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Bone marrow derived cells in adult skeletal muscle tissue in humans

Bone marrow derived cells in adult skeletal muscle tissue in humans Background: During the past decade, several animal studies have demonstrated that in addition to local cells, cells from the bone marrow (BM) possess the ability to contribute to regeneration of injured skeletal muscle tissue. In addition, in mice, regular physical activity has been displayed to be a sufficient stimulus for BM-derived cell contribution to the muscle, indicating that this is part of the ongoing physiological remodeling of skeletal muscle. However, whether BM-derived cells participate in human skeletal muscle remodeling is not known. To this end, we analyzed the incorporation of BM-derived cells in healthy human skeletal muscle in women transplanted with male BM. Methods: Skeletal muscle biopsies were obtained from the m. vastus lateralis of women transplanted with male donor hematopoietic stem cells 6 to 12 years earlier. Healthy women served as controls. Immunohistochemical staining for skeletal muscle fibers, satellite cells (SCs) or endothelial cells (ECs) combined with fluorescent in situ hybridization (FISH) of X and Y chromosomes was used to identify cells of BM origin within the biopsies. Three dimensional confocal imaging was performed to demonstrate colocalization of Y chromosome and DAPI within muscle fibers. To further investigate whether BM-derived cells incorporate into the SC niche, myoblasts were extracted from the biopsies from the transplanted women, cultured, and analyzed using XY FISH and immunocytochemistry. Results: Three dimensional confocal imaging indisputably demonstrated colocalization of Y chromosome and DAPI within muscle fibers. Some Y chromosomes were found within centrally located nuclei. No Y chromosomes were detected in CD56+ SCs in the tissue sections nor in the myoblasts cultured from the extracted SCs. Y chromosome+ ECs were found in all sections from the transplanted subjects. No Y chromosomes were found in the skeletal muscle biopsies obtained from healthy control women. Conclusions: We demonstrate that BM-derived cells contribute to skeletal muscle fibers and ECs. Our results support that BM contribution to skeletal muscle occurs via direct fusion to muscle fibers, and that the contributing cells derive from the hematopoietic lineage. Thus, the present findings encourage further studies of the importance of this process for the physiological adaptation occurring throughout life. Keywords: Fluorescent in situ hybridization (FISH), Needle biopsies, Satellite cell niche, Vasculogenesis * Correspondence: anna.stromberg@ki.se Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86, Stockholm, Sweden Full list of author information is available at the end of the article © 2013 Strömberg et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Strömberg et al. Skeletal Muscle 2013, 3:12 Page 2 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 Background Our hypothesis was that BM-derived cells contribute to In recent years, multipotent cells have attracted consid- the SC pool, skeletal muscle fibers and ECs in skeletal erable interest because of their potential capacity to re- muscle tissue in humans. pair and remodel peripheral tissue. Skeletal muscle is an organ system that is under constant adaptation to chan- Methods ging work demands due to day-to-day physical activity Study subjects and experimental model and inactivity. This includes effects on the number of Three non-smoking, moderately active female subjects mitochondria, storage of substrates, enzyme activity transplanted with male haematopoietic stem cells 6 to levels, and changes in the contractility apparatus [1]. In 12 years previously participated in the study. Their age, addition to changes in muscle fibers, extracellular struc- height and weight ranged between 28 to 36 yrs, 161 to tures and supporting cells surrounding the fibers adapt 178 cm, and 69 to 83 kg, respectively. All patients were to altered work demands [2]. The mature skeletal muscle on post-transplant hormonal replacement therapy and in cell is postmitotic; thus, muscle regeneration depends on remission. Patient A had suffered from myelodysplastic the recruitment and fusion of progenitor cells with syndrome, and was transplanted with BM from an unre- preexisting mature skeletal muscle fibers. The satellite lated donor. She had two children, a son and a daughter cell (SC), which was identified in 1961 [3], is the residen- aged 11 and 13, respectively, at the time of obtaining the tial stem cell of skeletal muscle tissue. However, the im- biopsy. Patient B had acute myeloid leukaemia, and had portance of this cell in the remodeling process was received BM-derived peripheral blood stem cells from seriously recognized only recently [4,5]. It has been pro- her brother. Patient C had chronic lymphatic leukaemia, posed that the SC pool is supplemented with progenitor and was transplanted with BM from her brother. Neither cells from other sources, such as the bone marrow (BM) patient B nor C had biological children. Two healthy, [6-8]. An alternative route suggested for BM-derived cell non-smoking, moderately active women served as con- contribution to skeletal muscle is via direct fusion to trols. Their age, height and weight ranged between 35 to muscle fibers in response to a physiological stimulus 36 yrs, 164 to 170 cm, and 58 to 65 kg, respectively. such as injury [9,10]. One of the control subjects had two sons aged 4 and 7, Another auxiliary structure with critical importance respectively, at the time of obtaining the biopsy. The for skeletal muscle function is the capillary network sur- other control subject had two children, a son and a rounding each fiber, and alteration of the number of ca- daughter, aged 3 and 5. Prior to the study, the experi- pillaries is a well-characterized extracellular remodeling mental protocol was explained to all subjects and in- process that occurs during skeletal muscle adaptation, formed written consent for participation was obtained. both in humans and animals [1,11-13]. The earlier pre- The study was approved by the Ethics Committee at dominant view was that any changes in the number of Karolinska Institutet and conformed to the Declaration vessels in adults were related to the proliferation of of Helsinki. existing vessels, i.e., angiogenesis or arteriogenesis. Cur- Muscle biopsy samples were obtained using the percu- rently, it has been shown that BM-derived endothelial taneous needle biopsy technique from the vastus lateralis progenitor cells (EPCs) contribute to vascular growth muscle at rest. The biopsies were immediately divided in (vasculogenesis) in numerous animal models [14-16] and two pieces; one piece was frozen within 10 to 15 s in that the circulating level of these cells changes in human isopentane cooled to freezing by liquid nitrogen and conditions of enhanced vascular formation [17-19]. Fur- stored at −80°C until analysis, and the other was stored thermore, several factors that stimulate vascular growth overnight in PBS/1% penicillin streptomycin at 4°C. For in skeletal muscle (e.g., vascular endothelial growth the control subjects the whole biopsy was frozen and factor-A, the angiopoietins, and erythropoietin) have stored at −80°C until analysis. been shown to stimulate both the angiogenic and vasculogenic processes [20-23]. Nevertheless, there are no data on the contribution of BM-derived cells to Satellite cell isolation physiological vascular remodeling of skeletal muscle About 30 mg of muscle tissue was put in a tube tissue in humans; however, data demonstrating that containing sterile PBS with 1% penicillin-streptomycin BM-derived cells differentiate into endometrial endothe- (Invitrogen AB, Stockholm, Sweden) and stored overnight lial cells (ECs) in a human female after BM transplant- at 4°C. After ~20 h, the biopsy was washed twice with ation [24] support such hypothesis. PBS, cut in small pieces and digested in 5 mL 0.25% tryp- The aim of the current study was to investigate whether sin EDTA in 37°C, 5% CO with gentle agitation for 20 cells from the BM incorporate into skeletal muscle tissue min. Undigested tissue was allowed to settle for 5 min and in adult humans. This was achieved by analyzing skeletal the supernatant was collected in DMEM-F12, with 20% muscle biopsies from women transplanted with male BM. FCS and 1% penicillin streptomycin. The cells were Strömberg et al. Skeletal Muscle 2013, 3:12 Page 3 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 cultured until reaching ~70% confluence when a fraction X (DXZ1) SpectrumGreen or SpectrumOrange Probe of the cells was obtained for cytocentrifugation. and CEP Y (DYZ1) SpectrumAqua or SpectrumGreen Probe), were mixed with hybridization buffer according Cytocentrifugation to the manufacturer (Abbott-Vysis Inc Downers Grove, The cells were suspended at a concentration of 10 cells/ IL, USA); 1.5 μL of the probe solution were added to a mL and 100 μL were spun onto each glass slide round cover slip (Thermo Fischer Scientific, Menzel (Superfrost/Plus microscope slides, Fisher Scientific, GmbH & Co KG, Braunschweig, Germany), and the Pittsburgh, PA, USA). The slides were fixed in −20°C glass slide was then put on top of the cover slip. The acetone for 10 min and then washed in PBS. Slides were cover slip was sealed around the edges with rubber ce- blocked in PBS/4% BSA for 30 min in a humid chamber. ment, to prevent the probe solution from drying out A mouse anti-human desmin antibody (M0760, clone during hybridization. Glass slides and probe mixture D33, Dako, Glostrup, Denmark) was added at a concen- were denatured together in a Vysis HYBrite (Abbot tration of 1:200 in 1% BSA/PBS and the slides were in- Diagnostics). The melt temperature was set to 73°C for 2 cubated overnight in the cold room. The slides were min and hybridization temperature 38°C for 20 hours. washed in PBS before staining with the secondary When the hybridization was complete, the rubber ce- rabbit-anti mouse Alexa488 antibody (A11059, Molecu- ment was removed from the cover slips, and the slides lar Probes, Eugene, OR, USA) at 1:1000 in 1% BSA/PBS were immersed in 2×SSC until the cover slips fell off. for 60 min in the humid chamber. After washing in PBS, The slides were then washed in 0.4×SSC/0.3% Igepal, the slides were mounted in Vectashield DAPI (406- 72°C for 2 min and then 2×SSC/0.1% Igepal at room diamidine-2-phenylidole-dihydrochloride)/antifade (Vec- temperature for 30 s, and subsequently allowed to air- tor Laboratories Inc. Burlingame, CA, USA) and then dry in the dark before mounting in VectaShield antifade analyzed for percentage of desmin positive cells. (Vector Laboratories) with DAPI or propidium iodide as nuclear staining. Immunohistochemistry The frozen muscle biopsies were cut into 4 μm sections Evaluation of immunohistochemistry and FISH staining and placed on Superfrost/Plus microscope slides (Fisher Evaluation and cell identification was done using an Scientific). For serial sections, one section was put on Olympus fluorescence microscope BH60 with appropriate each glass slide in a series of slides. The sections were filter set equipped with a CCD camera and connected to a fixed for 10 min in −20°C acetone, and then dried before Cyto-Vision image system (Applied Imaging Corp., San three 3 min washes in PBS. The slides were blocked in Jose, CA, USA) in which the results also were docu- PBS/4% BSA for 30 min in a humid chamber, and were mented. Contribution of Y+ nuclei to skeletal muscle then incubated with the primary antibody overnight at fibers was analyzed in sections stained with caveolin-3, 4°C in the humid chamber. The antibodies used were while sections double-stained with laminin-1 and CD56 mouse anti-human CD31 at 1:500 (M0823, Dako), were analyzed for contribution to the SC pool. A cell mouse anti-human CD56 (NCAM) at 1:50, (347740, stained with the marker CD56 containing a nucleus lying Becton Dickinson, San Jose, CA, USA), sheep anti- inside laminin-1 positive lamina was identified as an SC. human laminin at 1:10000 (PC128, The Binding Site, Three sections from each subject, at least 80 μmapart, Birmingham, UK), mouse anti-human Caveolin-3 (A-3) were completely examined by two independent investiga- at 1:500 (sc-5310, Santa Cruz Biotechnology Inc. Santa tors using an oil immersion objective with magnification Cruz, CA, USA) and mouse anti-human CD68 at 1:500 100×1.3. Some of the skeletal muscle fibers containing Y (M0718, Dako). After washing the slides in PBS, they were chromosomes were also evaluated using confocal micros- incubated in the secondary rabbit-anti mouse Alexa488 copy. For evaluation of Y+ nuclei within ECs, two sections antibody (A11059, Molecular Probes) at 1:500 and/or the from each subject stained with CD31 were examined. To donkey anti-goat TexasRed at 1:300 (ab6883, Abcam, exclude for the possibility of the Y chromosome belonging Cambridge, UK) for 60 min in room temperature, washed to a leukocyte travelling within the vessel, serial sections again and mounted in VectaShield with or without DAPI stained with CD68 were performed and examined. After (Vector Laboratories). The staining was evaluated before evaluating the sections for Y chromosomes, the sections fluorescent in situ hybridization. were photographed using a Leica DMLA microscope equipped with a Leica DFC 420 C digital camera Fluorescent in situ hybridization (FISH) (Leica Microsystems AB, Sweden). The amount of After antibody staining, the glass slides were put in 2× myofibers and ECs per section was then calculated using saline sodium-citrate (SSC) buffer, and the cover slips the software Leica Qwin V. For myofibers, the tissue sec- were allowed to fall off. The chromosome enumeration tions were photographed using the 5× objective. For ECs, probes (CEP) for the X and Y chromosomes (Vysis CEP the 20× objective was used and a picture was taken in a Strömberg et al. Skeletal Muscle 2013, 3:12 Page 4 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 AB C Figure 1 Y chromosome-positive nuclei are incorporated into host muscle fibers. Microphotographs show confocal images of combined FISH (Y chromosome, bright green, arrow) and indirect, fluorescent immunohistochemistry for caveolin-3 (green). Nuclear DNA was counterstained by DAPI. (A) Microphotograph of a muscle fiber section (width × height; 134.95 × 134.95 μm) depicting Y chromosome-negative host nuclei and a single Y chromosome-positive donor nucleus (arrow). Caveolin-3 staining is used to visualize muscle fiber membranes. (B) provides a Z-stack (width × height × depth; 67.48 × 67.48 × 5.148 μm; 13 optical sections) of the muscle fiber in (A). Dashed lines mark muscle fiber membranes indicating that the DAPI/Y chromosome nucleus is localized inside the muscle fiber. Size bar = 10 μm. (C) A high magnification Z-stack (15.83 × 15.83 × 6.007 μm; 15 optical sections) of the double-stained nucleus depicted in (B) clearly shows the integration of the Y chromosome DNA in the nucleus. random location of the section. The area of the field was centrally located nuclei (Figure 2). Laminin-1 together measured and the number of ECs was counted using Leica with CD56 staining showed SCs underneath the basal Qwin V. The area of the whole section was then measured membrane of muscle fibers. No Y chromosomes were at 5× magnification, and the number of ECs for the whole detected in CD56+ SC in the tissue sections analyzed section was then estimated based on the assumption of (Figure 3). Y chromosome+ cells stained with CD31 were equal distribution of ECs across the sections. The found in all sections from the transplanted subjects counting of ECs was performed in two different fields by (Figure 4). In the sections from the skeletal muscle biop- two independent persons. For evaluation of Y chromo- sies retrieved from the healthy controls, no Y chromo- somes in the cytocentrifuged cells, the preparations were somes could be detected (no data shown). completely examined using an oil immersion objective with magnification 100×1.3. For each slide (n = 10 per Number of Y chromosomes in skeletal muscle fibers and subject), 4 visual fields were analyzed using the 40× ECs objective for expression of desmin, a myogenic marker. For subject A and B, 0.6% of the skeletal muscle fibers counted contained a Y chromosome within a nucleus. In Confocal microscopy subject C, 0.7% of the fibers had a Y chromosome inside Tissue sections were visualized with a Zeiss LSM710 a nucleus (Table 1). For ECs, some of the cells were cut confocal system using a Plan-Apochromat 63×/1.40 Oil objective. Z-stacks were acquired sequentially using a 405 diode laser and the 488 laser line of the Argon laser to excite DAPI and SpectrumGreen, respectively. To determine whether a Y chromosome belonged to a muscle fiber nucleus, Y chromosome staining and DAPI nuclear staining had to colocalize and the nucleus of interest had to be on the same focal plane as the muscle membrane staining. Z-stacks are presented as maxi- mum intensity projections. Three-dimensional render- ing, maximum intensity projections and deconvolution were done with the AutoQuant X3 software. Results Immunohistochemistry and XY-FISH With caveolin-3 the sarcolemma was visualized, while Figure 2 Donor nucleus centrally located in host muscle fiber. Y chromosome (turquoise, arrow) and X chromosome (green, laminin-1 stained the basal membranes. Three dimen- arrowhead) inside host skeletal muscle fiber. Nuclei counterstained sional confocal imaging indisputably demonstrated with DAPI. The section was examined by fluorescent microscopy colocalization of Y chromosome and DAPI within muscle with a 100× objective. Size bar = 10 μm. fibers (Figure 1). Some Y chromosomes were found within Strömberg et al. Skeletal Muscle 2013, 3:12 Page 5 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 positive SCs were detected in the tissue sections or in the A B cytocentrifuged cell population derived from SC isolation. In tissue sections stained using XY FISH together with either caveolin-3, which stained the sarcolemma, or laminin-1, which stained the basal membrane, epifluor- escence microscopy showed Y chromosomes present within myofibers. To further demonstrate colocalization of Y chromosome and DAPI nuclear staining within muscle fibers, three dimensional confocal imaging was performed. Interestingly, some of the Y chromosomes found within myofibers were observed in centrally lo- cated nuclei, which is indicative of a regenerating fiber [25]. In the present study, no Y chromosomes were CD detected in the SC niche in tissue sections stained with laminin-1 together with CD56. Further, not a single Y chromosome was detected in desmin positive myoblasts in the cell cultures from the isolated SCs. In a study performed in mice, BM-derived cells were shown to be incorporated into the SC niche [8]. The current data does not support such a mechanism; instead it suggests that BM-derived cells fuse directly with the muscle fibers ra- ther than passing through a myogenic stem cell stage. This is also coherent with a very well performed study in mice, were an estimated number of 375,000 SCs were assayed to Figure 3 Satellite cell containing two X chromosomes. determine the mechanism behind haematopoietic stem Immunofluorescent staining visualized by fluorescent microscopy cell contribution to skeletal muscle [26]. In their experi- shows XY-FISH combined with staining for the SC marker CD56. The ments not a single SC was shown to derive from the BM, section was examined with a 100× objective. (A,B) CD56 (green), and BM contribution to skeletal muscle occurred through together with laminin-1 (red), with and without DAPI. The CD56 positive satellite cell is located just beneath the basal lamina of the myeloid cell fusion to muscle fibers [26]. muscle fiber. (C,D) The same SC after XY-FISH, with and without The observation of Y chromosomes in cells stained with DAPI, showing the presence of two X chromosomes (bright green, CD31 support the notion that circulating cells incorporate arrows). into the skeletal muscle endothelium in humans. Import- antly, to exclude the possibility of the Y chromosome so that the nucleus was not visible but the cells were belonging to a leukocyte travelling within the vessel only positive for CD31. For subject A, 0.6% of the ECs contained a Y chromosome (0.9% per EC with visual DAPI staining). For subject B, 0.3% (0.5% of ECs with DAPI) of ECs had a Y chromosome. Subject C had 0.4% Y chromosome containing ECs (0.6% of ECs with DAPI) (Table 1). Cytocentrifugation About 80% of the cytocentrifuged cells from the SC extraction were positive for desmin. These cells are myo- blasts; no intact muscle fibers were obtained from the biopsies. None of the slides analyzed had a single cell Figure 4 Y chromosome-positive nuclei in endothelial cell. containing Y chromosomes (Figure 5). Immunofluorescent staining visualized by fluorescent microscopy shows XY-FISH combined with staining for the endothelial marker CD31 (green). The section was examined with a 100× objective. Discussion (A) Y chromosome (bright green, arrow), together with X chromosome (red, arrowhead) inside endothelial cell stained for In the current study, we demonstrated that Y chromo- CD31 (green). Nuclear DNA was counterstained by DAPI. Size bar = somes are present within myofibers and in cells with an 10 μm. (B) Same image without CD31-staining for better endothelial phenotype/morphology in women transplanted visualization of chromosome staining. with male hematopoietic stem cells. No Y chromosome Strömberg et al. Skeletal Muscle 2013, 3:12 Page 6 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 Table 1 Detection of Y chromosomes in host skeletal muscle fiber and endothelial cell nuclei No. of skeletal muscle fibers No. of Y chromosomes in fibers No. of ECs No. of Y chromosomes in ECs Subjects (n = 3) 2796 (2287–3384) 18 (17–21) 3060 (2260–3982) 14 (9–24) Number of skeletal muscle fibers and ECs counted per subject, together with the corresponding number of Y chromosomes. The total number of fibers, ECs and Y chromosomes is reported as an average (range) for the three subjects analyzed. CD31+ Y chromosome+ cells without a CD68+ cell on the themselves? For instance, the beneficial effects of the same localization in a serial section were counted. Since injection of EPCs into patients with myocardial infarction the discovery by Asahara et al. in 1997 of the formation of are measured using markers of cardiac function, such as ECs from human BM mononuclear cells in mice, a large left ventricular ejection fraction, which does not reveal number of articles on EPCs have been published in vari- whether the injected cells have actually formed ECs ous animal settings. This includes the contribution of [30,31]. EPCs to neovascularization of myocardium, brain and One could claim that the XY cells found in these hindlimb in rodents after induced ischemia [16,27,28]. women derive from male offspring. However, only one Still, even though the circulating level of EPCs is known to of the women had biological children and she gave birth change in human conditions of enhanced vascular forma- to her son 11 years before the beginning of this study. In tion, to our knowledge only one earlier report exists of addition, not a single Y-chromosome containing cell was actual contribution to endothelium in humans, where found in sections from the control women that had BM-derived cells were shown to differentiate into endo- given birth to boys. Furthermore, as the women who metrial ECs in a human female [24]. Our study provides participated in our study had received either BM trans- novel data regarding the contribution of BM-derived cells plantation or peripheral blood stem cells, the exact to skeletal muscle tissue in humans. phenotype/origin of the BM-derived stem cells incorpo- In thepresent studywehavenot been ableto elabor- rated into muscle fibers remains undetermined. Regard- ate on the functionality of these incorporated cells. Be- ing the BM-transplanted women, donor-derived cells cause of the low number of BM-derived cells found in may be mesenchymal stem cells derived from the BM our study, one may think that this is a process with no stroma or hematopoietic stem cells. The woman trans- physiological relevance. However, based on earlier find- planted with peripheral blood stem cells, however, ings in mice [8,29], it is tempting to speculate on exhibited the same level of donor-derived cells in her whether their number would have been higher if these muscle tissue, indicating that the ability to form women had been subjected to remodeling stimuli, such myonuclei or ECs lies in the hematopoietic lineage. as habitual endurance-type exercise. Importantly, the Nevertheless, the contribution of mesenchymal stem number of skeletal muscle fibers containing Y chromo- cells to skeletal muscle tissue in the BM-transplanted somes in our study is consistent with levels found in women cannot be excluded in this study. Another issue non-exercised mice [8,26], in which the number of cells is whether irradiation administered prior to BM trans- showntoincreaseinresponsetoincreased muscle plantation affects the tissues and the systemic environ- activity from muscle overload, forced downhill running ment in a manner that causes the incorporation of [29], and voluntary exercise [8]. Moreover, the role of BM-derived cells into tissues. However, irradiation was EPCs in vascular formation and repair is a largely unre- shown not to be a prerequisite for BM-derived cell con- solved question; do they act in a paracrine fashion to sup- tribution to myofiber regeneration in a study performed ply local cells with growth factors, or do they form ECs in mice [29]. A B C Figure 5 Immunofluorescent and FISH analysis of cytocentrifuged myoblasts from satellite cell extraction. (A) Desmin staining (green) before XY-FISH, 20× magnification. (B) Myoblasts after XY FISH. X chromosomes were stained red and Y chromosomes green. All cells were negative for the Y chromosome, 20× magnification. (C) Myoblasts stained with desmin (green) containing two X chromosomes (red), 100× magnification. The nuclei were counterstained with DAPI. Strömberg et al. Skeletal Muscle 2013, 3:12 Page 7 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 Conclusions References 1. Saltin B, Gollnick PD: Skeletal muscle adaptability: significance for In conclusion, the contribution of BM-derived cells to metabolism and performance.In Handbook of Physiology. Section 10, skeletal muscle fibers and ECs was demonstrated by Skeletal Muscle. Edited by Peachey LD. Bethesda: American Physiological obtaining skeletal muscle biopsies from women trans- Society; 1983:555–631. 2. Timmons JA, Jansson E, Fischer H, Gustafsson T, Greenhaff PL, Ridden J, planted with male BM. 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Gill M, Dias S, Hattori K, Rivera ML, Hicklin D, Witte L, Girardi L, Yurt R, Himel Grants H, Rafii S: Vascular trauma induces rapid but transient mobilization of This study was supported by grants from the Swedish Medical Research VEGFR2(+)AC133(+) endothelial precursor cells. Circ Res 2001, 88:167–174. Council, the Wallenberg foundation, the Swedish National Centre for 18. Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Research in Sports, the Swedish Medical Association and the Karolinska Shimada T, Oike Y, Imaizumi T: Mobilization of endothelial progenitor cells Institutet Foundation. in patients with acute myocardial infarction. Circulation 2001, 103:2776–2779. Author details 19. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Department of Laboratory Medicine, Division of Clinical Physiology, Dimmeler S: Number and migratory activity of circulating endothelial Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86, progenitor cells inversely correlate with risk factors for coronary artery Stockholm, Sweden. Department of Medicine, Center for Hematology and disease. Circ Res 2001, 89:E1–E7. Regenerative Medicine, Karolinska Institutet, Karolinska University Hospital 20. Moore MA, Hattori K, Heissig B, Shieh JH, Dias S, Crystal RG, Rafii S: Huddinge, 141 86, Stockholm, Sweden. Department of Medicine, Mobilization of endothelial and hematopoietic stem and progenitor cells Hematology Center, Karolinska Institutet, Karolinska University Hospital by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and Huddinge, 141 86, Stockholm, Sweden. angiopoietin-1. Ann N Y Acad Sci 2001, 938:36–45. Discussion 45–37. 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Mints M, Jansson M, Sadeghi B, Westgren M, Uzunel M, Hassan M, Palmblad J: Endometrial endothelial cells are derived from donor stem cells in a bone marrow transplant recipient. Hum Reprod 2008, 23:139–143. 25. Charge SB, Rudnicki MA: Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004, 84:209–238. 26. Camargo FD, Green R, Capetanaki Y, Jackson KA, Goodell MA: Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003, 9:1520–1527. 27. Zhang ZG, Zhang L, Jiang Q, Chopp M: Bone marrow-derived endothelial progenitor cells participate in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res 2002, 90:284–288. 28. Yamahara K, Sone M, Itoh H, Yamashita JK, Yurugi-Kobayashi T, Homma K, Chao TH, Miyashita K, Park K, Oyamada N, Sawada N, Taura D, Fukunaga Y, Tamura N, Nakao K: Augmentation of neovascularization [corrected] in hindlimb ischemia by combined transplantation of human embryonic stem cells-derived endothelial and mural cells. PLoS One 2008, 3:e1666. 29. Palermo AT, Labarge MA, Doyonnas R, Pomerantz J, Blau HM: Bone marrow contribution to skeletal muscle: a physiological response to stress. Dev Biol 2005, 279:336–344. 30. Yousef M, Schannwell CM, Kostering M, Zeus T, Brehm M, Strauer BE: The BALANCE study: clinical benefit and long-term outcome after intracoronary autologous bone marrow cell transplantation in patients with acute myocardial infarction. J Am Coll Cardiol 2009, 53:2262–2269. 31. Leistner DM, Fischer-Rasokat U, Honold J, Seeger FH, Schachinger V, Lehmann R, Martin H, Burck I, Urbich C, Dimmeler S, Zeiher AM, Assmus B: Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI): final 5-year results suggest long-term safety and efficacy. Clin Res Cardiol 2011, 100:925–934. doi:10.1186/2044-5040-3-12 Cite this article as: Strömberg et al.: Bone marrow derived cells in adult skeletal muscle tissue in humans. Skeletal Muscle 2013 3:12. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Skeletal Muscle Springer Journals

Bone marrow derived cells in adult skeletal muscle tissue in humans

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Copyright © 2013 by Strömberg et al.; licensee BioMed Central Ltd.
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Life Sciences; Cell Biology; Developmental Biology; Biochemistry, general; Systems Biology; Biotechnology
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10.1186/2044-5040-3-12
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23680018
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Abstract

Background: During the past decade, several animal studies have demonstrated that in addition to local cells, cells from the bone marrow (BM) possess the ability to contribute to regeneration of injured skeletal muscle tissue. In addition, in mice, regular physical activity has been displayed to be a sufficient stimulus for BM-derived cell contribution to the muscle, indicating that this is part of the ongoing physiological remodeling of skeletal muscle. However, whether BM-derived cells participate in human skeletal muscle remodeling is not known. To this end, we analyzed the incorporation of BM-derived cells in healthy human skeletal muscle in women transplanted with male BM. Methods: Skeletal muscle biopsies were obtained from the m. vastus lateralis of women transplanted with male donor hematopoietic stem cells 6 to 12 years earlier. Healthy women served as controls. Immunohistochemical staining for skeletal muscle fibers, satellite cells (SCs) or endothelial cells (ECs) combined with fluorescent in situ hybridization (FISH) of X and Y chromosomes was used to identify cells of BM origin within the biopsies. Three dimensional confocal imaging was performed to demonstrate colocalization of Y chromosome and DAPI within muscle fibers. To further investigate whether BM-derived cells incorporate into the SC niche, myoblasts were extracted from the biopsies from the transplanted women, cultured, and analyzed using XY FISH and immunocytochemistry. Results: Three dimensional confocal imaging indisputably demonstrated colocalization of Y chromosome and DAPI within muscle fibers. Some Y chromosomes were found within centrally located nuclei. No Y chromosomes were detected in CD56+ SCs in the tissue sections nor in the myoblasts cultured from the extracted SCs. Y chromosome+ ECs were found in all sections from the transplanted subjects. No Y chromosomes were found in the skeletal muscle biopsies obtained from healthy control women. Conclusions: We demonstrate that BM-derived cells contribute to skeletal muscle fibers and ECs. Our results support that BM contribution to skeletal muscle occurs via direct fusion to muscle fibers, and that the contributing cells derive from the hematopoietic lineage. Thus, the present findings encourage further studies of the importance of this process for the physiological adaptation occurring throughout life. Keywords: Fluorescent in situ hybridization (FISH), Needle biopsies, Satellite cell niche, Vasculogenesis * Correspondence: anna.stromberg@ki.se Department of Laboratory Medicine, Division of Clinical Physiology, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86, Stockholm, Sweden Full list of author information is available at the end of the article © 2013 Strömberg et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Strömberg et al. Skeletal Muscle 2013, 3:12 Page 2 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 Background Our hypothesis was that BM-derived cells contribute to In recent years, multipotent cells have attracted consid- the SC pool, skeletal muscle fibers and ECs in skeletal erable interest because of their potential capacity to re- muscle tissue in humans. pair and remodel peripheral tissue. Skeletal muscle is an organ system that is under constant adaptation to chan- Methods ging work demands due to day-to-day physical activity Study subjects and experimental model and inactivity. This includes effects on the number of Three non-smoking, moderately active female subjects mitochondria, storage of substrates, enzyme activity transplanted with male haematopoietic stem cells 6 to levels, and changes in the contractility apparatus [1]. In 12 years previously participated in the study. Their age, addition to changes in muscle fibers, extracellular struc- height and weight ranged between 28 to 36 yrs, 161 to tures and supporting cells surrounding the fibers adapt 178 cm, and 69 to 83 kg, respectively. All patients were to altered work demands [2]. The mature skeletal muscle on post-transplant hormonal replacement therapy and in cell is postmitotic; thus, muscle regeneration depends on remission. Patient A had suffered from myelodysplastic the recruitment and fusion of progenitor cells with syndrome, and was transplanted with BM from an unre- preexisting mature skeletal muscle fibers. The satellite lated donor. She had two children, a son and a daughter cell (SC), which was identified in 1961 [3], is the residen- aged 11 and 13, respectively, at the time of obtaining the tial stem cell of skeletal muscle tissue. However, the im- biopsy. Patient B had acute myeloid leukaemia, and had portance of this cell in the remodeling process was received BM-derived peripheral blood stem cells from seriously recognized only recently [4,5]. It has been pro- her brother. Patient C had chronic lymphatic leukaemia, posed that the SC pool is supplemented with progenitor and was transplanted with BM from her brother. Neither cells from other sources, such as the bone marrow (BM) patient B nor C had biological children. Two healthy, [6-8]. An alternative route suggested for BM-derived cell non-smoking, moderately active women served as con- contribution to skeletal muscle is via direct fusion to trols. Their age, height and weight ranged between 35 to muscle fibers in response to a physiological stimulus 36 yrs, 164 to 170 cm, and 58 to 65 kg, respectively. such as injury [9,10]. One of the control subjects had two sons aged 4 and 7, Another auxiliary structure with critical importance respectively, at the time of obtaining the biopsy. The for skeletal muscle function is the capillary network sur- other control subject had two children, a son and a rounding each fiber, and alteration of the number of ca- daughter, aged 3 and 5. Prior to the study, the experi- pillaries is a well-characterized extracellular remodeling mental protocol was explained to all subjects and in- process that occurs during skeletal muscle adaptation, formed written consent for participation was obtained. both in humans and animals [1,11-13]. The earlier pre- The study was approved by the Ethics Committee at dominant view was that any changes in the number of Karolinska Institutet and conformed to the Declaration vessels in adults were related to the proliferation of of Helsinki. existing vessels, i.e., angiogenesis or arteriogenesis. Cur- Muscle biopsy samples were obtained using the percu- rently, it has been shown that BM-derived endothelial taneous needle biopsy technique from the vastus lateralis progenitor cells (EPCs) contribute to vascular growth muscle at rest. The biopsies were immediately divided in (vasculogenesis) in numerous animal models [14-16] and two pieces; one piece was frozen within 10 to 15 s in that the circulating level of these cells changes in human isopentane cooled to freezing by liquid nitrogen and conditions of enhanced vascular formation [17-19]. Fur- stored at −80°C until analysis, and the other was stored thermore, several factors that stimulate vascular growth overnight in PBS/1% penicillin streptomycin at 4°C. For in skeletal muscle (e.g., vascular endothelial growth the control subjects the whole biopsy was frozen and factor-A, the angiopoietins, and erythropoietin) have stored at −80°C until analysis. been shown to stimulate both the angiogenic and vasculogenic processes [20-23]. Nevertheless, there are no data on the contribution of BM-derived cells to Satellite cell isolation physiological vascular remodeling of skeletal muscle About 30 mg of muscle tissue was put in a tube tissue in humans; however, data demonstrating that containing sterile PBS with 1% penicillin-streptomycin BM-derived cells differentiate into endometrial endothe- (Invitrogen AB, Stockholm, Sweden) and stored overnight lial cells (ECs) in a human female after BM transplant- at 4°C. After ~20 h, the biopsy was washed twice with ation [24] support such hypothesis. PBS, cut in small pieces and digested in 5 mL 0.25% tryp- The aim of the current study was to investigate whether sin EDTA in 37°C, 5% CO with gentle agitation for 20 cells from the BM incorporate into skeletal muscle tissue min. Undigested tissue was allowed to settle for 5 min and in adult humans. This was achieved by analyzing skeletal the supernatant was collected in DMEM-F12, with 20% muscle biopsies from women transplanted with male BM. FCS and 1% penicillin streptomycin. The cells were Strömberg et al. Skeletal Muscle 2013, 3:12 Page 3 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 cultured until reaching ~70% confluence when a fraction X (DXZ1) SpectrumGreen or SpectrumOrange Probe of the cells was obtained for cytocentrifugation. and CEP Y (DYZ1) SpectrumAqua or SpectrumGreen Probe), were mixed with hybridization buffer according Cytocentrifugation to the manufacturer (Abbott-Vysis Inc Downers Grove, The cells were suspended at a concentration of 10 cells/ IL, USA); 1.5 μL of the probe solution were added to a mL and 100 μL were spun onto each glass slide round cover slip (Thermo Fischer Scientific, Menzel (Superfrost/Plus microscope slides, Fisher Scientific, GmbH & Co KG, Braunschweig, Germany), and the Pittsburgh, PA, USA). The slides were fixed in −20°C glass slide was then put on top of the cover slip. The acetone for 10 min and then washed in PBS. Slides were cover slip was sealed around the edges with rubber ce- blocked in PBS/4% BSA for 30 min in a humid chamber. ment, to prevent the probe solution from drying out A mouse anti-human desmin antibody (M0760, clone during hybridization. Glass slides and probe mixture D33, Dako, Glostrup, Denmark) was added at a concen- were denatured together in a Vysis HYBrite (Abbot tration of 1:200 in 1% BSA/PBS and the slides were in- Diagnostics). The melt temperature was set to 73°C for 2 cubated overnight in the cold room. The slides were min and hybridization temperature 38°C for 20 hours. washed in PBS before staining with the secondary When the hybridization was complete, the rubber ce- rabbit-anti mouse Alexa488 antibody (A11059, Molecu- ment was removed from the cover slips, and the slides lar Probes, Eugene, OR, USA) at 1:1000 in 1% BSA/PBS were immersed in 2×SSC until the cover slips fell off. for 60 min in the humid chamber. After washing in PBS, The slides were then washed in 0.4×SSC/0.3% Igepal, the slides were mounted in Vectashield DAPI (406- 72°C for 2 min and then 2×SSC/0.1% Igepal at room diamidine-2-phenylidole-dihydrochloride)/antifade (Vec- temperature for 30 s, and subsequently allowed to air- tor Laboratories Inc. Burlingame, CA, USA) and then dry in the dark before mounting in VectaShield antifade analyzed for percentage of desmin positive cells. (Vector Laboratories) with DAPI or propidium iodide as nuclear staining. Immunohistochemistry The frozen muscle biopsies were cut into 4 μm sections Evaluation of immunohistochemistry and FISH staining and placed on Superfrost/Plus microscope slides (Fisher Evaluation and cell identification was done using an Scientific). For serial sections, one section was put on Olympus fluorescence microscope BH60 with appropriate each glass slide in a series of slides. The sections were filter set equipped with a CCD camera and connected to a fixed for 10 min in −20°C acetone, and then dried before Cyto-Vision image system (Applied Imaging Corp., San three 3 min washes in PBS. The slides were blocked in Jose, CA, USA) in which the results also were docu- PBS/4% BSA for 30 min in a humid chamber, and were mented. Contribution of Y+ nuclei to skeletal muscle then incubated with the primary antibody overnight at fibers was analyzed in sections stained with caveolin-3, 4°C in the humid chamber. The antibodies used were while sections double-stained with laminin-1 and CD56 mouse anti-human CD31 at 1:500 (M0823, Dako), were analyzed for contribution to the SC pool. A cell mouse anti-human CD56 (NCAM) at 1:50, (347740, stained with the marker CD56 containing a nucleus lying Becton Dickinson, San Jose, CA, USA), sheep anti- inside laminin-1 positive lamina was identified as an SC. human laminin at 1:10000 (PC128, The Binding Site, Three sections from each subject, at least 80 μmapart, Birmingham, UK), mouse anti-human Caveolin-3 (A-3) were completely examined by two independent investiga- at 1:500 (sc-5310, Santa Cruz Biotechnology Inc. Santa tors using an oil immersion objective with magnification Cruz, CA, USA) and mouse anti-human CD68 at 1:500 100×1.3. Some of the skeletal muscle fibers containing Y (M0718, Dako). After washing the slides in PBS, they were chromosomes were also evaluated using confocal micros- incubated in the secondary rabbit-anti mouse Alexa488 copy. For evaluation of Y+ nuclei within ECs, two sections antibody (A11059, Molecular Probes) at 1:500 and/or the from each subject stained with CD31 were examined. To donkey anti-goat TexasRed at 1:300 (ab6883, Abcam, exclude for the possibility of the Y chromosome belonging Cambridge, UK) for 60 min in room temperature, washed to a leukocyte travelling within the vessel, serial sections again and mounted in VectaShield with or without DAPI stained with CD68 were performed and examined. After (Vector Laboratories). The staining was evaluated before evaluating the sections for Y chromosomes, the sections fluorescent in situ hybridization. were photographed using a Leica DMLA microscope equipped with a Leica DFC 420 C digital camera Fluorescent in situ hybridization (FISH) (Leica Microsystems AB, Sweden). The amount of After antibody staining, the glass slides were put in 2× myofibers and ECs per section was then calculated using saline sodium-citrate (SSC) buffer, and the cover slips the software Leica Qwin V. For myofibers, the tissue sec- were allowed to fall off. The chromosome enumeration tions were photographed using the 5× objective. For ECs, probes (CEP) for the X and Y chromosomes (Vysis CEP the 20× objective was used and a picture was taken in a Strömberg et al. Skeletal Muscle 2013, 3:12 Page 4 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 AB C Figure 1 Y chromosome-positive nuclei are incorporated into host muscle fibers. Microphotographs show confocal images of combined FISH (Y chromosome, bright green, arrow) and indirect, fluorescent immunohistochemistry for caveolin-3 (green). Nuclear DNA was counterstained by DAPI. (A) Microphotograph of a muscle fiber section (width × height; 134.95 × 134.95 μm) depicting Y chromosome-negative host nuclei and a single Y chromosome-positive donor nucleus (arrow). Caveolin-3 staining is used to visualize muscle fiber membranes. (B) provides a Z-stack (width × height × depth; 67.48 × 67.48 × 5.148 μm; 13 optical sections) of the muscle fiber in (A). Dashed lines mark muscle fiber membranes indicating that the DAPI/Y chromosome nucleus is localized inside the muscle fiber. Size bar = 10 μm. (C) A high magnification Z-stack (15.83 × 15.83 × 6.007 μm; 15 optical sections) of the double-stained nucleus depicted in (B) clearly shows the integration of the Y chromosome DNA in the nucleus. random location of the section. The area of the field was centrally located nuclei (Figure 2). Laminin-1 together measured and the number of ECs was counted using Leica with CD56 staining showed SCs underneath the basal Qwin V. The area of the whole section was then measured membrane of muscle fibers. No Y chromosomes were at 5× magnification, and the number of ECs for the whole detected in CD56+ SC in the tissue sections analyzed section was then estimated based on the assumption of (Figure 3). Y chromosome+ cells stained with CD31 were equal distribution of ECs across the sections. The found in all sections from the transplanted subjects counting of ECs was performed in two different fields by (Figure 4). In the sections from the skeletal muscle biop- two independent persons. For evaluation of Y chromo- sies retrieved from the healthy controls, no Y chromo- somes in the cytocentrifuged cells, the preparations were somes could be detected (no data shown). completely examined using an oil immersion objective with magnification 100×1.3. For each slide (n = 10 per Number of Y chromosomes in skeletal muscle fibers and subject), 4 visual fields were analyzed using the 40× ECs objective for expression of desmin, a myogenic marker. For subject A and B, 0.6% of the skeletal muscle fibers counted contained a Y chromosome within a nucleus. In Confocal microscopy subject C, 0.7% of the fibers had a Y chromosome inside Tissue sections were visualized with a Zeiss LSM710 a nucleus (Table 1). For ECs, some of the cells were cut confocal system using a Plan-Apochromat 63×/1.40 Oil objective. Z-stacks were acquired sequentially using a 405 diode laser and the 488 laser line of the Argon laser to excite DAPI and SpectrumGreen, respectively. To determine whether a Y chromosome belonged to a muscle fiber nucleus, Y chromosome staining and DAPI nuclear staining had to colocalize and the nucleus of interest had to be on the same focal plane as the muscle membrane staining. Z-stacks are presented as maxi- mum intensity projections. Three-dimensional render- ing, maximum intensity projections and deconvolution were done with the AutoQuant X3 software. Results Immunohistochemistry and XY-FISH With caveolin-3 the sarcolemma was visualized, while Figure 2 Donor nucleus centrally located in host muscle fiber. Y chromosome (turquoise, arrow) and X chromosome (green, laminin-1 stained the basal membranes. Three dimen- arrowhead) inside host skeletal muscle fiber. Nuclei counterstained sional confocal imaging indisputably demonstrated with DAPI. The section was examined by fluorescent microscopy colocalization of Y chromosome and DAPI within muscle with a 100× objective. Size bar = 10 μm. fibers (Figure 1). Some Y chromosomes were found within Strömberg et al. Skeletal Muscle 2013, 3:12 Page 5 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 positive SCs were detected in the tissue sections or in the A B cytocentrifuged cell population derived from SC isolation. In tissue sections stained using XY FISH together with either caveolin-3, which stained the sarcolemma, or laminin-1, which stained the basal membrane, epifluor- escence microscopy showed Y chromosomes present within myofibers. To further demonstrate colocalization of Y chromosome and DAPI nuclear staining within muscle fibers, three dimensional confocal imaging was performed. Interestingly, some of the Y chromosomes found within myofibers were observed in centrally lo- cated nuclei, which is indicative of a regenerating fiber [25]. In the present study, no Y chromosomes were CD detected in the SC niche in tissue sections stained with laminin-1 together with CD56. Further, not a single Y chromosome was detected in desmin positive myoblasts in the cell cultures from the isolated SCs. In a study performed in mice, BM-derived cells were shown to be incorporated into the SC niche [8]. The current data does not support such a mechanism; instead it suggests that BM-derived cells fuse directly with the muscle fibers ra- ther than passing through a myogenic stem cell stage. This is also coherent with a very well performed study in mice, were an estimated number of 375,000 SCs were assayed to Figure 3 Satellite cell containing two X chromosomes. determine the mechanism behind haematopoietic stem Immunofluorescent staining visualized by fluorescent microscopy cell contribution to skeletal muscle [26]. In their experi- shows XY-FISH combined with staining for the SC marker CD56. The ments not a single SC was shown to derive from the BM, section was examined with a 100× objective. (A,B) CD56 (green), and BM contribution to skeletal muscle occurred through together with laminin-1 (red), with and without DAPI. The CD56 positive satellite cell is located just beneath the basal lamina of the myeloid cell fusion to muscle fibers [26]. muscle fiber. (C,D) The same SC after XY-FISH, with and without The observation of Y chromosomes in cells stained with DAPI, showing the presence of two X chromosomes (bright green, CD31 support the notion that circulating cells incorporate arrows). into the skeletal muscle endothelium in humans. Import- antly, to exclude the possibility of the Y chromosome so that the nucleus was not visible but the cells were belonging to a leukocyte travelling within the vessel only positive for CD31. For subject A, 0.6% of the ECs contained a Y chromosome (0.9% per EC with visual DAPI staining). For subject B, 0.3% (0.5% of ECs with DAPI) of ECs had a Y chromosome. Subject C had 0.4% Y chromosome containing ECs (0.6% of ECs with DAPI) (Table 1). Cytocentrifugation About 80% of the cytocentrifuged cells from the SC extraction were positive for desmin. These cells are myo- blasts; no intact muscle fibers were obtained from the biopsies. None of the slides analyzed had a single cell Figure 4 Y chromosome-positive nuclei in endothelial cell. containing Y chromosomes (Figure 5). Immunofluorescent staining visualized by fluorescent microscopy shows XY-FISH combined with staining for the endothelial marker CD31 (green). The section was examined with a 100× objective. Discussion (A) Y chromosome (bright green, arrow), together with X chromosome (red, arrowhead) inside endothelial cell stained for In the current study, we demonstrated that Y chromo- CD31 (green). Nuclear DNA was counterstained by DAPI. Size bar = somes are present within myofibers and in cells with an 10 μm. (B) Same image without CD31-staining for better endothelial phenotype/morphology in women transplanted visualization of chromosome staining. with male hematopoietic stem cells. No Y chromosome Strömberg et al. Skeletal Muscle 2013, 3:12 Page 6 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 Table 1 Detection of Y chromosomes in host skeletal muscle fiber and endothelial cell nuclei No. of skeletal muscle fibers No. of Y chromosomes in fibers No. of ECs No. of Y chromosomes in ECs Subjects (n = 3) 2796 (2287–3384) 18 (17–21) 3060 (2260–3982) 14 (9–24) Number of skeletal muscle fibers and ECs counted per subject, together with the corresponding number of Y chromosomes. The total number of fibers, ECs and Y chromosomes is reported as an average (range) for the three subjects analyzed. CD31+ Y chromosome+ cells without a CD68+ cell on the themselves? For instance, the beneficial effects of the same localization in a serial section were counted. Since injection of EPCs into patients with myocardial infarction the discovery by Asahara et al. in 1997 of the formation of are measured using markers of cardiac function, such as ECs from human BM mononuclear cells in mice, a large left ventricular ejection fraction, which does not reveal number of articles on EPCs have been published in vari- whether the injected cells have actually formed ECs ous animal settings. This includes the contribution of [30,31]. EPCs to neovascularization of myocardium, brain and One could claim that the XY cells found in these hindlimb in rodents after induced ischemia [16,27,28]. women derive from male offspring. However, only one Still, even though the circulating level of EPCs is known to of the women had biological children and she gave birth change in human conditions of enhanced vascular forma- to her son 11 years before the beginning of this study. In tion, to our knowledge only one earlier report exists of addition, not a single Y-chromosome containing cell was actual contribution to endothelium in humans, where found in sections from the control women that had BM-derived cells were shown to differentiate into endo- given birth to boys. Furthermore, as the women who metrial ECs in a human female [24]. Our study provides participated in our study had received either BM trans- novel data regarding the contribution of BM-derived cells plantation or peripheral blood stem cells, the exact to skeletal muscle tissue in humans. phenotype/origin of the BM-derived stem cells incorpo- In thepresent studywehavenot been ableto elabor- rated into muscle fibers remains undetermined. Regard- ate on the functionality of these incorporated cells. Be- ing the BM-transplanted women, donor-derived cells cause of the low number of BM-derived cells found in may be mesenchymal stem cells derived from the BM our study, one may think that this is a process with no stroma or hematopoietic stem cells. The woman trans- physiological relevance. However, based on earlier find- planted with peripheral blood stem cells, however, ings in mice [8,29], it is tempting to speculate on exhibited the same level of donor-derived cells in her whether their number would have been higher if these muscle tissue, indicating that the ability to form women had been subjected to remodeling stimuli, such myonuclei or ECs lies in the hematopoietic lineage. as habitual endurance-type exercise. Importantly, the Nevertheless, the contribution of mesenchymal stem number of skeletal muscle fibers containing Y chromo- cells to skeletal muscle tissue in the BM-transplanted somes in our study is consistent with levels found in women cannot be excluded in this study. Another issue non-exercised mice [8,26], in which the number of cells is whether irradiation administered prior to BM trans- showntoincreaseinresponsetoincreased muscle plantation affects the tissues and the systemic environ- activity from muscle overload, forced downhill running ment in a manner that causes the incorporation of [29], and voluntary exercise [8]. Moreover, the role of BM-derived cells into tissues. However, irradiation was EPCs in vascular formation and repair is a largely unre- shown not to be a prerequisite for BM-derived cell con- solved question; do they act in a paracrine fashion to sup- tribution to myofiber regeneration in a study performed ply local cells with growth factors, or do they form ECs in mice [29]. A B C Figure 5 Immunofluorescent and FISH analysis of cytocentrifuged myoblasts from satellite cell extraction. (A) Desmin staining (green) before XY-FISH, 20× magnification. (B) Myoblasts after XY FISH. X chromosomes were stained red and Y chromosomes green. All cells were negative for the Y chromosome, 20× magnification. (C) Myoblasts stained with desmin (green) containing two X chromosomes (red), 100× magnification. The nuclei were counterstained with DAPI. Strömberg et al. Skeletal Muscle 2013, 3:12 Page 7 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 Conclusions References 1. Saltin B, Gollnick PD: Skeletal muscle adaptability: significance for In conclusion, the contribution of BM-derived cells to metabolism and performance.In Handbook of Physiology. Section 10, skeletal muscle fibers and ECs was demonstrated by Skeletal Muscle. Edited by Peachey LD. Bethesda: American Physiological obtaining skeletal muscle biopsies from women trans- Society; 1983:555–631. 2. Timmons JA, Jansson E, Fischer H, Gustafsson T, Greenhaff PL, Ridden J, planted with male BM. 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Physiol Rev 1992, 72:369–417. Authors’ contributions 12. Gustafsson T, Kraus WE: Exercise-induced angiogenesis-related growth AS designed the study, collected and analyzed the material, interpreted the and transcription factors in skeletal muscle, and their modification in data and drafted the manuscript. MJ contributed to the study design, and muscle pathology. Front Biosci 2001, 6:D75–D89. participated in the acquisition and interpretation of data. HF analyzed and 13. Gustafsson T: Vascular remodelling in human skeletal muscle. interpreted the data. ER obtained the muscle biopsies and helped to draft Biochem Soc Trans 2011, 39:1628–1632. the manuscript. HH recruited the study subjects and contributed to the study design. TG conceived the study, and contributed to the design and 14. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, coordination of the study and drafted the manuscript. All authors read and Witzenbichler B, Schatteman G, Isner JM: Isolation of putative progenitor approved the final manuscript. endothelial cells for angiogenesis. Science 1997, 275:964–967. 15. Kalka C, Masuda H, Takahashi T, Kalka-Moll WM, Silver M, Kearney M, Li T, Isner JM, Asahara T: Transplantation of ex vivo expanded endothelial Acknowledgments progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA We thank associate professor Gunnar Schulte, Karolinska Institutet who 2000, 97:3422–3427. performed the confocal imaging. His work is supported by grants from the 16. Kawamoto A, Gwon HC, Iwaguro H, Yamaguchi JI, Uchida S, Masuda H, Knut & Alice Wallenberg Foundation, the Swedish Medical Research Council Silver M, Ma H, Kearney M, Isner JM, Asahara T: Therapeutic potential of and Karolinska Institutet. ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 2001, 103:634–637. 17. Gill M, Dias S, Hattori K, Rivera ML, Hicklin D, Witte L, Girardi L, Yurt R, Himel Grants H, Rafii S: Vascular trauma induces rapid but transient mobilization of This study was supported by grants from the Swedish Medical Research VEGFR2(+)AC133(+) endothelial precursor cells. Circ Res 2001, 88:167–174. Council, the Wallenberg foundation, the Swedish National Centre for 18. Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Research in Sports, the Swedish Medical Association and the Karolinska Shimada T, Oike Y, Imaizumi T: Mobilization of endothelial progenitor cells Institutet Foundation. in patients with acute myocardial infarction. Circulation 2001, 103:2776–2779. Author details 19. Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Department of Laboratory Medicine, Division of Clinical Physiology, Dimmeler S: Number and migratory activity of circulating endothelial Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86, progenitor cells inversely correlate with risk factors for coronary artery Stockholm, Sweden. Department of Medicine, Center for Hematology and disease. Circ Res 2001, 89:E1–E7. Regenerative Medicine, Karolinska Institutet, Karolinska University Hospital 20. Moore MA, Hattori K, Heissig B, Shieh JH, Dias S, Crystal RG, Rafii S: Huddinge, 141 86, Stockholm, Sweden. Department of Medicine, Mobilization of endothelial and hematopoietic stem and progenitor cells Hematology Center, Karolinska Institutet, Karolinska University Hospital by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and Huddinge, 141 86, Stockholm, Sweden. angiopoietin-1. Ann N Y Acad Sci 2001, 938:36–45. Discussion 45–37. Received: 17 December 2012 Accepted: 10 April 2013 21. Hiratsuka S: Vasculogenensis, angiogenesis and special features of tumor Published: 16 May 2013 blood vessels. Front Biosci 2011, 16:1413–1427. Strömberg et al. Skeletal Muscle 2013, 3:12 Page 8 of 8 http://www.skeletalmusclejournal.com/content/3/1/12 22. Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rihm C, Martin H, Zeiher AM, Dimmeler S: Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 2003, 102:1340–1346. 23. Hattori K, Dias S, Heissig B, Hackett NR, Lyden D, Tateno M, Hicklin DJ, Zhu Z, Witte L, Crystal RG, Moore MA, Rafii S: Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells. J Exp Med 2001, 193:1005–1014. 24. Mints M, Jansson M, Sadeghi B, Westgren M, Uzunel M, Hassan M, Palmblad J: Endometrial endothelial cells are derived from donor stem cells in a bone marrow transplant recipient. Hum Reprod 2008, 23:139–143. 25. Charge SB, Rudnicki MA: Cellular and molecular regulation of muscle regeneration. Physiol Rev 2004, 84:209–238. 26. Camargo FD, Green R, Capetanaki Y, Jackson KA, Goodell MA: Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates. Nat Med 2003, 9:1520–1527. 27. Zhang ZG, Zhang L, Jiang Q, Chopp M: Bone marrow-derived endothelial progenitor cells participate in cerebral neovascularization after focal cerebral ischemia in the adult mouse. Circ Res 2002, 90:284–288. 28. Yamahara K, Sone M, Itoh H, Yamashita JK, Yurugi-Kobayashi T, Homma K, Chao TH, Miyashita K, Park K, Oyamada N, Sawada N, Taura D, Fukunaga Y, Tamura N, Nakao K: Augmentation of neovascularization [corrected] in hindlimb ischemia by combined transplantation of human embryonic stem cells-derived endothelial and mural cells. PLoS One 2008, 3:e1666. 29. Palermo AT, Labarge MA, Doyonnas R, Pomerantz J, Blau HM: Bone marrow contribution to skeletal muscle: a physiological response to stress. Dev Biol 2005, 279:336–344. 30. Yousef M, Schannwell CM, Kostering M, Zeus T, Brehm M, Strauer BE: The BALANCE study: clinical benefit and long-term outcome after intracoronary autologous bone marrow cell transplantation in patients with acute myocardial infarction. J Am Coll Cardiol 2009, 53:2262–2269. 31. Leistner DM, Fischer-Rasokat U, Honold J, Seeger FH, Schachinger V, Lehmann R, Martin H, Burck I, Urbich C, Dimmeler S, Zeiher AM, Assmus B: Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI): final 5-year results suggest long-term safety and efficacy. Clin Res Cardiol 2011, 100:925–934. doi:10.1186/2044-5040-3-12 Cite this article as: Strömberg et al.: Bone marrow derived cells in adult skeletal muscle tissue in humans. Skeletal Muscle 2013 3:12. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

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Skeletal MuscleSpringer Journals

Published: May 16, 2013

References