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. 2004 Dec;114(11):1577-85.
doi: 10.1172/JCI23071.

Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle

Karen A Lapidos  1 , Yiyin E Chen, Judy U Earley, Ahlke Heydemann, Jill M Huber, Marcia Chien, Averil Ma, Elizabeth M McNally
Affiliations

Transplanted hematopoietic stem cells demonstrate impaired sarcoglycan expression after engraftment into cardiac and skeletal muscle

Karen A Lapidos et al. J Clin Invest. 2004 Dec.

Abstract

Pluripotent bone marrow-derived side population (BM-SP) stem cells have been shown to repopulate the hematopoietic system and to contribute to skeletal and cardiac muscle regeneration after transplantation. We tested BM-SP cells for their ability to regenerate heart and skeletal muscle using a model of cardiomyopathy and muscular dystrophy that lacks delta-sarcoglycan. The absence of delta-sarcoglycan produces microinfarcts in heart and skeletal muscle that should recruit regenerative stem cells. Additionally, sarcoglycan expression after transplantation should mark successful stem cell maturation into cardiac and skeletal muscle lineages. BM-SP cells from normal male mice were transplanted into female delta-sarcoglycan-null mice. We detected engraftment of donor-derived stem cells into skeletal muscle, with the majority of donor-derived cells incorporated within myofibers. In the heart, donor-derived nuclei were detected inside cardiomyocytes. Skeletal muscle myofibers containing donor-derived nuclei generally failed to express sarcoglycan, with only 2 sarcoglycan-positive fibers detected in the quadriceps muscle from all 14 mice analyzed. Moreover, all cardiomyocytes with donor-derived nuclei were sarcoglycan-negative. The absence of sarcoglycan expression in cardiomyocytes and skeletal myofibers after transplantation indicates impaired differentiation and/or maturation of bone marrow-derived stem cells. The inability of BM-SP cells to express this protein severely limits their utility for cardiac and skeletal muscle regeneration.

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Figures

Figure 1
Figure 1
Sgcd–/– mice develop focal degeneration in heart and skeletal muscle that leads to cardiomyopathy and muscular dystrophy. (A_D) The focal degeneration in δ sarcoglycan_null myocardium (A and B) and striated muscle (C and D) can be seen with Masson trichrome staining. (E_H) Also shown is normal cardiac (E and F) and skeletal muscle (G and H). B, D, F, and H show higher-magnification views of the boxed regions in A, C, E, and G, respectively.
Figure 2
Figure 2
Hoechst dye exclusion technique isolates SP cells. Whole bone marrow was incubated with Hoechst 33342 with or without verapamil and then subjected to FACS analysis. (A and B) Typical FACS profiles. A shows that the SP cells represent approximately 0.04% of whole bone marrow. B indicates the expected verapamil sensitivity of Hoechst dye. (C_E) Whole bone marrow was incubated with Hoechst dye and then stained with a PE-conjugated Sca-1 antibody and a mouse lineage antibody cocktail. Lineage-positive cells were magnetically depleted from whole bone marrow, and lineage-negative cells were subjected to FACS analysis. (C) A FACS profile. The boxed region in C indicates that the SP represents 0.13% of lineage-negative cells. (D and E) The results of staining these cells with Sca-1. SP cells are 77% Sca-1_positive, and lineage-negative cells are 8% Sca-1_positive.
Figure 3
Figure 3
BM-SP cells engraft into skeletal muscle but have limited sarcoglycan expression. Normal male quadriceps (A_C) and female Sgcd–/– recipient quadriceps muscle sections (D_F) were stained for γ-sarcoglycan immunoreactivity (A and D, red) and then subjected to FISH with a Y chromosome_specific probe, Y-1 (B and E, green). The merged images along with DAPI staining (blue) in C and F show nuclear localization of the Y-1 signals. Arrows in D_F indicate Y-1_positive cells engrafted between myofibers. The arrowheads in D_F show a Y-1_positive nucleus inside a myofiber. Despite engraftment, sarcoglycan expression was not restored. (G) A single γ-sarcoglycan_positive cell detected in δ-sarcoglycan_null quadriceps after transplantation with BM-SP cells. (H) A serial section of the same donor-derived cell also expresses δ-sarcoglycan. (I) Immunostaining of a transplant-recipient muscle with dystrophin and concomitant FISH with the Y-1 probe to detect donor nuclei (dystrophin, red; Y-1, green; DAPI, blue). Arrowheads in I indicate 3 Y chromosome signals in the central or peripheral nucleus position. Scale bars: 50 μm.
Figure 4
Figure 4
Irradiation increases total engraftment into skeletal muscle. The number of Y-1_positive nuclei per square millimeter was quantified in recipient mice irradiated before transplantation, or not irradiated, and sacrificed 3 weeks after transplantation (3-wk IRRAD and 3-wk NON, respectively; n = 5 of each). The number of Y-1_positive nuclei per square millimeter was also quantified in recipient mice irradiated before transplantation and sacrificed 3 months after transplantation (3-mo IRRAD; n = 4). The 3-week irradiated group showed a significantly higher number of Y-1_positive nuclei per square millimeter compared with the 3-week nonirradiated group (Kruskal-Wallis test followed by Dunn’s multiple-comparison test, P < 0.05).
Figure 5
Figure 5
BM-SP cells engraft into myocardium but fail to express sarcoglycan. (A) Sections from a normal male heart (top panels) and a transplanted female δ-sarcoglycan_null heart (bottom panels) were stained for γ-sarcoglycan (γ-SG) and then subjected to FISH with the Y-1 probe. The left panels show γ-sarcoglycan staining in red, the middle panels show Y-1 in green, and the right panels show the merged image with DAPI in blue. Y chromosome_positive cells engrafted in the recipient myocardium between (arrow) or inside (arrowhead) cardiomyocytes but failed to restore sarcoglycan expression. YChr, Y chromosome. Scale bar: 50 μm. (B) Cross section through a cardiomyocyte containing 2 nuclei (arrows), where 1 is donor-derived. Cardiomyocytes are outlined with dystrophin (red), and the Y-1 probe (green) is found in the left nucleus but not the right. Scale bars: 50 μm.
Figure 6
Figure 6
Irradiation and a longer time interval after transplantation increase overall engraftment into the myocardium. The number of Y-1_positive nuclei per square millimeter was quantified for mice preirradiated or not preirradiated and sacrificed 3 weeks after transplantation (3-wk IRRAD and 3-wk NON, respectively; n = 5 of each). The number of Y-1_positive nuclei per square millimeter was quantified for mice preirradiated and sacrificed 3 months after transplantation (3-mo IRRAD; n = 4). A significantly higher number of Y-1_positive nuclei per square millimeter was detected in the 3-week irradiated group versus the 3-week nonirradiated group (Mann-Whitney test, P < 0.01). The 3-month irradiated group showed a significantly increased number of Y-1_positive nuclei per square millimeter compared with the 3-week irradiated group (Mann-Whitney test, P < 0.05).
Figure 7
Figure 7
BM-SP cells in between myocytes express CD45 as a marker of hematopoietic differentiation. Heart and quadriceps sections were processed for IFM with an antibody to CD45 followed by FISH with the Y-1 probe. Y chromosome hybridization signals are shown in green, CD45 is shown in red, and DAPI is shown in blue. Y chromosome_positive cells that are CD45-positive are readily detected in between skeletal myofibers as isolated cells (arrow, A) or in large clusters (B). CD45-positive donor-derived cells are also found in between cardiomyocytes in the heart of recipient mice after transplantation (arrows, C and D). Scale bar: 50 μm.
Figure 8
Figure 8
Transplantation of normal primary myoblasts restores the sarcoglycan complex in Sgcd–/– mice. The top panels represent normal control muscle without transplant. The middle panels are from an Sgcd–/– mouse. The bottom panels show serial sections from an Sgcd–/– mouse that underwent direct injection of wild-type myoblasts. Sections were immunostained with δ-sarcoglycan (δ-SG), γ-sarcoglycan (γ-SG), or β-sarcoglycan (β-SG). Myoblast transplantation can be effective to restore the sarcoglycan complex in δ-sarcoglycan_null muscle. Scale bar: 25 μm.
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