Human Umbilical Cord Blood Cells Differentiate into Muscle in sjl Muscular Dystrophy Mice
http://www.100md.com
《干细胞学杂志》
a Day Neuromuscular Research Laboratory, Massachusetts General Hospital-East, Harvard Medical School, Charlestown, Massachusetts, USA;
b ViaCell, Inc., Worcester, Massachusetts, USA
Key Words. Dysferlin ? Miyoshi myopathy ? Limb girdle muscular dystrophy type 2B sjl mice ? Human umbilical cord blood
Correspondence: Robert H. Brown Jr., M.D., D.Phil., Day Neuromuscular Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Building 114, Room 3125, 16th Street, Charlestown, Massachusetts 02129, USA. Telephone: 617-726-5750; Fax: 617-726-8543; e-mail: rhbrown@partners.org
ABSTRACT
Progressive muscle wasting and weakness characterize muscular dystrophies. A subgroup of autosomal recessive dystrophies, the limb girdle muscular dystrophies (LGMDs), is characterized by weakness and wasting of muscles of the pelvic and shoulder girdle . Another dystrophy, Miyoshi myopathy (MM), affects distal muscles at onset, with preferential early involvement of the gastrocnemius muscle. Mutations in a novel muscle gene, dysferlin, cause the type 2B form of LGMD (LGMD-2B) and MM; dysferlin expression is reduced or absent in these patients . A 171-bp deletion in the murine dysf gene was detected in the sjl mice, with a corresponding reduction in dysferlin levels to 15% of normal . Thus, the sjl mouse is a natural model of LGMD-2B/MM.
Muscle cells are formed developmentally by the fusion of multiple muscle precursor cells and are therefore multinucleated. Moreover, normal adult muscle retains a population of satellite cells that are capable of differentiating into myoblasts and fusing to regenerate muscle after injury. These observations led to the hypothesis that creation of chimeric muscle cells by introduction of wild-type, nondystrophic nuclei at the time of fusion could correct the deficiency of a protein in a dystrophic muscle cell. Although early studies of engrafting muscle precursor cells into dystrophic mdx host mouse muscle were promising , attempts to use human myoblasts to restore dystrophin expression in muscles of patients with Becker and Duchenne dystrophy were not successful despite the long-term survival of some donor myoblasts . One interpretation of these studies was that the donor myoblasts were at an inappropriate developmental stage to reproduce the behavior of satellite cells; conceivably, less-differentiated but muscle tissue–specific stem cells might engraft into host muscle more successfully. In the past 5 years, several studies have identified populations of undifferentiated cells within bone marrow that demonstrate properties of stem cells, including myogenic stem cells with the capacity to proliferate as myoblasts and participate in normal muscle regeneration . Hoechst-stained side populations of cells (SP cells) isolated from either bone marrow or muscle and a subpopulation of long-time proliferating cells or Sca-1+/CD34+ muscle-derived cells also engraft muscle and restore expression of a muscle-specific protein after local or systemic administration in recipient animals. These reports argue that systemic administration of muscle stem cells may provide an effective form of gene and protein replacement therapy for recessively inherited, loss-of-function muscular dystrophies.
If this therapy is to be clinically practical, one of many issues that must be addressed is the availability of appropriate numbers of muscle stem cells. Although xenogeneic transplantation strategies might be considered, it can also be argued that an additional constraint is that the cells should, optimally, be human in origin. Indeed, if human-derived donor stem cells can be obtained that immunomatch the recipient, it is conceivable that immune barriers to successful muscle cell therapy can be minimized. As an alternative to generating large numbers of immunomatched donor human muscle stem cells from sources such as donor biopsies, we have considered the possibility that human umbilical cord blood (HUCB) cells might be a renewable and noncontroversial source of stem cells containing myogenic precursors.
Since transplantation of HUCB cells was successfully performed for the first time in 1988 to treat Fanconi’s anemia , there has been extensive experience in the use of HUCB cells to reconstitute the hematopoietic system in both humans and mice . It is now apparent that the proliferative capacity of hematopoietic stem cells in cord blood is superior to that of cells in the marrow of blood from adults . Moreover, the use of HUCB reduces risk of graft-versus-host disease . Some studies suggest that HUCB-derived cells can differentiate into nonhematopoietic cells. Thus, cells derived from HUCB can differentiate into neurons or glia both in vitro and in vivo . Mesenchymal precursor cells have also been obtained from HUCB . Recent studies document that subpopulations of HUCB cells can transdifferentiate into hepatic, endothelial, or muscle cells .
The present investigation was performed to test the concept that populations of HUCB cells can engraft into recipient dystrophic muscle cells after systemic administration, fuse, and become part of myofibers in muscle cells, thereby allowing expression of muscle proteins absent in the dystrophic host. We have tested this hypothesis using sjl mouse as a model of LGMD-2B/MM in which dysferlin is absent. In these studies, the end point for successful engraftment and myogenic differentiation of subsets of HUCB was the presence of dysferlin or, as a second marker of myogenicity, human-specific dystrophin in muscle that is normally devoid of these proteins.
MATERIALS AND METHODS
Specific Markers to Distinguish Human Origin Donor Cells
We used immunostaining with three different antibodies to determine whether HUCB cells survive and undergo myogenic differentiation within the sjl recipients after intravenous administration. The control experiments documented that anti-NuMA and anti-dystrophin antibodies bind specifically to human NuMA and dystrophin (Figs. 1A, 1C). The anti-dysferlin antibody interacts strongly with both human and murine dysferlin antigens, but, as expected, it does not detect dysferlin in dysferlin-deficient sjl mouse muscle (Fig. 1B). Other control experiments showed excellent costaining of NuMA and dysferlin proteins or dystrophin and dysferlin proteins (Figs. 2A, 2B). These three antibodies (NuMA, dysferlin, and dystrophin) thus clearly allow detection of engrafted, myogenic human donor cells and the restoration of dysferlin expression in the recipient animals.
Figure 1. Specific markers identify human donor cells in recipient sjl muscle. (A): Immunostaining with human-specific anti-NuMA primary monoclonal antibody using an FITC-conjugated goat anti-mouse IgG second antibody. (B): Anti-Dysf immunostaining, detected with Cy3-conjugated goat anti-mouse IgG. (C): Immunostaining with human-specific anti-Dys monoclonal antibody, counterstained with FITC-conjugated goat anti-mouse IgG. All sections were counterstained with DAPI. In A, B, and C, the top row is human skeletal muscle (i); the bottom row is sjl mouse skeletal muscle (ii). Scale bar = 50 μm.Abbreviations: Cy3, cyanine dye; DAPI, 4,6-diamidino-2-phenylindole; Dys, dystrophin; Dysf, dysferlin; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; NuMA, nuclear-mitotic apparatus protein.
Figure 2. Dysf-positive fibers in human skeletal muscle are positive for NuMA and human Dys immunofluorescence. (A): Sequential staining with human-specific anti-NuMA antibody, FITC-conjugated goat anti-mouse Ig, anti-Dysf antibody, and Cy3-conjugated goat anti-mouse Ig. (B): Sequential staining with human-specific anti-Dys antibody, FITC-conjugated goat anti-mouse Ig, anti-Dysf antibody, and TRITC-conjugated goat anti-mouse Ig. All sections were counterstained with DAPI. Abbreviations: Cy3, cyanine dye; DAPI, 4,6-diamidino-2-phenylindole; Dysf, dysferlin; Dys, dystrophin; FITC, fluorescein isothiocyanate; NuMA, nuclear-mitotic apparatus protein; TRITC, tetramethylrhodamine isothiocyanate.
Human Donor Cells Were Detected in sjl Mice Infused with Two Cell Types
We have divided the sjl mice into three groups, group A (n = 8), group B (n = 8), and group C (n = 8), corresponding to the transplantation with the following three different types of donor cells: whole HUCB, LIN–CD34+/– subpopulation of HUCB, and irradiated spleen cells. Each mouse was injected with 1 x 106 HUCB cells or control spleen cells systemically. Two animals from each group (A, B, or C) were euthanized at each time point after the transplantation (1, 4, 8, or 12 weeks). Two skeletal muscles, gastrocnemius and quadriceps, were harvested from each animal for immunohisto-chemical analyses. In this initial study, we did not evaluate time points after 12 weeks, because this was the longest point analyzed in previous studies of systemic delivery of muscle stem cells .
The first set of experiments analyzed the survival and engraftment levels of donor cells in the recipient animals. We used the human-specific antibody NuMA to distinguish the donor stem cells (human origin) from the host cells (mouse). Ten sections from each muscle (gastrocnemius and quadriceps) collected at the above four different time points were examined for NuMA immunofluorescent staining. This was readily detectable in animals infused with either the whole HUCB (group A) or the LIN–CD34+/– HUCB (group B) and colocalized with nuclear staining by 4,6-diamidino-2-phenylindole (DAPI) (Figs. 3A, 3B). By contrast, no NuMA staining was seen from muscles in animals infused with the irradiated mouse spleen cells (group C).
Figure 3. Human donor cells are detected in gastrocnemius muscle of sjl mice at 1 and 12 weeks after infusion of whole HUCB cells (A) or LIN–CD34+/– HUCB cells (B). NuMA immunofluorescence is evident in sjl mouse muscle after administration of whole HUCB cells (A) or fractionated LIN–CD34+/– HUCB cells (B). All sections were counterstained with DAPI. In both (A) and (B), the top row (i) is muscle examined at 1 week after HUCB infusion; the bottom row (ii) is muscle immunostained at 12 weeks after HUCB infusion. Scale bar = 25 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; NuMA, nuclear-mitotic apparatus protein.
Expression of Dysferlin within NuMA-Positive Muscle Cells
A small number of the NuMA-positive muscle cells, <1% per muscle section, expressed dysferlin in both group A and B animals (Fig. 4). Although NuMA immunofluorescent staining was readily detected from all postinfusion muscle samples (1, 4, 8, and 12 weeks) in both group A and B animals, dysferlin expression was only detected at 12 weeks after transplantation (Fig. 4).
Figure 4. Dysf is detected in NuMA-positive muscle cells from sjl mice 12 weeks after infusion of whole and fractionated LIN–CD34+/–HUCB cells. (A): Coimmunostaining for NuMA and Dysf in sjl muscle after infusion of unfractionated HUCB cells. (B, C): Coimmunofluorescent staining for NuMA and Dysf in sjl muscle after administration of LIN–CD34+/– HUCB cells. All sections were counterstained with DAPI. (A, B): Gastrocnemius muscle. (C): Quadriceps muscle. Scale bar = 25 μm.Abbreviations: Cy3, cyanine dye; DAPI, 4,6-diamidino-2-phenylindole; Dysf, dysferlin; Dys, dystrophin; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; NuMA, nuclear-mitotic apparatus protein.
Muscle-Specific Protein Is Synthesized by Human Donor Cells 12 Weeks after Infusion
To exclude the possibility that the dysferlin-positive fibers are endogenous sjl muscle fibers that have undergone spontaneous mutational reversion that permits expression of mouse dysferlin, we have repeated the immunohistochemical analyses to survey for fibers that are positive for both human-specific dystrophin and dysferlin. In these studies, colocalization of dystrophin and dysferlin was evident at 12 weeks after transplantation (Fig. 5). The positive immunostaining for human-specific dystrophin clearly designates these muscle cells as human. Thus, these dystrophin-positive fibers constitute additional evidence that a subpopulation of the HUCB cells is capable of undergoing myogenesis.
Figure 5. Human-specific Dys and Dysf are detected in sjl mice 12 weeks after infusion of unfractionated and LIN–CD34+/– HUCB cells. (A): Coimmunofluorescent staining for Dysf and human-specific Dys after administration of unfractionated HUCB. (B, C): Coimmunofluorescent staining for Dys and human-specific Dys after infusions of LIN–CD34+/– HUCB cells. All sections were counterstained with DAPI. (A, B): Gastrocnemius muscle. (C): Quadriceps muscle. Scale bar = 25 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; Dysf, dysferlin; Dys, dystrophin; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; TRITC, tetramethylrhodamine isothiocyanate.
Semi-Quantitative Analysis of HUCB Donor Cell Engraftment and Restoration of Dysferlin Expression in sjl Recipients
Table 1 summarizes our results. It is clear that at 12 weeks after transplantation, there are detectable cells and myofibers of human origin specifically in those sjl mice that received preparations either of whole HUCB or LIN–CD34+/– HUCB. Because no dysferlin or human dystrophin was detected at weeks 1, 4, and 8, Table 1 only summarizes the 12-week data.
Table 1. Semiquantification of human umbilical cord blood (HUCB)cells in sjl recipient skeletal muscle
As indicated, quantitation of immunopositivity in multiple sections revealed that the numbers of immunopositive cells (NuMA, dysferlin, and dystrophin) were very small. However, approximately half of the NuMA-positive cells were associated with dysferlin staining. Moreover, every cell that expressed dysferlin also expressed human dystrophin and reciprocally. Both human-specific markers (NuMA and dystrophin) and the dysferlin immunostaining were negative from all 10 sections examined from animals infused with the mouse spleen cell preparation. It was our impression that the selected LIN–CD34+/– cells were more favorable for the myogenic differentiation, in part because dysferlin-positive cells of LIN–CD34+/– origin were slightly more frequent and because they sometimes appeared in clusters, as if they had undergone local proliferation. It is clear that additional studies will be needed to confirm this observation, because the number of surviving and differentiated HUCB cells is small.
Human Donor Cells Were Detected in Liver and Kidney of sjl Mice Infused with Two Cell Types
Because intravenous administration results in a more systemic distribution of donor cells within the recipient animals, various organs were also collected and analyzed to account for the possibility of donor cell engraftment within organs other than the skeletal muscle in these animals.
Four nonserial sections from both liver and kidney were examined for NuMA immunofluorescent staining. Once again, this was readily detectable in animals infused with the whole HUCB (group A) or the LIN–CD34+/–-enriched HUCB (group B) and colocalized with nuclear staining by DAPI (Fig. 6). No NuMA staining was seen from livers and kidneys collected from animals infused with the irradiated mouse spleen cells (group C).
Figure 6. Human donor cells are detected in liver and kidney of sjl mice at 12 weeks after infusion. NuMA immunofluorescence is evident in sjl mouse kidney (A) or liver (B) after administration of either whole HUCB cell (i) or fractionated LIN–CD34+/– HUCB cells (ii). All sections are counterstained with DAPI. In both (A) and (B), the samples were collected 12 weeks after HUCB cell infusion. Scale bar = 25 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; NuMA, nuclear-mitotic apparatus protein.
Low Levels of T-Lymphocyte Infiltration Were Detected in FK506/Leflunomide-Treated sjl Mice
Assessing the infiltration of immune cells in the muscles of the injected animals allows us to evaluate the immune rejection against transplanted cells. Only one muscle (gastrocnemius) from the 12-week time point of both animals from all three groups was examined for CD4 or CD8 immunoperoxidase staining. A total of three nonserial sections were analyzed, and an average of ~30 to 100 CD4+ or CD8+ cells per section were seen from all of the animals that were immunosuppressed with both FK506 and leflunomide (Fig. 7).
Figure 7. T-lymphocyte infiltration is present in gastrocnemius samples collected from immunosuppressed sjl mice 12 weeks after infusion. Immunoperoxidase staining for the presence of immune cells in gastrocnemius samples collected from group A (A), group B (B), or group C (C) animals. Row (i) shows immunostaining with anti-CD4 antibody. CD4+ cells are indicated by arrowheads. Row (ii) shows immunostaining with anti-CD8a antibody. CD8+ cells are indicated by arrowheads. All sections are counterstained with hematoxylin. Scale bar = 50 μm.
DISCUSSION
This work was supported by the C. B. Day Investment Company, ViaCell, Inc., the Thayer and Jain families, and National Institutes of Health award 5P01BS40828. K.Y.K. was supported by a development grant from Muscular DystrophyAssociation.
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b ViaCell, Inc., Worcester, Massachusetts, USA
Key Words. Dysferlin ? Miyoshi myopathy ? Limb girdle muscular dystrophy type 2B sjl mice ? Human umbilical cord blood
Correspondence: Robert H. Brown Jr., M.D., D.Phil., Day Neuromuscular Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Building 114, Room 3125, 16th Street, Charlestown, Massachusetts 02129, USA. Telephone: 617-726-5750; Fax: 617-726-8543; e-mail: rhbrown@partners.org
ABSTRACT
Progressive muscle wasting and weakness characterize muscular dystrophies. A subgroup of autosomal recessive dystrophies, the limb girdle muscular dystrophies (LGMDs), is characterized by weakness and wasting of muscles of the pelvic and shoulder girdle . Another dystrophy, Miyoshi myopathy (MM), affects distal muscles at onset, with preferential early involvement of the gastrocnemius muscle. Mutations in a novel muscle gene, dysferlin, cause the type 2B form of LGMD (LGMD-2B) and MM; dysferlin expression is reduced or absent in these patients . A 171-bp deletion in the murine dysf gene was detected in the sjl mice, with a corresponding reduction in dysferlin levels to 15% of normal . Thus, the sjl mouse is a natural model of LGMD-2B/MM.
Muscle cells are formed developmentally by the fusion of multiple muscle precursor cells and are therefore multinucleated. Moreover, normal adult muscle retains a population of satellite cells that are capable of differentiating into myoblasts and fusing to regenerate muscle after injury. These observations led to the hypothesis that creation of chimeric muscle cells by introduction of wild-type, nondystrophic nuclei at the time of fusion could correct the deficiency of a protein in a dystrophic muscle cell. Although early studies of engrafting muscle precursor cells into dystrophic mdx host mouse muscle were promising , attempts to use human myoblasts to restore dystrophin expression in muscles of patients with Becker and Duchenne dystrophy were not successful despite the long-term survival of some donor myoblasts . One interpretation of these studies was that the donor myoblasts were at an inappropriate developmental stage to reproduce the behavior of satellite cells; conceivably, less-differentiated but muscle tissue–specific stem cells might engraft into host muscle more successfully. In the past 5 years, several studies have identified populations of undifferentiated cells within bone marrow that demonstrate properties of stem cells, including myogenic stem cells with the capacity to proliferate as myoblasts and participate in normal muscle regeneration . Hoechst-stained side populations of cells (SP cells) isolated from either bone marrow or muscle and a subpopulation of long-time proliferating cells or Sca-1+/CD34+ muscle-derived cells also engraft muscle and restore expression of a muscle-specific protein after local or systemic administration in recipient animals. These reports argue that systemic administration of muscle stem cells may provide an effective form of gene and protein replacement therapy for recessively inherited, loss-of-function muscular dystrophies.
If this therapy is to be clinically practical, one of many issues that must be addressed is the availability of appropriate numbers of muscle stem cells. Although xenogeneic transplantation strategies might be considered, it can also be argued that an additional constraint is that the cells should, optimally, be human in origin. Indeed, if human-derived donor stem cells can be obtained that immunomatch the recipient, it is conceivable that immune barriers to successful muscle cell therapy can be minimized. As an alternative to generating large numbers of immunomatched donor human muscle stem cells from sources such as donor biopsies, we have considered the possibility that human umbilical cord blood (HUCB) cells might be a renewable and noncontroversial source of stem cells containing myogenic precursors.
Since transplantation of HUCB cells was successfully performed for the first time in 1988 to treat Fanconi’s anemia , there has been extensive experience in the use of HUCB cells to reconstitute the hematopoietic system in both humans and mice . It is now apparent that the proliferative capacity of hematopoietic stem cells in cord blood is superior to that of cells in the marrow of blood from adults . Moreover, the use of HUCB reduces risk of graft-versus-host disease . Some studies suggest that HUCB-derived cells can differentiate into nonhematopoietic cells. Thus, cells derived from HUCB can differentiate into neurons or glia both in vitro and in vivo . Mesenchymal precursor cells have also been obtained from HUCB . Recent studies document that subpopulations of HUCB cells can transdifferentiate into hepatic, endothelial, or muscle cells .
The present investigation was performed to test the concept that populations of HUCB cells can engraft into recipient dystrophic muscle cells after systemic administration, fuse, and become part of myofibers in muscle cells, thereby allowing expression of muscle proteins absent in the dystrophic host. We have tested this hypothesis using sjl mouse as a model of LGMD-2B/MM in which dysferlin is absent. In these studies, the end point for successful engraftment and myogenic differentiation of subsets of HUCB was the presence of dysferlin or, as a second marker of myogenicity, human-specific dystrophin in muscle that is normally devoid of these proteins.
MATERIALS AND METHODS
Specific Markers to Distinguish Human Origin Donor Cells
We used immunostaining with three different antibodies to determine whether HUCB cells survive and undergo myogenic differentiation within the sjl recipients after intravenous administration. The control experiments documented that anti-NuMA and anti-dystrophin antibodies bind specifically to human NuMA and dystrophin (Figs. 1A, 1C). The anti-dysferlin antibody interacts strongly with both human and murine dysferlin antigens, but, as expected, it does not detect dysferlin in dysferlin-deficient sjl mouse muscle (Fig. 1B). Other control experiments showed excellent costaining of NuMA and dysferlin proteins or dystrophin and dysferlin proteins (Figs. 2A, 2B). These three antibodies (NuMA, dysferlin, and dystrophin) thus clearly allow detection of engrafted, myogenic human donor cells and the restoration of dysferlin expression in the recipient animals.
Figure 1. Specific markers identify human donor cells in recipient sjl muscle. (A): Immunostaining with human-specific anti-NuMA primary monoclonal antibody using an FITC-conjugated goat anti-mouse IgG second antibody. (B): Anti-Dysf immunostaining, detected with Cy3-conjugated goat anti-mouse IgG. (C): Immunostaining with human-specific anti-Dys monoclonal antibody, counterstained with FITC-conjugated goat anti-mouse IgG. All sections were counterstained with DAPI. In A, B, and C, the top row is human skeletal muscle (i); the bottom row is sjl mouse skeletal muscle (ii). Scale bar = 50 μm.Abbreviations: Cy3, cyanine dye; DAPI, 4,6-diamidino-2-phenylindole; Dys, dystrophin; Dysf, dysferlin; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; NuMA, nuclear-mitotic apparatus protein.
Figure 2. Dysf-positive fibers in human skeletal muscle are positive for NuMA and human Dys immunofluorescence. (A): Sequential staining with human-specific anti-NuMA antibody, FITC-conjugated goat anti-mouse Ig, anti-Dysf antibody, and Cy3-conjugated goat anti-mouse Ig. (B): Sequential staining with human-specific anti-Dys antibody, FITC-conjugated goat anti-mouse Ig, anti-Dysf antibody, and TRITC-conjugated goat anti-mouse Ig. All sections were counterstained with DAPI. Abbreviations: Cy3, cyanine dye; DAPI, 4,6-diamidino-2-phenylindole; Dysf, dysferlin; Dys, dystrophin; FITC, fluorescein isothiocyanate; NuMA, nuclear-mitotic apparatus protein; TRITC, tetramethylrhodamine isothiocyanate.
Human Donor Cells Were Detected in sjl Mice Infused with Two Cell Types
We have divided the sjl mice into three groups, group A (n = 8), group B (n = 8), and group C (n = 8), corresponding to the transplantation with the following three different types of donor cells: whole HUCB, LIN–CD34+/– subpopulation of HUCB, and irradiated spleen cells. Each mouse was injected with 1 x 106 HUCB cells or control spleen cells systemically. Two animals from each group (A, B, or C) were euthanized at each time point after the transplantation (1, 4, 8, or 12 weeks). Two skeletal muscles, gastrocnemius and quadriceps, were harvested from each animal for immunohisto-chemical analyses. In this initial study, we did not evaluate time points after 12 weeks, because this was the longest point analyzed in previous studies of systemic delivery of muscle stem cells .
The first set of experiments analyzed the survival and engraftment levels of donor cells in the recipient animals. We used the human-specific antibody NuMA to distinguish the donor stem cells (human origin) from the host cells (mouse). Ten sections from each muscle (gastrocnemius and quadriceps) collected at the above four different time points were examined for NuMA immunofluorescent staining. This was readily detectable in animals infused with either the whole HUCB (group A) or the LIN–CD34+/– HUCB (group B) and colocalized with nuclear staining by 4,6-diamidino-2-phenylindole (DAPI) (Figs. 3A, 3B). By contrast, no NuMA staining was seen from muscles in animals infused with the irradiated mouse spleen cells (group C).
Figure 3. Human donor cells are detected in gastrocnemius muscle of sjl mice at 1 and 12 weeks after infusion of whole HUCB cells (A) or LIN–CD34+/– HUCB cells (B). NuMA immunofluorescence is evident in sjl mouse muscle after administration of whole HUCB cells (A) or fractionated LIN–CD34+/– HUCB cells (B). All sections were counterstained with DAPI. In both (A) and (B), the top row (i) is muscle examined at 1 week after HUCB infusion; the bottom row (ii) is muscle immunostained at 12 weeks after HUCB infusion. Scale bar = 25 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; NuMA, nuclear-mitotic apparatus protein.
Expression of Dysferlin within NuMA-Positive Muscle Cells
A small number of the NuMA-positive muscle cells, <1% per muscle section, expressed dysferlin in both group A and B animals (Fig. 4). Although NuMA immunofluorescent staining was readily detected from all postinfusion muscle samples (1, 4, 8, and 12 weeks) in both group A and B animals, dysferlin expression was only detected at 12 weeks after transplantation (Fig. 4).
Figure 4. Dysf is detected in NuMA-positive muscle cells from sjl mice 12 weeks after infusion of whole and fractionated LIN–CD34+/–HUCB cells. (A): Coimmunostaining for NuMA and Dysf in sjl muscle after infusion of unfractionated HUCB cells. (B, C): Coimmunofluorescent staining for NuMA and Dysf in sjl muscle after administration of LIN–CD34+/– HUCB cells. All sections were counterstained with DAPI. (A, B): Gastrocnemius muscle. (C): Quadriceps muscle. Scale bar = 25 μm.Abbreviations: Cy3, cyanine dye; DAPI, 4,6-diamidino-2-phenylindole; Dysf, dysferlin; Dys, dystrophin; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; NuMA, nuclear-mitotic apparatus protein.
Muscle-Specific Protein Is Synthesized by Human Donor Cells 12 Weeks after Infusion
To exclude the possibility that the dysferlin-positive fibers are endogenous sjl muscle fibers that have undergone spontaneous mutational reversion that permits expression of mouse dysferlin, we have repeated the immunohistochemical analyses to survey for fibers that are positive for both human-specific dystrophin and dysferlin. In these studies, colocalization of dystrophin and dysferlin was evident at 12 weeks after transplantation (Fig. 5). The positive immunostaining for human-specific dystrophin clearly designates these muscle cells as human. Thus, these dystrophin-positive fibers constitute additional evidence that a subpopulation of the HUCB cells is capable of undergoing myogenesis.
Figure 5. Human-specific Dys and Dysf are detected in sjl mice 12 weeks after infusion of unfractionated and LIN–CD34+/– HUCB cells. (A): Coimmunofluorescent staining for Dysf and human-specific Dys after administration of unfractionated HUCB. (B, C): Coimmunofluorescent staining for Dys and human-specific Dys after infusions of LIN–CD34+/– HUCB cells. All sections were counterstained with DAPI. (A, B): Gastrocnemius muscle. (C): Quadriceps muscle. Scale bar = 25 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; Dysf, dysferlin; Dys, dystrophin; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; TRITC, tetramethylrhodamine isothiocyanate.
Semi-Quantitative Analysis of HUCB Donor Cell Engraftment and Restoration of Dysferlin Expression in sjl Recipients
Table 1 summarizes our results. It is clear that at 12 weeks after transplantation, there are detectable cells and myofibers of human origin specifically in those sjl mice that received preparations either of whole HUCB or LIN–CD34+/– HUCB. Because no dysferlin or human dystrophin was detected at weeks 1, 4, and 8, Table 1 only summarizes the 12-week data.
Table 1. Semiquantification of human umbilical cord blood (HUCB)cells in sjl recipient skeletal muscle
As indicated, quantitation of immunopositivity in multiple sections revealed that the numbers of immunopositive cells (NuMA, dysferlin, and dystrophin) were very small. However, approximately half of the NuMA-positive cells were associated with dysferlin staining. Moreover, every cell that expressed dysferlin also expressed human dystrophin and reciprocally. Both human-specific markers (NuMA and dystrophin) and the dysferlin immunostaining were negative from all 10 sections examined from animals infused with the mouse spleen cell preparation. It was our impression that the selected LIN–CD34+/– cells were more favorable for the myogenic differentiation, in part because dysferlin-positive cells of LIN–CD34+/– origin were slightly more frequent and because they sometimes appeared in clusters, as if they had undergone local proliferation. It is clear that additional studies will be needed to confirm this observation, because the number of surviving and differentiated HUCB cells is small.
Human Donor Cells Were Detected in Liver and Kidney of sjl Mice Infused with Two Cell Types
Because intravenous administration results in a more systemic distribution of donor cells within the recipient animals, various organs were also collected and analyzed to account for the possibility of donor cell engraftment within organs other than the skeletal muscle in these animals.
Four nonserial sections from both liver and kidney were examined for NuMA immunofluorescent staining. Once again, this was readily detectable in animals infused with the whole HUCB (group A) or the LIN–CD34+/–-enriched HUCB (group B) and colocalized with nuclear staining by DAPI (Fig. 6). No NuMA staining was seen from livers and kidneys collected from animals infused with the irradiated mouse spleen cells (group C).
Figure 6. Human donor cells are detected in liver and kidney of sjl mice at 12 weeks after infusion. NuMA immunofluorescence is evident in sjl mouse kidney (A) or liver (B) after administration of either whole HUCB cell (i) or fractionated LIN–CD34+/– HUCB cells (ii). All sections are counterstained with DAPI. In both (A) and (B), the samples were collected 12 weeks after HUCB cell infusion. Scale bar = 25 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; FITC, fluorescein isothiocyanate; HUCB, human umbilical cord blood; NuMA, nuclear-mitotic apparatus protein.
Low Levels of T-Lymphocyte Infiltration Were Detected in FK506/Leflunomide-Treated sjl Mice
Assessing the infiltration of immune cells in the muscles of the injected animals allows us to evaluate the immune rejection against transplanted cells. Only one muscle (gastrocnemius) from the 12-week time point of both animals from all three groups was examined for CD4 or CD8 immunoperoxidase staining. A total of three nonserial sections were analyzed, and an average of ~30 to 100 CD4+ or CD8+ cells per section were seen from all of the animals that were immunosuppressed with both FK506 and leflunomide (Fig. 7).
Figure 7. T-lymphocyte infiltration is present in gastrocnemius samples collected from immunosuppressed sjl mice 12 weeks after infusion. Immunoperoxidase staining for the presence of immune cells in gastrocnemius samples collected from group A (A), group B (B), or group C (C) animals. Row (i) shows immunostaining with anti-CD4 antibody. CD4+ cells are indicated by arrowheads. Row (ii) shows immunostaining with anti-CD8a antibody. CD8+ cells are indicated by arrowheads. All sections are counterstained with hematoxylin. Scale bar = 50 μm.
DISCUSSION
This work was supported by the C. B. Day Investment Company, ViaCell, Inc., the Thayer and Jain families, and National Institutes of Health award 5P01BS40828. K.Y.K. was supported by a development grant from Muscular DystrophyAssociation.
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