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. 2014 Feb 12;5(1):21.
doi: 10.1186/scrt411.

Human adipose-derived stem cell transplantation as a potential therapy for collagen VI-related congenital muscular dystrophy

Vitali Alexeev, Machiko Arita, Adele Donahue, Paolo Bonaldo, Mon-Li Chu, Olga Igoucheva

Human adipose-derived stem cell transplantation as a potential therapy for collagen VI-related congenital muscular dystrophy

Vitali Alexeev et al. Stem Cell Res Ther. .

Abstract

Introduction: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness within the first two years of life. Collagen VI-related muscle disorders have recently emerged as one of the most common types of CMD. COL6 CMD is caused by deficiency and/or dysfunction of extracellular matrix (ECM) protein collagen VI. Currently, there is no specific treatment for this disabling and life-threatening disease. The primary cellular targets for collagen VI CMD therapy are fibroblasts in muscle, tendon and skin, as opposed to muscle cells for other types of muscular dystrophies. However, recent advances in stem cell research have raised the possibility that use of adult stem cells may provide dramatic new therapies for treatment of COL6 CMD.

Methods: Here, we developed a procedure for isolation of human stem cells from the adipose layer of neonatal skin. The adipose-derived stem cells (ADSC) were examined for expression of ECM and related genes using gene expression array analysis. The therapeutic potential of ADSC was assessed after a single intramuscular transplantation in collagen VI-deficient mice.

Results: Analysis of primary cultures confirmed that established ADSC represent a morphologically homogenous population with phenotypic and functional features of adult mesenchymal stem cells. A comprehensive gene expression analysis showed that ADSC express a vast array of ECM genes. Importantly, it was observed that ADSC synthesize and secrete all three collagen VI chains, suggesting suitability of ADSC for COL6 CMD treatment. Furthermore, we have found that a single intramuscular transplantation of ADSC into Col6a1-/-Rag1-/- mice under physiological and cardiotoxin-induced injury/regeneration conditions results in efficient engraftment and migration of stem cells within the skeletal muscle. Importantly, we showed that ADSC can survive long-term and continuously secrete the therapeutic collagen VI protein missing in the mutant mice.

Conclusions: Overall, our findings suggest that stem cell therapy can potentially provide a new avenue for the treatment of COL6 CMD and other muscular disorders and injuries.

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Figures

Figure 1
Figure 1
Phenotypic analysis of primary human neonatal ADSC. (A) Immunophenotypic analysis was performed using fluorescein-conjugated antibodies. For each sample, 10,000 events were read and the percent of positive cells expressing the respective surface marker is listed in the box. (B) Multilineage differentiation capacity of neonatal ADSC. Cells treated with osteogenic supplements showed an increased number of alkaline phosphatase-positive cells. Treatment of cells with adipogenic supplements resulted in the formation of adipocytic cells containing intracellular lipid droplets as detected by oil red staining. ADSC cultured in micromass showed a chondrogenic phenotype as detected by Alcian blue staining. (C) RT-PCR analysis of lineage-specific genes. Alkaline phosphatase (ALP) and osteocalcin (OCN) were used to indicate commitment to the osteogenic lineage. Lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor gamma transcript variant 2 (PPARγ2) were used to indicate cells of an adipogenic lineage. SRY (sex determining region Y)-box 9 (SOX9), collagen type II (COLII), collagen type X (COLX), collagen type XI (COLXI), and aggrecan (AGN) were used to show commitment to the chondrogenic lineage. Top row shows the basal expression of these genes in control ADSC at 0 weeks. Bottom row shows expression of genes after three weeks of culturing in inductive media. (D) RT-PCR analysis for the expression of COL6A1, COL6A2 and COL6A3 genes in cultured ADSC using gene-specific primers. (E) Western blot analysis of individual (α1, α2, α3) collagen VI chains in ADSC using chain-specific antibodies. (F) Immunofluorescence analysis of individual (α1, α2, α3) chains of collagen VI was performed using chain-specific antibodies (red) and co-stained with human lamin A/C (green). ADSC, adipose-derived stem cells.
Figure 2
Figure 2
Intramuscular transplantation of human neonatal ADSC into Col6a1−/−Rag1−/−mice. (A) Representative in vivo images showing the hindlimbs that received a single intramuscular injection of ADSC. Cells were cultured in the presence of a red fluorescent lipophilic dye (DiOC18), and 0.5 × 106 DiOC18-ADSC were injected into the left GCM. The right GCM served as the control. IVIS imaging was performed at one, two, three, four and six weeks post-transplantation. (B) Morphometric analysis of DiOC18-ADSC engraftment into the GCM was performed at one, two, three, four and six weeks post-transplantation. The percentage of engrafted cells was determined by staining of transplanted GCM with lamin A/C antibody as a marker for donor cells and by counting lamin A/C positive-cells on at least fifty 7 μm sections. As an additional approach, the total number of DiOC18-positive ADSC was calculated using FACS analysis of a single-cell suspension of the GCM containing ADSC transplant. The vertical axis shows the percentage (%) of engrafted ADSC per injected muscle. The horizontal axis shows the time point after transplantation in weeks (w). (C) Morphometric analysis of α1(VI)-positive myofibers in the GCM transplanted with ADSC. The number of α1(VI)-positive myofibers was determined by counting a minimum of fifty 7 μm sections per animal. The vertical axis shows the number of α1(VI)-positive myofibers per section. The horizontal axis shows the time point after transplantation in weeks (w). In all studies, at least five animals were analyzed per time point. Error bars represent means ± SEM. ADSC, adipose-derived stem cells; FACS, fluorescence-activated cell sorting; GCM, gastrocnemius muscle; SEM, standard error of the mean.
Figure 3
Figure 3
Histochemical and indirect immunofluorescence analyses of GCM biopsies after transplantation of human neonatal ADSC into Col6a1−/−Rag1−/−mice. Muscle biopsies were collected at one, two, three, four, and six weeks after transplantation under homeostatic conditions. Time points are indicated to the left of the panels. (A) Hematoxylin and eosin staining (H & E) of the ADSC-treated GCM. (B, C) Indirect immunofluorescence detection of ADSC-derived collagen VI in the transplanted GCM. Donor cells were detected with anti-human lamin A/C antibodies (AlexaFluor488, green) and collagen VI-positive myofibers were detected with anti-α1(VI)-collagen antibodies (AlexaFluor594, red). Images were taken from representative sections at low (B) and high (C) magnification, respectively. (D, E) Co-localization of the ADSC-donated α1(VI)-collagen (AlexaFluor488, green) and basement-membrane-associated type IV collagen (AlexaFluor594, red) in ADSC-treated muscles. Images were taken from representative sections at low (D) and high (E) magnification, respectively. Nuclei were stained with DAPI (blue). Scale bar, 100 μm (low magnification) and 25 μm (high magnification), respectively. ADSC, adipose-derived stem cells; DAPI, 4′,6-diamidino-2-phenyl indol; GCM, gastrocnemius muscle.
Figure 4
Figure 4
Analysis of muscle tissue infiltration with CD11b-positive cells after ADSC transplantation. (A) Indirect immunofluorescence assessment of the infiltrating CD11b-positive leukocytes (green) in untreated and ADSC-treated muscles of Col6a1−/−Rag1−/− mice at different time points after transplantation. (B) Indirect immunofluorescence assessment of the infiltrating CD11b-positive leukocytes (green) in CTX-injured muscles with or without ADSC transplantation. In all cases, ADSC were detected with anti-human lamin A/C antibodies (red), whereas infiltrating CD11b-positive cells were detected with anti-CD11b antibodies (green). Time points are indicated to the left of the panels. Corresponding treatment is shown on top of the panels. Nuclei were stained with DAPI (blue). Scale bar, 100 μm. ADSC, adipose-derived stem cells; CTX, cardiotoxin; DAPI, 4′,6-diamidino-2-phenyl indol.
Figure 5
Figure 5
Histochemical and indirect immunofluorescent analyses of GCM biopsies after ADSC transplantation and CTX injury. Biopsies were collected at one, two, three, four and six weeks after ADSC transplantation (indicated to the left of the panels). (A) Hematoxylin and eosin staining (H & E) staining of ADSC-treated and CTX-injured GCM. (B, C) Indirect immunofluorescent detection of ADSC-derived collagen VI was performed using anti-lamin A/C (green) and anti-α1(VI) collagen (red) antibodies. Images were taken from representative sections at low (B) and high (C) magnification, respectively. Progressive, time-dependent spreading of transplanted ADSC within muscle tissue and donation of type VI collagen into perimysium and endomysium is apparent. (D, E) Indirect immunofluorescence detection of the α1(VI) collagen (green) and type IV collagen (red) co-localization at the basement membrane of the ADSC-treated, CTX-injured GCM. Images were taken from representative sections at low (D) and high (E) magnification, respectively. Nuclei were stained with DAPI (blue). Scale bar, 100 μm (low magnification) and 25 μm (high magnification), respectively. (F, G) Indirect immunofluorescence detection of lamin A/C (green) and type IV collagen (blue) in muscle biopsy after 30 days. Arrow points to donor cells (dc). P, perimysium; e, endomysium. (H) Indirect immunofluorescence detection of Pax7 (green), lamin A/C (red) and type IV collagen (blue) in muscle biopsy after 30 days. (I) Indirect immunofluorescence detection of Pax7 (green), α1(VI) collagen (red) and type IV collagen (blue) in muscle biopsy after 30 days. Arrows point to Pax7-positive (green) satellite cells. Scale bar, 100 μm. ADSC, adipose-derived stem cells; CTX, cardiotoxin; DAPI, 4′,6-diamidino-2-phenyl indol; GCM, gastrocnemius muscle.
Figure 6
Figure 6
Comparative morphometric analysis of ADSC engraftment and type VI collagen distribution in homeostatic and CTX-treated GCM, respectively. Analysis was performed at one, two, three, four and six weeks post-transplantation and CTX injury. (A) Engraftment of ADSC into the GCM with or without CTX treatment. The vertical axis shows the percentage (%) of engrafted ADSC per injected muscle. The horizontal axis shows the time point after transplantation in weeks (w). (B) Morphometric analysis of α1(VI)-positive myofibers in the transplanted GCM with or without CTX treatment. The vertical axis shows the number of α1(VI)-positive myofibers per section. The horizontal axis shows the time point after transplantation in weeks (w). In all cases, ADSC were detected with anti-lamin A/C antibodies. The percentage of engrafted cells and the number of α1(VI)-positive myofibers were determined on at least fifty 7 μm cryosections, respectively, covering approximately 125 mm3 of muscle tissue. In all studies, at least five animals were analyzed per time point. Data is presented as average ± SD. ADSC, adipose-derived stem cells; CTX, cardiotoxin; GCM, gastrocnemius muscle; SD, standard deviation.
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