Two Different Roles of Purified CD45+c-Kit+Sca-1+Lin– Cells After Transplantation in Muscles
http://www.100md.com
《干细胞学杂志》
a Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan;
b Department of Biological Science, Graduate School of Sciences, University of Tokyo, Tokyo, Japan
Key Words. Hematopoietic stem cells ? Transplantation ? c-Kit+Sca-1+Lin– ? Muscle stem cells
Correspondence: Tatsutoshi Nakahata, M.D., Ph.D., Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: 81-75-751-3290; Fax: 81-75-752-2361; e-mail: tnakaha@kuhp.kyoto-u.ac.jp
ABSTRACT
Various tissue-specific stem cells have been identified in epidermis , intestinal epithelium , testis , liver , brain , and muscle . Until recently, it was thought that tissue-specific stem cells could only differentiate into their original tissue, but it has been demonstrated that they can also differentiate into other lineages. For example, cells of donor origin have been detected in liver, heart, vascular endothelium, skeletal muscles, and other organs after bone marrow (BM) transplantation . Of special interest for our study is that BM-derived cells have been shown to participate in the regeneration of chemically damaged fibers in skeletal muscle . Subsequent studies showed that dystrophin-positive myofibers were restored in mdx mice, an animal model of Duchenne’s muscular dystrophy, after transplantation of stem cells purified by fluorescence-activated cell sorting with Hoechst 33342 low-stained cells, also known as side population (SP) cells . In short, stem cells in BM, comprising hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), have been found to be capable of regenerating damaged muscle fibers after BM transplantation.
It has also been demonstrated, however, that BM cells can adopt the phenotype of other cells by means of cell fusion . These investigators warned that in vivo "transdifferentiation" might result from cell fusion. The differentiation potential of BM cells beyond the lineage restriction of stem cells remains to be determined.
In addition to BM cells, SP cells have also been identified in muscle tissues . These muscle SP cells are reported to have hematopoietic as well as myogenic potential and to express CD45 antigen, which is recognized as a hematopoietic cell marker. Another study gave evidence that CD45+ cells in skeletal muscle are of BM origin . These reports thus indicate that hematopoietic cells of BM origin seem to be present in skeletal muscles. However, the correlations among HSCs, CD45+ cells in skeletal muscle, satellite cells, myogenic precursors, and muscle-derived stem cells have not yet been determined. It is important to clarify these relationships, both for scientific research and for the application of stem cell therapy.
To investigate the potential and the kinetics of HSCs in skeletal muscles, we transplanted c-Kit+Sca-1+Lin– (KSL) cells as enriched HSC fraction from green fluorescent protein (GFP) transgenic mice into lethally irradiated C57BL/6 mice or nonirradiated W/Wv neonates that can accept HSCs without myeloablation. We examined the time-course behavior of GFP+ cells in recipient muscles with a fluorescent stereo microscope and immunohistochemical staining during the early and late phases after transplantation. Our visualization system makes it possible to detect transplanted cells with GFP signals in intact organs without the need to make sections first and can easily trace their kinetics throughout the entire body . With this system, we found that myeloablation enables KSL cells to migrate into damaged muscles and to regenerate muscle fibers in the whole body during the early phase following transplantation. In the late phase, progenies of KSL cells had remained in muscle tissues and gave rise to satellite cells with myogenic potential, regardless of whether the mice had been irradiated.
MATERIALS AND METHODS
KSL Cells Can Repair Muscle Damage in Early Phase After Transplantation
To investigate the process of settlement of donor hematopoietic cells into skeletal muscles, HSC fraction, KSL cells , derived from BM of GFP-transgenic mice, were transplanted into lethally irradiated adult mice by tail vein injection. The entire KSL cell fraction was hematopoietic and expressed CD45 (Fig. 1). The purity of KSL cells after sorting was 98%. To exclude the possibility of contamination of mesenchymal cells, we cultured 1 to 5 x 103 per well of KSL cells with Mesen Cult medium for 14 days. No adherent cells were observed (data not shown).
Figure 1. Purification of KSL cells. After collecting Lin– cells by auto-MACS, KSL cells were sorted by FACS Vantage. (A): Lin– gating. (B): Sorting gate for KSL cells. (C): All KSL gated cells express CD45. (D): The purity of KSL cells after sorting was 98%. Abbreviation: FSC, forward scatter.
After transplantation, the muscles in the entire body were examined, first with a fluorescent stereomicroscope and then with immunohistochemical staining, to detect the GFP signals. On day 3 after transplantation, no GFP signals could be detected in any muscles. On days 10 and 20, powerful GFP signals in the muscle fibers throughout the body were observed under a fluorescent stereomicroscope such as in the intercostal, thigh, abdominal, greater pectoral, and external ocular muscles (Fig. 2A). Transverse sections showed muscle fibers of variable sizes with centrally localized nuclei, which represents the regenerative status (Fig. 2B). Among these muscle fibers, GFP+ region–like muscle fibers were detected (Fig. 2B, b, arrowheads; faintly red region). There were mainly two types of GFP+ regions. One region consisted of GFP+ fibers (Fig. 3B), which were confirmed by typical cross-striations (Fig. 3A) and by immunostaining with anti-myosin antibody (Fig. 3C, D). And the other consisted of GFP+ mononuclear cells (Fig. 3E), most of which were not stained with anti-myosin (Fig. 3G, H) but stained with anti-CD45 antibody (Fig. 3F, H). In the region of GFP+ fibers, myogenin+ myoblasts or myotubes were detected around the fibers (Fig.4D, E, arrowheads). Interestingly, CD45low myogenin+ GFP+ cells were also detected (Fig. 4B–E, arrows) but only very few in number. This replacement by GFP+ fibers could be observed only from day 10 to day 20 and had dramatically decreased on day 30 (Fig. 2A, g–i). FISH analysis revealed a small number of GFP-DNAs in myonuclei (data not shown). These results indicated that the irradiation for myeloablation evoked muscle injury and that KSL cells engrafted in damaged muscles, fusing the host’s muscle fibers, and participated in muscle regeneration in the early phase of transplantation.
Figure 2. (A): Visualization of transplanted GFP+ cells in muscles on days 10, 20, and 30. Appearances of various muscles under a fluorescent stereomicroscope are shown. Muscle tissues were removed from recipient mice and observed under a fluorescent stereomicroscope on days 10, 20, and 30 after transplantation. a, d, g: Intercostal muscle (the white lines indicate the shape of ribs). b: Abdominal muscle. c: Thigh muscle. e: Greater pectoral muscle. f: External ocular muscle (the white line indicates the shape of the eyeball). h: Dorsal muscle. Bars: a–c, e, 2 mm; d, 500 μm; f–g, i, 1 mm; h, 200 μm. (B): Immunohistostaining of the section of muscle tissues on day 10. a: Negative control. b: The section was stained with anti-GFP antibodies (alkaline phosphatase, faintly red region; arrowheads), and hematoxylin was used for nuclear staining.
Figure 3. GFP+ regions in intercostal muscle on day 20. Intercostal muscles were removed from recipient mice and fixed with 4% paraformaldehyde and made into frozen sections, as described in Materials and Methods. There were two types of GFP regions: (A–D) GFP+ fibers and (E–H) GFP+ mononuclear cells. Each section was stained with anti-GFP antibody (B, D, E, H, green), anti-myosin antibody (C, D, Cy3 red; G, H, Alexa350 blue), and anti-CD45 antibody (F, H, Cy3 red). (A): Phase contrast (D, H): Merge. Bars: D, 10 μm; H, 20 μm.
Figure 4. Myogenic phenotype of a CD45+ cell in muscle tissues on day 20. (A, B): GFP+ muscle structure showed cross striations. These sections were stained with anti-CD45 antibody (C, Cy3 red) and anti-myogenin antibody (D, blue). (E): Merge. Myogenin+ myoblast- or myotube-like cells were detected (D, E, arrowhead), and an elongated GFP+ cell coexpressed myogenin and CD45 (B–E, arrow).
KSL Cells Settle in Muscle Tissue Like a Satellite Cell
Sections of the muscle tissues obtained 30 days and 6 months after transplantation were stained with anti-GFP and anti-laminin (a marker of the basal lamina) antibodies, showing that several GFP+ cells were located inside the basal lamina with laminin expression (Fig. 5A), which is where satellite cells are usually found. GFP+ cells under the basal lamina were coexpressed with c-Met antigen (a marker of satellite cells) (Fig. 5E, arrows). Furthermore, we isolated single fibers from soleus muscles 2 months after transplantation and stained them with anti-PAX7, MyoD, or Myf5, which are specific satellite cell markers. We detected Myf5+ GFP+ satellite cells on the fibers (Fig. 6A–D), but neither PAX7+ GFP+ cells nor MyoD+ GFP+ cells (data not shown). These results suggested that GFP+ KSL cells migrate into muscle tissues, with some of them localizing beneath the basal lamina expressing satellite cell markers.
Figure 5. GFP+ cells localized like satellite cells long term after transplantation. Sections from 6 months after transplantation were stained with laminin (A, Cy3 red; D, E, Alexa350 blue) and c-Met (C, E, Cy3 red) GFP+ cells localized under the basal lamina (A, E, arrows) and were costained with c-Met (E, arrows). Bar: 20 μm.
Figure 6. Immunostaining of single fibers. Single fibers were isolated from (A–D) irradiated mice 2 months after transplantation and from (E–P) W/Wv mice 1 or 2 months after transplantation and were stained with satellite cell–specific markers. (A, E, I, M): Anti-GFP antibody (FITC green, arrowheads). (B, N): Anti-Myf5 antibody (Cy3 red, arrowheads). (F): Anti-MyoD1 antibody (Cy3 red, arrowhead). (J): Anti-PAX7 antibody (Cy3 red, arrowhead). (D, H, L, P): Merge. Bars: D, P, 50 μm; H, L, 100 μm.
KSL Cells Can Settle in Muscle Tissues Without Muscle Damage
Tissue-specific stem cells occupy niches—microenvironments that maintain self-renewal activity and multipotency of stem cells. Since it is known that irradiation depletes endogenous satellite cells and that injected muscle precursors can replace them , we used lethal irradiation for the transplantation assay. However, lethal irradiation also evokes various responses in the body. To exclude the influence of irradiation damage, we next transplanted KSL cells into W/Wv neonates. W/Wv mice possess a c-Kit gene mutation and can accept transplanted HSCs in a BM niche without irradiation . Moreover, transplantation into W/Wv neonates results in a higher chimeric ratio than does transplantation into adults . Our experiments using transplantation into W/Wv neonates showed no evidence of GFP+ muscle fibers at any time, whereas BM cells were almost entirely replaced. However, small GFP+ mononuclear cells could be detected between muscle fibers as early as 30 days after transplantation (Fig. 7A). Some GFP+ cells were also detected beneath the laminin-positive basement membrane (Fig. 7B), and some of them were also stained with anti-c-Met antibody (Fig. 7C). We also examined isolated single fibers 1 month after transplantation and detected MyoD+ GFP+ cells and PAX7+ GFP+ cells on the fibers (Fig. 6H, L). We could also detect Myf5+ GFP+ cells on the fibers 2 months after transplantation (Fig. 6P). These results showed that KSL cells and/or their progenies could migrate into undamaged muscle tissues expressing satellite cell–specific markers also, and that a suitable microenvironment for them might exist in skeletal muscles.
Figure 7. Analysis of the potential of GFP+ satellite-like cells in muscle 6 months after transplantation. Thirty days and 6 months after nonirradiated transplantation into W/Wv neonates, GFP+ mononuclear cells were detected between muscle fibers under fluorescent stereomicroscope (A, arrows). In the sections, immunostaining for laminin and c-Met was performed (B, C). (A): Appearance of rib and rib muscle under fluorescent stereomicroscope on day 30. Some GFP+ mononuclear cells were detected (arrows). The white lines indicate the shape of ribs. (B): Immunohistochemistry with anti-laminin (Cy3 red) and anti-GFP antibodies. A confocal microscope was used to determine the precise location of GFP+ cells. GFP+ cells beneath basal lamina (arrow) were detected on day 30 and 6 months later. (C): GFP+ cell under laminin-positive basal lamina (Alexa350 blue) was also stained with anti c-Met antibody (Cy3 red, arrow). (D–F): Single-fiber culture was performed with single fibers isolated from recipient mice 6 months after transplantation, followed by immunostaining. C, negative control; D, anti-myosin (Cy3 red) staining; E, anti-GFP-ALP (red) staining. D and E were same position. GFP-ALP+ fibers were confirmed to be muscle fibers using myosin Cy3 staining. (G): PCR analysis of extracted DNA from single-fiber culture. DNA was extracted from the single-fiber culture followed by PCR for GFP DNA, as described under Materials and Methods. 1: negative control; 2: peripheral blood of GFP transgenic mouse; 3: single fiber culture. Bars: A, 1 mm; B, C, 20 μm; D, 200 μm; E, F, 50 μm.
KSL-Derived Cells in Muscle Can Generate Muscle Fibers In Vitro in Long Term After Transplantation
So far it has been reported that damaged muscles by chemical agent or stress were regenerated by donor cells after BM transplantation . However, these muscle-regeneration assays in vivo cannot clarify whether donor cells participate in regenerating muscles directly from the settled muscle or indirectly from settled BM. To determine whether GFP+ mononuclear cells in muscle tissues can differentiate into muscle fibers, a single-fiber culture was performed. Six months after transplantation with KSL cells, muscle fibers were isolated from the hind limb muscles of W/Wv mice or lethally irradiated mice transplanted, and single fibers were cultured onto matrigel-coated plates. After 14 days, most of the satellite cells had migrated from the fibers, proliferated, and formed new muscle fibers, which were confirmed to be myosin-positive by immunohistochemical staining (Fig. 7D, E). Since the GFP signal in vitro was too weak to be detected by fluorescent microscope, we determined the presence of GFP+ cells by means of immunohistochemical staining using the anti-GFP antibody or by GFP DNA amplification with the aid of PCR. Staining with the anti-GFP antibody proved the presence of GFP+ myofibers (Fig. 7F), which were also myosin-positive. In addition, GFP DNA was also confirmed by PCR to be present in samples extracted from culture dishes of single-muscle fiber (Fig. 7G). No GFP+ fibers were generated in the single-fiber culture in the early phase of transplantation in irradiated mice (data not shown). In brief, GFP+ KSL cell that engrafted muscle tissues could repair damaged muscle but not produce muscle fibers in vitro in the early phase. In the long term, however, they acquired the potential to differentiate into muscle fibers like satellite cells also in vitro. Since the single-fiber culture is a functional assay of the presence of satellite cells, it can be said that GFP+ KSL cells can give rise to satellite cells with myogenic potential in the long term after transplantation.
DISCUSSION
This study was supported by Health and Labour Science Research Grant, Research on Human Genome, Tissue Engineering Food Biotechnology, Ministry of Health, Labour and Welfare, Tokyo; by Grant-in-Aid for Science Research on Priority Areas no. 122150-67; and by Grant-in-Aid for Creative Scientific Research no. 13GS-0009. This study was also supported by Research Grant no. 13B-1 for Nervous and Mental Disorders and no. H13-iyaku-043, both from the Ministry of Health, Labour and Welfare.
REFERENCES
Taylor G, Lehrer MS, Jensen PJ et al. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 2000;102:451–461.
Mills JC, Gordon JI. The intestinal stem cell niche: there grows the neighborhood. Proc Natl Acad Sci U S A 2001;98:12334–12336.
Shinohara T, Orwig KE, Avarbock MR et al. Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. Proc Natl Acad Sci U S A 2001;98:6186–6191.
Suzuki A, Zheng Yw YW, Kaneko S et al. Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver. J Cell Biol 2002;156:173–184.
Rietze RL, Valcanis H, Brooker GF et al. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 2001;412:736–739.
Qu-Petersen Z, Deasy B, Jankowski R et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002;157:851–864.
Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168–1170.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Brazelton TR, Rossi FM, Keshet GI et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:1775–1779.
Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782.
Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.
Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528–1530.
Bittner RE, Schofer C, Weipoltshammer K et al. Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anat Embryol (Berl) 1999;199:391–396.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
Gussoni E, Soneoka Y, Strickland CD et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999;401:390–394.
Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545.
Ying QL, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.
Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci U S A 1999;96:14482–14486.
Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123–134.
Kawada H, Ogawa M. Bone marrow origin of hematopoietic progenitors and stem cells in murine muscle. Blood 2001;98:2008–2013.
Yoshimoto M, Shinohara T, Heike T et al. Direct visualization of reconstitution process of transplanted hematopoietic cells in intact mouse organs indicates the presence of a niche. Exp Hematol 2003;31:733–740.
Zammit PS, Golding JP, Nagata Y et al. Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 2004;166:347–357.
Kawakami A, Kimura-Kawakami M, Nomura T et al. Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development. Mech Dev 1997;66:119–130.
Rosenblatt JD, Lunt AI, Parry DJ et al. Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 1995;31:773–779.
Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34–low/negative hematopoietic stem cell. Science 1996;273:242–245.
Blaveri K, Heslop L, Yu DS et al. Patterns of repair of dystrophic mouse muscle: studies on isolated fibers. Dev Dyn 1999;216:244–256.
Mintz B, Anthony K, Litwin S. Monoclonal derivation of mouse myeloid and lymphoid lineages from totipotent hematopoietic stem cells experimentally engrafted in fetal hosts. Proc Natl Acad Sci U S A 1984;81:7835–7839.
Capel B, Mintz B. Neonatal W-mutant mice are favorable hosts for tracking development of marked hematopoietic stem cells. Exp Hematol 1989;17:872–876.
Fukada S, Miyagoe-Suzuki Y, Tsukihara H et al. Muscle regeneration by reconstitution with bone marrow or fetal liver cells from green fluorescent protein-gene transgenic mice. J Cell Sci 2002;115:1285–1293.
LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002;111:589–601.
Camargo FD, Green R, Capetenaki Y et al. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003;9:1520–1527.
Corbel SY, Lee A, Yi L et al. Contribution of hematopoietic stem cells to skeletal muscle. Nat Med 2003;9:1528–1532.
McKinney-Freeman SL, Jackson KA, Camargo FD et al. Muscle-derived hematopoietic stem cells are hematopoietic in origin. Proc Natl Acad Sci U S A 2002;99:1341–1346.
Heslop L, Morgan JE, Partridge TA. Evidence for a myogenic stem cell that is exhausted in dystrophic muscle. J Cell Sci 2000;113( Pt 12):2299–2308.
Schweitzer KM, Drager AM, van der Valk P et al. Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues. Am J Pathol 1996;148:165–175.
Jacobsen K, Kravitz J, Kincade PW et al. Adhesion receptors on bone marrow stromal cells: in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice. Blood 1996;87:73–82.
Williams DA, Rios M, Stephens C et al. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991;352:438–441.(Momoko Yoshimotoa, Hsi Ch)
b Department of Biological Science, Graduate School of Sciences, University of Tokyo, Tokyo, Japan
Key Words. Hematopoietic stem cells ? Transplantation ? c-Kit+Sca-1+Lin– ? Muscle stem cells
Correspondence: Tatsutoshi Nakahata, M.D., Ph.D., Department of Pediatrics, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: 81-75-751-3290; Fax: 81-75-752-2361; e-mail: tnakaha@kuhp.kyoto-u.ac.jp
ABSTRACT
Various tissue-specific stem cells have been identified in epidermis , intestinal epithelium , testis , liver , brain , and muscle . Until recently, it was thought that tissue-specific stem cells could only differentiate into their original tissue, but it has been demonstrated that they can also differentiate into other lineages. For example, cells of donor origin have been detected in liver, heart, vascular endothelium, skeletal muscles, and other organs after bone marrow (BM) transplantation . Of special interest for our study is that BM-derived cells have been shown to participate in the regeneration of chemically damaged fibers in skeletal muscle . Subsequent studies showed that dystrophin-positive myofibers were restored in mdx mice, an animal model of Duchenne’s muscular dystrophy, after transplantation of stem cells purified by fluorescence-activated cell sorting with Hoechst 33342 low-stained cells, also known as side population (SP) cells . In short, stem cells in BM, comprising hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), have been found to be capable of regenerating damaged muscle fibers after BM transplantation.
It has also been demonstrated, however, that BM cells can adopt the phenotype of other cells by means of cell fusion . These investigators warned that in vivo "transdifferentiation" might result from cell fusion. The differentiation potential of BM cells beyond the lineage restriction of stem cells remains to be determined.
In addition to BM cells, SP cells have also been identified in muscle tissues . These muscle SP cells are reported to have hematopoietic as well as myogenic potential and to express CD45 antigen, which is recognized as a hematopoietic cell marker. Another study gave evidence that CD45+ cells in skeletal muscle are of BM origin . These reports thus indicate that hematopoietic cells of BM origin seem to be present in skeletal muscles. However, the correlations among HSCs, CD45+ cells in skeletal muscle, satellite cells, myogenic precursors, and muscle-derived stem cells have not yet been determined. It is important to clarify these relationships, both for scientific research and for the application of stem cell therapy.
To investigate the potential and the kinetics of HSCs in skeletal muscles, we transplanted c-Kit+Sca-1+Lin– (KSL) cells as enriched HSC fraction from green fluorescent protein (GFP) transgenic mice into lethally irradiated C57BL/6 mice or nonirradiated W/Wv neonates that can accept HSCs without myeloablation. We examined the time-course behavior of GFP+ cells in recipient muscles with a fluorescent stereo microscope and immunohistochemical staining during the early and late phases after transplantation. Our visualization system makes it possible to detect transplanted cells with GFP signals in intact organs without the need to make sections first and can easily trace their kinetics throughout the entire body . With this system, we found that myeloablation enables KSL cells to migrate into damaged muscles and to regenerate muscle fibers in the whole body during the early phase following transplantation. In the late phase, progenies of KSL cells had remained in muscle tissues and gave rise to satellite cells with myogenic potential, regardless of whether the mice had been irradiated.
MATERIALS AND METHODS
KSL Cells Can Repair Muscle Damage in Early Phase After Transplantation
To investigate the process of settlement of donor hematopoietic cells into skeletal muscles, HSC fraction, KSL cells , derived from BM of GFP-transgenic mice, were transplanted into lethally irradiated adult mice by tail vein injection. The entire KSL cell fraction was hematopoietic and expressed CD45 (Fig. 1). The purity of KSL cells after sorting was 98%. To exclude the possibility of contamination of mesenchymal cells, we cultured 1 to 5 x 103 per well of KSL cells with Mesen Cult medium for 14 days. No adherent cells were observed (data not shown).
Figure 1. Purification of KSL cells. After collecting Lin– cells by auto-MACS, KSL cells were sorted by FACS Vantage. (A): Lin– gating. (B): Sorting gate for KSL cells. (C): All KSL gated cells express CD45. (D): The purity of KSL cells after sorting was 98%. Abbreviation: FSC, forward scatter.
After transplantation, the muscles in the entire body were examined, first with a fluorescent stereomicroscope and then with immunohistochemical staining, to detect the GFP signals. On day 3 after transplantation, no GFP signals could be detected in any muscles. On days 10 and 20, powerful GFP signals in the muscle fibers throughout the body were observed under a fluorescent stereomicroscope such as in the intercostal, thigh, abdominal, greater pectoral, and external ocular muscles (Fig. 2A). Transverse sections showed muscle fibers of variable sizes with centrally localized nuclei, which represents the regenerative status (Fig. 2B). Among these muscle fibers, GFP+ region–like muscle fibers were detected (Fig. 2B, b, arrowheads; faintly red region). There were mainly two types of GFP+ regions. One region consisted of GFP+ fibers (Fig. 3B), which were confirmed by typical cross-striations (Fig. 3A) and by immunostaining with anti-myosin antibody (Fig. 3C, D). And the other consisted of GFP+ mononuclear cells (Fig. 3E), most of which were not stained with anti-myosin (Fig. 3G, H) but stained with anti-CD45 antibody (Fig. 3F, H). In the region of GFP+ fibers, myogenin+ myoblasts or myotubes were detected around the fibers (Fig.4D, E, arrowheads). Interestingly, CD45low myogenin+ GFP+ cells were also detected (Fig. 4B–E, arrows) but only very few in number. This replacement by GFP+ fibers could be observed only from day 10 to day 20 and had dramatically decreased on day 30 (Fig. 2A, g–i). FISH analysis revealed a small number of GFP-DNAs in myonuclei (data not shown). These results indicated that the irradiation for myeloablation evoked muscle injury and that KSL cells engrafted in damaged muscles, fusing the host’s muscle fibers, and participated in muscle regeneration in the early phase of transplantation.
Figure 2. (A): Visualization of transplanted GFP+ cells in muscles on days 10, 20, and 30. Appearances of various muscles under a fluorescent stereomicroscope are shown. Muscle tissues were removed from recipient mice and observed under a fluorescent stereomicroscope on days 10, 20, and 30 after transplantation. a, d, g: Intercostal muscle (the white lines indicate the shape of ribs). b: Abdominal muscle. c: Thigh muscle. e: Greater pectoral muscle. f: External ocular muscle (the white line indicates the shape of the eyeball). h: Dorsal muscle. Bars: a–c, e, 2 mm; d, 500 μm; f–g, i, 1 mm; h, 200 μm. (B): Immunohistostaining of the section of muscle tissues on day 10. a: Negative control. b: The section was stained with anti-GFP antibodies (alkaline phosphatase, faintly red region; arrowheads), and hematoxylin was used for nuclear staining.
Figure 3. GFP+ regions in intercostal muscle on day 20. Intercostal muscles were removed from recipient mice and fixed with 4% paraformaldehyde and made into frozen sections, as described in Materials and Methods. There were two types of GFP regions: (A–D) GFP+ fibers and (E–H) GFP+ mononuclear cells. Each section was stained with anti-GFP antibody (B, D, E, H, green), anti-myosin antibody (C, D, Cy3 red; G, H, Alexa350 blue), and anti-CD45 antibody (F, H, Cy3 red). (A): Phase contrast (D, H): Merge. Bars: D, 10 μm; H, 20 μm.
Figure 4. Myogenic phenotype of a CD45+ cell in muscle tissues on day 20. (A, B): GFP+ muscle structure showed cross striations. These sections were stained with anti-CD45 antibody (C, Cy3 red) and anti-myogenin antibody (D, blue). (E): Merge. Myogenin+ myoblast- or myotube-like cells were detected (D, E, arrowhead), and an elongated GFP+ cell coexpressed myogenin and CD45 (B–E, arrow).
KSL Cells Settle in Muscle Tissue Like a Satellite Cell
Sections of the muscle tissues obtained 30 days and 6 months after transplantation were stained with anti-GFP and anti-laminin (a marker of the basal lamina) antibodies, showing that several GFP+ cells were located inside the basal lamina with laminin expression (Fig. 5A), which is where satellite cells are usually found. GFP+ cells under the basal lamina were coexpressed with c-Met antigen (a marker of satellite cells) (Fig. 5E, arrows). Furthermore, we isolated single fibers from soleus muscles 2 months after transplantation and stained them with anti-PAX7, MyoD, or Myf5, which are specific satellite cell markers. We detected Myf5+ GFP+ satellite cells on the fibers (Fig. 6A–D), but neither PAX7+ GFP+ cells nor MyoD+ GFP+ cells (data not shown). These results suggested that GFP+ KSL cells migrate into muscle tissues, with some of them localizing beneath the basal lamina expressing satellite cell markers.
Figure 5. GFP+ cells localized like satellite cells long term after transplantation. Sections from 6 months after transplantation were stained with laminin (A, Cy3 red; D, E, Alexa350 blue) and c-Met (C, E, Cy3 red) GFP+ cells localized under the basal lamina (A, E, arrows) and were costained with c-Met (E, arrows). Bar: 20 μm.
Figure 6. Immunostaining of single fibers. Single fibers were isolated from (A–D) irradiated mice 2 months after transplantation and from (E–P) W/Wv mice 1 or 2 months after transplantation and were stained with satellite cell–specific markers. (A, E, I, M): Anti-GFP antibody (FITC green, arrowheads). (B, N): Anti-Myf5 antibody (Cy3 red, arrowheads). (F): Anti-MyoD1 antibody (Cy3 red, arrowhead). (J): Anti-PAX7 antibody (Cy3 red, arrowhead). (D, H, L, P): Merge. Bars: D, P, 50 μm; H, L, 100 μm.
KSL Cells Can Settle in Muscle Tissues Without Muscle Damage
Tissue-specific stem cells occupy niches—microenvironments that maintain self-renewal activity and multipotency of stem cells. Since it is known that irradiation depletes endogenous satellite cells and that injected muscle precursors can replace them , we used lethal irradiation for the transplantation assay. However, lethal irradiation also evokes various responses in the body. To exclude the influence of irradiation damage, we next transplanted KSL cells into W/Wv neonates. W/Wv mice possess a c-Kit gene mutation and can accept transplanted HSCs in a BM niche without irradiation . Moreover, transplantation into W/Wv neonates results in a higher chimeric ratio than does transplantation into adults . Our experiments using transplantation into W/Wv neonates showed no evidence of GFP+ muscle fibers at any time, whereas BM cells were almost entirely replaced. However, small GFP+ mononuclear cells could be detected between muscle fibers as early as 30 days after transplantation (Fig. 7A). Some GFP+ cells were also detected beneath the laminin-positive basement membrane (Fig. 7B), and some of them were also stained with anti-c-Met antibody (Fig. 7C). We also examined isolated single fibers 1 month after transplantation and detected MyoD+ GFP+ cells and PAX7+ GFP+ cells on the fibers (Fig. 6H, L). We could also detect Myf5+ GFP+ cells on the fibers 2 months after transplantation (Fig. 6P). These results showed that KSL cells and/or their progenies could migrate into undamaged muscle tissues expressing satellite cell–specific markers also, and that a suitable microenvironment for them might exist in skeletal muscles.
Figure 7. Analysis of the potential of GFP+ satellite-like cells in muscle 6 months after transplantation. Thirty days and 6 months after nonirradiated transplantation into W/Wv neonates, GFP+ mononuclear cells were detected between muscle fibers under fluorescent stereomicroscope (A, arrows). In the sections, immunostaining for laminin and c-Met was performed (B, C). (A): Appearance of rib and rib muscle under fluorescent stereomicroscope on day 30. Some GFP+ mononuclear cells were detected (arrows). The white lines indicate the shape of ribs. (B): Immunohistochemistry with anti-laminin (Cy3 red) and anti-GFP antibodies. A confocal microscope was used to determine the precise location of GFP+ cells. GFP+ cells beneath basal lamina (arrow) were detected on day 30 and 6 months later. (C): GFP+ cell under laminin-positive basal lamina (Alexa350 blue) was also stained with anti c-Met antibody (Cy3 red, arrow). (D–F): Single-fiber culture was performed with single fibers isolated from recipient mice 6 months after transplantation, followed by immunostaining. C, negative control; D, anti-myosin (Cy3 red) staining; E, anti-GFP-ALP (red) staining. D and E were same position. GFP-ALP+ fibers were confirmed to be muscle fibers using myosin Cy3 staining. (G): PCR analysis of extracted DNA from single-fiber culture. DNA was extracted from the single-fiber culture followed by PCR for GFP DNA, as described under Materials and Methods. 1: negative control; 2: peripheral blood of GFP transgenic mouse; 3: single fiber culture. Bars: A, 1 mm; B, C, 20 μm; D, 200 μm; E, F, 50 μm.
KSL-Derived Cells in Muscle Can Generate Muscle Fibers In Vitro in Long Term After Transplantation
So far it has been reported that damaged muscles by chemical agent or stress were regenerated by donor cells after BM transplantation . However, these muscle-regeneration assays in vivo cannot clarify whether donor cells participate in regenerating muscles directly from the settled muscle or indirectly from settled BM. To determine whether GFP+ mononuclear cells in muscle tissues can differentiate into muscle fibers, a single-fiber culture was performed. Six months after transplantation with KSL cells, muscle fibers were isolated from the hind limb muscles of W/Wv mice or lethally irradiated mice transplanted, and single fibers were cultured onto matrigel-coated plates. After 14 days, most of the satellite cells had migrated from the fibers, proliferated, and formed new muscle fibers, which were confirmed to be myosin-positive by immunohistochemical staining (Fig. 7D, E). Since the GFP signal in vitro was too weak to be detected by fluorescent microscope, we determined the presence of GFP+ cells by means of immunohistochemical staining using the anti-GFP antibody or by GFP DNA amplification with the aid of PCR. Staining with the anti-GFP antibody proved the presence of GFP+ myofibers (Fig. 7F), which were also myosin-positive. In addition, GFP DNA was also confirmed by PCR to be present in samples extracted from culture dishes of single-muscle fiber (Fig. 7G). No GFP+ fibers were generated in the single-fiber culture in the early phase of transplantation in irradiated mice (data not shown). In brief, GFP+ KSL cell that engrafted muscle tissues could repair damaged muscle but not produce muscle fibers in vitro in the early phase. In the long term, however, they acquired the potential to differentiate into muscle fibers like satellite cells also in vitro. Since the single-fiber culture is a functional assay of the presence of satellite cells, it can be said that GFP+ KSL cells can give rise to satellite cells with myogenic potential in the long term after transplantation.
DISCUSSION
This study was supported by Health and Labour Science Research Grant, Research on Human Genome, Tissue Engineering Food Biotechnology, Ministry of Health, Labour and Welfare, Tokyo; by Grant-in-Aid for Science Research on Priority Areas no. 122150-67; and by Grant-in-Aid for Creative Scientific Research no. 13GS-0009. This study was also supported by Research Grant no. 13B-1 for Nervous and Mental Disorders and no. H13-iyaku-043, both from the Ministry of Health, Labour and Welfare.
REFERENCES
Taylor G, Lehrer MS, Jensen PJ et al. Involvement of follicular stem cells in forming not only the follicle but also the epidermis. Cell 2000;102:451–461.
Mills JC, Gordon JI. The intestinal stem cell niche: there grows the neighborhood. Proc Natl Acad Sci U S A 2001;98:12334–12336.
Shinohara T, Orwig KE, Avarbock MR et al. Remodeling of the postnatal mouse testis is accompanied by dramatic changes in stem cell number and niche accessibility. Proc Natl Acad Sci U S A 2001;98:6186–6191.
Suzuki A, Zheng Yw YW, Kaneko S et al. Clonal identification and characterization of self-renewing pluripotent stem cells in the developing liver. J Cell Biol 2002;156:173–184.
Rietze RL, Valcanis H, Brooker GF et al. Purification of a pluripotent neural stem cell from the adult mouse brain. Nature 2001;412:736–739.
Qu-Petersen Z, Deasy B, Jankowski R et al. Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration. J Cell Biol 2002;157:851–864.
Petersen BE, Bowen WC, Patrene KD et al. Bone marrow as a potential source of hepatic oval cells. Science 1999;284:1168–1170.
Orlic D, Kajstura J, Chimenti S et al. Bone marrow cells regenerate infarcted myocardium. Nature 2001;410:701–705.
Brazelton TR, Rossi FM, Keshet GI et al. From marrow to brain: expression of neuronal phenotypes in adult mice. Science 2000;290:1775–1779.
Mezey E, Chandross KJ, Harta G et al. Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 2000;290:1779–1782.
Lagasse E, Connors H, Al-Dhalimy M et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000;6:1229–1234.
Ferrari G, Cusella-De Angelis G, Coletta M et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 1998;279:1528–1530.
Bittner RE, Schofer C, Weipoltshammer K et al. Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anat Embryol (Berl) 1999;199:391–396.
Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105:369–377.
Gussoni E, Soneoka Y, Strickland CD et al. Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 1999;401:390–394.
Terada N, Hamazaki T, Oka M et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 2002;416:542–545.
Ying QL, Nichols J, Evans EP et al. Changing potency by spontaneous fusion. Nature 2002;416:545–548.
Jackson KA, Mi T, Goodell MA. Hematopoietic potential of stem cells isolated from murine skeletal muscle. Proc Natl Acad Sci U S A 1999;96:14482–14486.
Asakura A, Seale P, Girgis-Gabardo A et al. Myogenic specification of side population cells in skeletal muscle. J Cell Biol 2002;159:123–134.
Kawada H, Ogawa M. Bone marrow origin of hematopoietic progenitors and stem cells in murine muscle. Blood 2001;98:2008–2013.
Yoshimoto M, Shinohara T, Heike T et al. Direct visualization of reconstitution process of transplanted hematopoietic cells in intact mouse organs indicates the presence of a niche. Exp Hematol 2003;31:733–740.
Zammit PS, Golding JP, Nagata Y et al. Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? J Cell Biol 2004;166:347–357.
Kawakami A, Kimura-Kawakami M, Nomura T et al. Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development. Mech Dev 1997;66:119–130.
Rosenblatt JD, Lunt AI, Parry DJ et al. Culturing satellite cells from living single muscle fiber explants. In Vitro Cell Dev Biol Anim 1995;31:773–779.
Osawa M, Hanada K, Hamada H et al. Long-term lymphohematopoietic reconstitution by a single CD34–low/negative hematopoietic stem cell. Science 1996;273:242–245.
Blaveri K, Heslop L, Yu DS et al. Patterns of repair of dystrophic mouse muscle: studies on isolated fibers. Dev Dyn 1999;216:244–256.
Mintz B, Anthony K, Litwin S. Monoclonal derivation of mouse myeloid and lymphoid lineages from totipotent hematopoietic stem cells experimentally engrafted in fetal hosts. Proc Natl Acad Sci U S A 1984;81:7835–7839.
Capel B, Mintz B. Neonatal W-mutant mice are favorable hosts for tracking development of marked hematopoietic stem cells. Exp Hematol 1989;17:872–876.
Fukada S, Miyagoe-Suzuki Y, Tsukihara H et al. Muscle regeneration by reconstitution with bone marrow or fetal liver cells from green fluorescent protein-gene transgenic mice. J Cell Sci 2002;115:1285–1293.
LaBarge MA, Blau HM. Biological progression from adult bone marrow to mononucleate muscle stem cell to multinucleate muscle fiber in response to injury. Cell 2002;111:589–601.
Camargo FD, Green R, Capetenaki Y et al. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003;9:1520–1527.
Corbel SY, Lee A, Yi L et al. Contribution of hematopoietic stem cells to skeletal muscle. Nat Med 2003;9:1528–1532.
McKinney-Freeman SL, Jackson KA, Camargo FD et al. Muscle-derived hematopoietic stem cells are hematopoietic in origin. Proc Natl Acad Sci U S A 2002;99:1341–1346.
Heslop L, Morgan JE, Partridge TA. Evidence for a myogenic stem cell that is exhausted in dystrophic muscle. J Cell Sci 2000;113( Pt 12):2299–2308.
Schweitzer KM, Drager AM, van der Valk P et al. Constitutive expression of E-selectin and vascular cell adhesion molecule-1 on endothelial cells of hematopoietic tissues. Am J Pathol 1996;148:165–175.
Jacobsen K, Kravitz J, Kincade PW et al. Adhesion receptors on bone marrow stromal cells: in vivo expression of vascular cell adhesion molecule-1 by reticular cells and sinusoidal endothelium in normal and gamma-irradiated mice. Blood 1996;87:73–82.
Williams DA, Rios M, Stephens C et al. Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions. Nature 1991;352:438–441.(Momoko Yoshimotoa, Hsi Ch)