remodel muscle efficiently, although several have been investigated, including cell surface marker expression, in vitro adhesion properties, Hoechst efflux, or a combination of these characteristics ." P! ?5 h" B: e& E# D: E

Although the use of Sca-1 cell surface antigen to identify cells with myoremodeling capacity has shown some variable success, it is still largely unclear whether Sca-1 expression is the most potent indicator of myoremodeling capacity in cells derived from BM or muscle itself . This study, therefore, initially investigated the myoremodeling capacity of BM and muscle sorted solely on the expression of Sca-1.
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In 1999, Gussoni et al. . They suggest that this population of cells may represent universal pluripotent SCs residing at different levels in multiple murine tissues. To date, myogenic differentiation of these cells has not been demonstrated in vivo. Furthermore, there is little clarity as to the true origin and identity of these cells. Nonetheless, these studies suggest that the BMCs with the optimal myoremodeling capacity may be elucidated by their cell surface marker expression profile, and CD45, Sca-1, and c-Kit may be important candidate markers for the isolation of these cells. This study was, therefore, extended to compare the relative myoremodeling capacity of various cellular components of WBM fractionated/enriched on the basis of their expression of cell surface antigens CD45, Sca-1, and c-Kit.
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MATERIALS AND METHODS1 M: G# c0 {$ x

Animals" H- x$ o3 B* }8 `" Q
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Wild-type (wt) male C57Bl/10 mice, 3¨C12 weeks of age, and male C57Bl/10 mdx mice, between 8 and 12 weeks of age, were obtained from Monash University Animal Services (Clayton, Victoria, Australia). All procedures were approved by the Animal Experimental Ethics Committee of St. Vincent's Hospital, Melbourne, Australia (protocol 01-11) and conformed to the guidelines for the care and use of experimental animals as described by the National Health and Medical Research Council of Australia.5 _/ I7 D7 s" D) v% i

BM Cell Suspension Extraction3 D$ i7 L) B6 W# J1 n( I

Femurs, tibiae, and iliac crests were dissected from 20 male C57Bl/10 wt mice (8¨C12 weeks of age) and cleaned of all soft tissue, and BM cells were harvested by crushing the bones using a mortar and pestle. The cell suspension was filtered through a 40-µm cell strainer (Falcon, Becton Dickinson, Franklin Lakes, NJ, http://www.bd.com) to remove bone debris, washed twice, and resuspended in phosphate-buffered saline (PBS) supplemented with 2% newborn calf serum (PBS-2% Se). Approximately 1 x 108 WBM cells were obtained from each mouse. Low-density (LD) BMCs were isolated by discontinuous density gradient centrifugation using Nycoprep Animal (density 1.077 g/cm3; Nycomed Pharma, Oslo, Norway, http://www.nycomed.com/en) and collected into PBS-2% Se. Chromogenic donor cells (e.g., GTRosa-26) were not used in this study because of difficulties in breeding sufficient numbers of mice to generate the cell numbers needed for these experiments.& t; g* F$ e) [8 B7 U1 Y
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Negative Immunomagnetic Selection5 V* U! ^/ g! u$ A1 Z
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LD BMCs were depleted of cells expressing mature hemopoietic cell lineage antigens by immunomagnetic selection using the MACS system (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com/en). The antibodies anti-B220, anti-CD4, anti-CD8, anti-Gr-1, anti-Mac-1, and anti-TER 119 were used as a cocktail, purified, or biotin-conjugated for MACS separation as previously described ) was collected by eluting the cells through a 20-G needle with 100 ml of PBS-EDTA-0.5% bovine serum albumin.0 M; B# r4 v7 S* p3 i' e

Flow Cytometry3 V; k$ M( f: Z! y3 W' r6 n

Lin¨C cells were resuspended at 5.0 x 106 cells per 100 µl, and labeled with 1 µl of fluorescein isothiocyanate (FITC)-conjugated rat anti-mouse Sca-1 and phycoerythrin (PE)-conjugated rat anti-mouse c-Kit. CD45  cells were identified by labeling with biotinylated rat anti-mouse CD45 antibody and streptavidin-RED670 for 20 minutes on ice. The cells were then washed, and Lin¨C, CD45¨C cells within a predetermined rectilinear forward scatter (FSC) and side scatter (SCC) blast cell region  using a FACStarPlus cell sorter (Becton Dickinson), equipped with a 5-W argon ion laser (Innova 90; Coherent, Palo Alto, CA, http://www.cohr.com) running at 200 mW power, and a Spectra-Physics ultraviolet (UV) laser (Mountain View, CA, http://www.spectra-physics.com), running at 50 mW power. Green fluorescence (FITC) was collected through an FITC 530-nm filter, with a bandwidth of ¡À15 nm. Red fluorescence (PE) was collected through a 575DF26 filter, and RED670 fluorescence was collected through a long-pass RG655 filter.0 [/ V+ J6 G; z) r/ v& b
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Sorted cells were collected into serum-coated tubes at 4¡ãC and reanalyzed to establish purity prior to storage at 4¡ãC overnight in PBS-2% Se until injection into recipient mdx mice. These cells were counted and washed in PBS to remove all traces of serum before injection into mdx recipients. Immediately before injection, viable cell counts using trypan blue nuclear exclusion showed an average excess of 90% of viable cells in the injected population.
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Isolation of Whole Muscle Cells from Fresh Muscle! U2 R% X% t) p' K/ J
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Male C57Bl/10 wt mice, 3 weeks of age, were killed by cervical dislocation. Skeletal muscle from the hind limbs was removed, and visible nerves and fat were separated from the muscle. The muscle was finely minced and then digested at 37¡ãC with 2 ml/g muscle of dispase (grade II, 2.4 U/ml; Roche Molecular Biomedicals, Indianapolis, http://www.roche-applied-science.com) and collagenase (class D, 1%; Roche Molecular Biomedicals) in 10 ml of Hams/F10 (Trace Biosystems, Sydney, Australia) supplemented with 2.5 mM CaCl2, 1x penicillin/streptomycin (Gibco Life Technologies, Gaithersburg, MD, http://www.invitrogen.com), and 25 ng/ml amphotericin. The muscle slurry was triturated every 20 minutes over 2 hours. After 2 hours, the muscle slurry was centrifuged at 840g for 10 minutes, and the supernatant was removed. Red blood cells were lysed with 17 mM Tris/144 mM NH4Cl, pH 7.2. The cells were washed twice in PBS by centrifugation before injection into mdx recipients.* ~9 o  ~+ o5 n9 C
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Cell Transplantation into Dystrophic mdx Muscle

The tibialis anterior (TA) muscle of 8¨C12-week-old male mdx mice was exposed by surgical incision. Cells were injected into the TA in a 10-µl volume (refer to Table 1 for injected cell types and cell numbers). In the experiment involving i.m. injection of Sca-1-sorted components of muscle or BM, mice were injected 2 days before injection of cells, with 50 µl of 0.5% bupivacaine hydrochloride (BUP; Marcaine; Astra, NSW, Australia, http://www.astrazeneca.com), because this myotoxic agent has been shown to promote equal myoregenerative activities in mdx and wt rodent muscle . Right TAs served as internal contralateral saline controls and were likewise preconditioned with BUP 2 days before saline injection. The TA muscles were all harvested after 6 weeks and snap frozen in liquid nitrogen-cooled isopentane.

Table 1. Percentage of each BMC fraction within WBM, actual number of cells injected, summary of myogenic engraftment by each BMC fraction, and their associated RMI# h% e/ V, g* }7 e, W" R% I; `

Dystrophin Immunohistochemistry9 x: _( y: a7 E$ x; ~* _7 {- h
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The TAs were sectioned and analyzed for dystrophin expression as described previously . Briefly, serial sections 8 µm thick were taken along the entire length of the muscles. Dystrophin expression was assessed with a 1:100 dilution of a polyclonal antibody raised in sheep against a 60-kDa dystrophin fusion protein (a gift from Prof. L. Kunkel, Children's Hospital and Harvard Medical School, Boston). The primary antibody was visualized with FITC-conjugated rabbit anti-sheep IgG (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com), diluted 1:100 in PBS. Where applicable, an area containing the maximum number of dystrophin  myofibers for each injection was used to determine the Relative Myoremodeling Index (RMI; derivation of the RMI is discussed in the next section), to better facilitate comparison. The RMI was calculated from dystrophin  myofiber numbers standardized for revertant fibers by subtracting the mean number of revertant fibers from the total number of dystrophin  myofibers observed in each TA muscle. The revertant fiber numbers in each treatment groups was assessed by counting dystrophin  fibers in TA muscles of littermates that were injected with saline (vehicle), but not cells. Statistical analysis between groups was analyzed by the Mann-Whitney U test (significance at p
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Derivation of the RMI

The relative abundance of the various cell types used in this study differ significantly within the BM cellular compartment (Table 1). As a consequence, the absolute numbers of each cell type injected into recipient host muscles differed accordingly. A calibration factor that reflected the relationship between numbers of injected cells and cellular integration into the host tissue was derived to accommodate these differences in cell numbers injected into recipient mice. On the basis of the well-established Regeneration Efficiency Index  that can be defined by:6 s) x7 `. V# o9 L
7 t' a! g* c( p" o0 k
where x = log10 n7 g3 |1 P) c# t$ Q
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where y is the number of dystrophin  myofibers, k (a constant which we have termed the "Regeneration Constant") is the rate of increase in the number of dystrophin  myofibers with an increase in injected cells on a semi-log10 plot, and x is the logarithm of n, the number of injected cells. This relationship holds true as long as the value of n is  106 (i.e., x  6), where 106 is the upper limit of the number of cells injected before a grafting plateau is reached .$ a0 ], n8 K, w

If the relationship between injected and engrafted cells of the various cell types used in this study assumes equivalence, then the relationship can be simply described by a "Myoremodeling Index" for each cell type, defined as the number of dystrophin  myofibers arising from the donor cells as a function of the number of donor cells injected:
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where x1 and y1 represent log10(number of cell-type 1 injected) and the number of dystrophin  myofibers resulting from engraftment of these cells, respectively. x2 and y2 represent similar parameters from grafted cell-type 2.

A RMI, which describes the relative capacities of two given cell types to engraft into muscle can thus be derived by:
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The third equation therefore reflects the ratio of dystrophin  fibers, y2 (generated by cell-type 2) as a function of the number of type 2 cells injected (x2) and number of injected type 1 cells required (x1) to generate the number of dystrophin  fibers (y1) by the type 1 cells. As such, this transform defines the myoengraftment (and therefore myoremodeling) capacity of type 1 cells as a function of the engraftment capacity of the type 2 cells.& q3 M1 Y( Q7 ~- o, C

Allele-Specific Polymerase Chain Reaction Detection of wt DMD Gene and Transcript4 |( |% w9 i6 u0 F

Genomic DNA (gDNA) was isolated from the cryosectioned right TAs (QIAamp DNA Mini Kit, Qiagen, Valencia, CA, http://www1.quiagen.com). A 892-base pair, double-stranded DMD product (Amplicon A) was amplified from the gDNA, using antisense primer Dys In23 AS-02 (5'-CAGACAATCCAAGAAGGTATGAC-3') and sense primer Dys In22 S-01 (5'-CACTATGATTAAATGCTTGATATTGAG-3') (Supplemental Fig. 1). Reactions (50 µl) consisted of 100 ng of gDNA, 0.4 µM each primer, 0.2 µM each dNTP, 3.5 mM Mg(OAc)2, and 1.25 units of KlenTaq LA DNA polymerase in buffer supplied by the manufacturer (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). The reactions were subjected to 29 cycles of 92¡ãC for 30 seconds, 62¡ãC for 2 minutes, with an initial cycle of 92¡ãC for 2 minutes and 65¡ãC for 2 minutes on a Sprint PCR thermocyler (Eppendorf South Pacific, North Ryde, Australia).8 S3 t+ {! ]" c; \2 r$ Q& U
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Allele-specific polymerase chain reaction (AS-PCR) for the detection of wt DMD gene was performed with antisense primer Dys-wt AS-01 (3' mismatch for the mdx nucleotide) and sense primer Dys In22 S-02, whereas wt DMD transcript was detected with antisense primer Dys wt AS-01 and sense primer C2917-S (supplemental online Fig. 1) as previously described .4 U& V9 }2 ]5 _8 |0 N

Dystrophin is exclusively expressed in mature terminally myodifferentiated multinucleated fibers. Detection of wt DMD transcript arising from donor cell nuclei is, therefore, a good indicator of their myoremodeling capacity. The significance of wt DMD transcript expression (or absence) in mdx muscles bearing wt DMD loci was assessed by 2 analysis (significance at p
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BMCs Contribute to Skeletal Myofibers

To assess the ability of BMCs to remodel muscle, recipient mdx mice each received i.m. injections of 3.0 x 106 WBM from C57Bl/10 wt donors. At 6 weeks post-transplant, their TAs were evaluated for the presence of dystrophin  myofibers. Dystrophin expression was observed in its typical sarcolemmal distribution around the periphery of myofibers (Fig. 1A, 1B). The morphology of dystrophin  myofibers was identical to the surrounding dystrophin¨C myofibers. In WBM-injected TAs, clusters of 2¨C5 myofibers of varying size and shape, scattered about the injection site, showed strong sarcolemmal expression of dystrophin (Fig. 1B). These dystrophin  fibers were interspersed with typical mdx myofibers lacking this intense sarcolemmal staining. Dystrophin  myofibers amounted to an average of 1.13% ¡À 0.13% of the total number of myofibers, whereas 0.74% ¡À 0.07% myofibers in saline-injected control TAs were found to express dystrophin (Fig. 1C). These revertant fibers arise from post-transcriptional splicing or second mutation events that bypass the mutation in the DMD gene and restore an open reading frame .$ m! N* G7 B7 h5 {$ n

Figure 1. Dystrophin expression in WBM-injected mdx muscle. Cryosections taken from mdx muscle were immunochemically stained to reveal dystrophin expression after i.m. injection of (A) saline and (B) WBM. (C): Graph of dystrophin  myofibers expressed as a percentage of total myofiber number in the tibialis anterior after i.m. injection of saline or WBM. Revertant fibers (arrows) occurred at a mean total number of 20 per muscle, representing a relative percentage of approximately 0.7% (determined from saline controls, ). Values are mean ¡À SEM. Abbreviations: WBM, whole bone marrow. *, Mann-Whitney p 4 u5 S6 ?' G: l5 n

Sca-1  Cells from BM or Muscle Sorted Solely on Sca-1 Expression Do Not Have a Myoremodeling Advantage Over the Sca-1¨C Subpopulations: X3 ?0 Y! `( g+ g& _8 D
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To determine whether and to what extent BMCs capable of remodeling muscle are characterized by the expression of Sca-1, WBM was sorted exclusively on the basis of Sca-1 expression. Unfractionated WBM, Sca-1-positive (Sca  ) and Sca-1-negative (Sca¨C) BMC populations (Fig. 2A, 2B) were injected into the TA muscle of mdx mice. If Sca-1 expression characterizes a subset of BMCs with optimal myoremodeling capacity, then the Sca-1 status of muscle-derived SCs may also coincide with their ability to remodel muscle. Therefore, whole muscle was similarly sorted on Sca-1 expression and unfractionated whole muscle, Sca  and Sca¨C muscle fractions were transplanted into the TA muscle of mdx mice. The Sca-1 profiles of muscle and WBM are shown in Figure 2.
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Figure 2. Sca-sorted muscle and bone marrow (BM). Sca¨C and Sca  BM cells were pre-enriched by immunomagnetic selection using biotinylated anti-Sca-1-FITC conjugated antibody and goat anti-rat microbeads. Sca¨C (A) and Sca  (B) cells were further purified by fluorescence-activated cell sorting (FACS) by setting the sort gates as shown with reference to the fluorescence profile of unlabeled BM cells (open histogram). (C): Sca¨C and Sca  muscle cells were purified by FACS by setting sort gates with reference to the histogram profile of unlabeled muscle cells (open histogram). Sorted (D) Sca¨C and (E) Sca  muscle cells were reanalyzed to establish the purity and resolution of target cell populations. Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin.
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Whole muscle was significantly more efficient (p ! O. `5 N( ~. u8 r# ~0 _

Figure 3. Efficiency of dystrophin restoration in mdx tibialis anterior (TA) by Sca-sorted muscle and BM at 6 weeks after i.m. injection. Injected were 3 x 105 of whole muscle and its Sca-sorted fractions, and 1 x 106 of WBM and it Sca-sorted fractions. (A): Graph of RMI, comparing the capacity of each cell fraction to remodel dystrophic mdx TA muscle relative to whole muscle (values are mean ¡À SEM). Cryosections taken from muscle after transplantation and immunochemically stained to reveal dystrophin expression after injection of (B) saline (control), (C) WBM, (D) whole muscle, (E) Sca  muscle and (F) Sca¨C muscle are shown. Dystrophin expression was also observed in the Sca  and Sca¨C BM fractions (data not shown here). Abbreviations: BM, bone marrow; RMI, Relative Myoremodeling Index; WBM, whole BM. *, p

Isolation and Characterization/Enrichment of BMC Populations within WBM
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Because of the lack of distinction in myoremodeling capacity observed in i.m.-injected Sca  and Sca¨C BMC fractions, WBM was sorted into various cellular fractions to further purify and enrich for cell populations that may be more efficient at myoremodeling.5 E9 V$ H; T" L3 D

Low-density BM cells were first depleted of mature hemopoietic cells using antibodies directed against hemopoietic lineage antigens, and the lineage antigen negative (Lin¨C) fraction was further fractionated into CD45  cells (enriched for HSCs) . The Lin¨C cell fraction comprised approximately 50% CD45  and 50% CD45¨C cells. The nonhemopoietic CD45¨CLin¨C fraction was then sorted into Sca¨CKit¨C, Sca¨CKit , Sca Kit¨C and Sca Kit  BMCs, each fraction representing approximately 85%, 12%, 1.5%, and 1.5%, respectively, of the entire CD45¨C BMC population. These cell fractions will be termed Sca¨CKit¨C, Sca¨CKit , Sca Kit¨C and Sca Kit  from this point forward. A schematic of the cellular fractions of BM that were sorted for and injected into mdx TAs is shown in Figure 4A, and a summary of the percentages of each cell fraction found within WBM is provided in Table 1. Sca Kit¨C and Sca Kit  BMCs are very rare, with each fraction representing only 1 in 3,000 WBM (Table 1). Representative FACS dotplots are shown in Figure 4B¨C4D.5 y( E. ?8 G1 L6 N: Q

Figure 4. Schematic of the purified bone marrow (BM) cellular fractions. (A): The BM fractions shaded in gray were fluorescence-activated cell sorted and injected (i.m.) into the right tibialis anteriors of dystrophic mdx mice. Lin¨C BM cells were labeled with CD45-R670, Sca-1-FITC and c-Kit-PE. CD45¨C cells (region M1, ). Abbreviations: FITC, fluorescein isothiocyanate; PE, phycoerythrin; WBM, whole bone marrow.9 {2 W- \6 z9 g& R, m/ {6 v9 [: y

Myoremodeling Capacity in BMC Subpopulations That Express c-Kit
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To investigate which BMC fraction possessed the highest myoremodeling capacity, we injected the various FAC-sorted BMCs into dystrophic mdx TAs and harvested them after 6 weeks. BMC contribution to skeletal muscle was assessed by dystrophin immunofluorescence.

As mentioned previously herein, the Sca Kit¨C and Sca Kit  cells are extremely rare cell populations within WBM. All of the cells acquired in each FACS fraction were used for transplantation. As a result, fewer cells from the rarer subpopulations were transplanted into recipient mdx mice. To account for the disparate cell numbers injected, an RMI was utilized for comparison of the BMC fractions  did not contribute significantly to the myoremodeling process (Fig. 5F). The number of dystrophin  myofibers observed in TAs injected with each of the sorted BMC fractions is summarized in Table 1.

Figure 5. Dystrophin expression in BMC-injected mdx muscle. Cryosections taken from mdx muscle immunochemically stained to reveal dystrophin expression after injection of (A) saline, (B) WBM, (C) CD45¨C, (D) Sca¨CKit  and, (E) Sca Kit  BMCs are shown. Arrows in (A) indicate revertant fibers in saline-injected mdx muscle. (F) Graph of RMI, comparing restoration of dystrophin by the various BMC fractions evaluated relative to WBM (values are mean ¡À SEM). Abbreviations: BMC, bone marrow-derived cells; RMI, Relative Myoremodeling Index; WBM, whole bone marrow. *, p - j' F/ A4 g! `. g

AS-PCR detected wt DMD gene in all four BMC fractions sorted for Sca-1 and c-Kit (Fig. 6A). wt DMD transcript was consistently detectable in all TAs injected with Sca¨CKit  and Sca Kit  BMC fractions. However, this was not so in those injected with Sca¨CKit¨C and Sca Kit¨C BMC fractions (Fig. 6A), supporting the likelihood that cells expressing c-Kit are attributed with a higher myoremodeling capacity than cells not expressing c-Kit (2 = 8.00; p 5 a! W7 H1 O, H8 q
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Figure 6. Detection of wt DMD gene and transcript using AS-polymerase chain reaction (PCR) and AS-reverse transcriptase (rt) PCR. (A): CD45¨CLin¨C BMC fractions from C57Bl/10 (wt) mice were fluorescence-activated cell-sorted using c-Kit and Sca-1 selective markers and injected into the tibialis anterior (TA) muscles of C57Bl/10DMD/mdx mice in the groups shown. With the exception of mouse 8 (Sca¨CKit¨C) and mouse 13 (Sca Kit¨C), all of the mdx mouse TAs injected with bone marrow-derived cell (BMC) fractions showed detectable levels of both wt DMD gene and transcript. Absence of wt DMD gene or transcript (Saline group) in saline-injected mdx muscle confirmed that all wt bands observed via AS-PCR or AS-rtPCR were attributed to donor BMC nucleic acid species. An 803-bp primary rtPCR amplicon (PRIMARY), shown at the bottom of the AS-rtPCR panel confirms that the mice (8 and 13, both injected with Kit¨C cells) in which no wt DMD transcript was detected were in fact expressing DMD transcript, but only from mdx gene loci. The presence of donor (wt) DMD gene loci in the muscle DNA of these two mice in turn indicated that although engrafted, cells containing these loci were not capable of contributing to the myoremodeling of the dystrophic mdx muscle, and consequently, expression of wt dystrophin. (B): The possibility that c-Kit could be used as a selectable marker was then evaluated by 2 analysis to 1 degree of freedom using null hypotheses (H0) as shown. The analyses were made with the assumption that if myoremodeling capacity was not selectable by c-Kit (presence or absence), then this would lead to an equal number of cases in which wt DMD transcript was present when the cells engrafted into the recipient mdx muscle. The resulting 2 statistic generated by H0 suggesting that c-Kit did not cofractionate with myoremodeling capacity, was not able to be retained (p ( R0 @$ `+ S) i" B3 r, ?1 B* q

DISCUSSION5 ]7 O% H- ?8 A3 r! R+ m' g

Using a systematic WBM FAC-sorting protocol incorporating lineage markers CD45, Sca-1, and c-Kit, this study showed that the Lin¨CCD45¨CSca¨CKit  (Sca¨CKit ) and Lin¨CCD45¨C Sca Kit  (Sca Kit ) cell fractions demonstrated the highest myoremodeling capacity when injected into the dystrophic muscle of mdx mice. Three independent lines of evidence (DNA, RNA, and dystrophin protein) were used to generate statistically significant cross-referential data that our injected cell fractions functionally remodeled dystrophic mdx muscle. These results build on several studies, including our own earlier findings indicating that hemopoietic (CD45 ) cell contribution to in vivo myogenesis is rare . Collectively, these observations give rise to the possibility that cells with efficient myoremodeling capacity may be enriched by FAC-sorting from the mesenchymal nonhemopoietic (CD45¨CLin¨C) BMC fraction using "rare" stem cell surface antigen markers such as c-Kit.1 w: H( p3 j# O3 V' I

A Lin¨C, CD45¨C cell surface antigen phenotype in adherent BM stromal cells has generally been accepted as defining a population rich in MSCs .$ L9 F. y3 M  i/ X, u6 O* r/ {
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After the initial observation of in vivo muscle remodeling by BMCs .
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Muscle cells displayed a significantly greater myoremodeling potential compared to corresponding BMC fractions (Fig. 3A). On investigating the relative myoremodeling capacity of Sca  and Sca¨C cell types, we observed no significant differences between transplanted Sca  and Sca¨C muscle cells (Fig. 3A, 3E, 3F) or BMCs (Fig. 3A). This may result from the fact that the Sca  population of BMCs contains within it CD45 Lin  cells that may influence the myoremodeling capacity of promyoremodeling cells in the Sca  fraction. Further subfractionation of the BMCs revealed that the Sca¨CKit  and Sca Kit  cells accounted for most of the myoremodeling activity of BMCs observed in this study (Figs. 5F, 6). This indicates an important role for c-Kit as a selectable marker for myoremodeling capacity in BMCs, particularly in conjunction with other selectable markers such as Sca-1.
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Our results build on findings that cells with myoremodeling potential can be isolated using Sca-1 cell surface antigen .

A number of factors potentially apply to our findings regarding Sca-1's contribution to the identification of cells with effective dystrophic mdx muscle myoremodeling capacity. Firstly, Sca-1 cell surface expression is dynamically modulated by the prevailing microenvironment . Thus, Sca  cells isolated from any given tissue/organ, may contain Sca  cells of different lineage(s) in addition to those of source tissue origin, which may in turn affect the net myoremodeling activity of the population.

Muscle progenitors are able to home to injured muscle when injected systemically . However, the myoremodeling potential demonstrated by the CD45¨C, Kit  BMC fractions in this study (Fig. 6; 2 p   A& }; M$ ^9 [0 B8 r# v) ^
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Myoremodeling in this study was achieved by all injected C57BL10/J wt BMCs with the exception of mature hemopoietic (Lin ) cells (Fig. 5F). From this perspective, our results promote c-Kit as a useful selectable FACS marker that complements existing cell isolation approaches for identifying cells with myoremodeling capacity from BM and possibly from other nonmuscle tissues. Nevertheless, it remains to be seen if these myogenic Sca¨CKit  or Sca Kit  BMCs can overcome the ceiling effect commonly observed with cell transplantation in dystrophic mdx muscle , the generally small myoremodeling increments achieved via these avenues suggests as yet unidentified, but powerful antagonistic mechanisms limiting muscle remodeling by injection of myogenic and nonmyogenic cells alike.
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Subfractionation of MSCs to identify cells enriched for myoremodeling potential from adult tissues is a promising step toward understanding and possibly developing the therapeutic potential of these cells in muscle cell replacement therapies. However, the existence of these cells as extremely low fractions of the BMC population gives rise to significant challenges for the development of their therapeutic potential. Further work is required to better understand and define the growth conditions by which these cells' myoremodeling characteristics are maximally retained  in addition to physical barriers (e.g., extracellular matrix) within the muscle microenvironment.! \3 k& W' }: }$ N4 J4 u* G: L; f) ~

CONCLUSION7 y* c, F) A" c7 @& b$ T

Cells derived from BM displaying a MSC/stromal Lin¨CCD45¨CSca¨CKit  and Lin¨CCD45¨CSca Kit  cell marker profile were shown to possess a potent myoremodeling potential, defined by restoration of wt dystrophin protein and transcript expression in dystrophic mdx mouse muscle. Future research aimed at the optimization, expansion, and in vivo delivery of these cells will more clearly identify their potential application as therapeutic agents for neuromuscular disorders.% b8 q$ n' E* E4 I1 h: ]
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors indicate no potential conflicts of interest.. u& h8 M, X: T' }6 w: j0 k
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ACKNOWLEDGMENTS
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We thank the Muscular Dystrophy Association (U.S.), National Health and Medical Research Council (Australia), Aktion Benni, and Muscular Dystrophy Australia for their support in funding the work communicated in this article; and Stephania Tombs, Kelly Steeper, and Judy Chin for their excellent technical support. This study was funded in part by the National Health and Medical Research Council of Australia, Muscular Dystrophy Association (Australia), Muscular Dystrophy Association (U.S.), and Aktion Benni and Co.- D# e! Y( |/ |- G$ X' w* w
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