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. 2020 Nov 2;11(1):463.
doi: 10.1186/s13287-020-01979-y.

Congenital muscular dystrophy-associated inflammatory chemokines provide axes for effective recruitment of therapeutic adult stem cell into muscles

Vitali Alexeev  1 , Jacquelyn Olavarria  1 , Paolo Bonaldo  2 , Luciano Merlini  3 , Olga Igoucheva  4
Affiliations

Congenital muscular dystrophy-associated inflammatory chemokines provide axes for effective recruitment of therapeutic adult stem cell into muscles

Vitali Alexeev et al. Stem Cell Res Ther. .

Abstract

Background: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin α2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1-/- mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue.

Methods: Proteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations.

Results: Comprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2, and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect.

Conclusions: Collectively, identified molecules provided insight into the mechanisms governing directional migration and intramuscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment.

Keywords: Adipose-derived stem cell; COL6A1; Cell-based therapy; Chemokines; Chemotaxis; Congenital muscular dystrophy; Type VI collagen.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Proteome analysis of chemokines in muscles of CMD-affected patients. The data were collected from independent arrays with duplicate measurements for each chemokine using 5 BM, 8 UCMD, and 5 MDC1A muscle samples (Table S1). Chemokines are listed below the columns. Data are presented as a mean pixel density ± SD. Statistical significance (p < 0.05) is indicated by asterisk. CMD types are indicated in the key. N, normal (control); BM, Bethlem myopathy; UCMD, Ulrich congenital muscular dystrophy; MDC1A, Merosin-deficient congenital muscular dystrophy type 1A
Fig. 2
Fig. 2
Proteomic screens of chemokines in muscles of dyW mice. Data were collected from independent arrays with duplicate measurements for each chemokine using muscle biopsies from wild-type (a) and dyW (b) mice, respectively, at 1, 2, 3, 4, 5, and 6 weeks after birth. Time points are indicated in the key. Chemokines are listed below the columns. Data are presented as a mean pixel density ± SD. w, week(s)
Fig. 3
Fig. 3
Proteomic screens of chemokines in muscles of Col6a1−/− mice under physiological and pro-inflammatory conditions. a Data were collected from independent arrays with duplicate measurements for each chemokine using muscle biopsies from wild-type and Col6a1−/− mice, respectively, under physiological conditions (−CTX). b, c Data were collected from independent arrays with duplicate measurements for each chemokine using muscle biopsies from wild-type (b) and Col6a1−/− (c) mice, respectively, under pro-inflammatory conditions (+CTX). Post-injury time points (days) are indicated in the key. Chemokines are listed below the columns. Data are presented as a mean pixel density ± SD. Statistical significance (p < 0.05) is indicated by asterisk. d, day(s)
Fig. 4
Fig. 4
Representative live imaging of Ccl2/Ccr2-mediated recruitment of systemically transplanted heterogeneous ADSC (a, c, e) and Ccr2-positive ADSC (b, d, f) into the GCM of NCr nude mice. Mouse recombinant Ccl2 chemokine was administered into the left GCM immediately after cell transplant. Cell recruitment to the muscle was assessed by luciferase fluorescence at 24, 48, and 72 h after transplantation, respectively. Oval demarcates luciferase-ADSC fluorescence. Asterisk in oval indicates Ccl2 chemokine injection site
Fig. 5
Fig. 5
Systemic transplantation of selected ADSC into Col6a1−/− mice. a Representative in vivo images showing the hindlimbs of mice receiving Ccc2-positive ADSC and Cxcr2-positive ADSC transplants under physiological (−CTX) and pro-inflammatory (+CTX) conditions, respectively. IVIS live imaging was performed at 4, 14, and 21 days post-transplant. b Quantitative analysis of fluorescence detected by live imaging from differently treated cohorts of mice (n = 3 per time point) at 3 time points (as indicated below the columns). Data are presented as radiant efficacy ± SD and as fluorescent area ± SD, respectively. Color-coding for Ccr2-positive ADSC and Cxcr2-positive ADSC is shown in the key. Dotted trend lines illustrate time-dependent changes in differently treated mice, as indicated in the key. Statistically significant differences in Ccr2- and Cxcr2-positive ADSC-treated mice (p < 0.05) are indicated with asterisks (***). c Immunofluorescent analysis of GCM tissue from untreated and CTX-treated muscle biopsies was performed 21 days after transplantation. Co-localization of the ADSC-donated COL6 and basement membrane-associated type IV collagen was detected with α1(VI) collagen (AlexaFluor488, green) and LAMA2 (AlexaFluor594, red) antibodies. Images were taken from representative sections at low and high (20x) magnification, respectively. Nuclei were stained with DAPI (blue). Scale bar, 100 μm (low magnification) and 25 μm (high magnification), respectively. d Quantitative assessment of COL6-positive myofibers on sections of muscle tissue from mice treated with unselected ADSC, Ccr2-positive ADSC, and Cxcr2-positive ADSC, respectively. Data are presented as the percentage of COL6-positive myofibers per microscopic field ± SD. Time points of tissue collection and treatments are indicted below the columns. Statistical significance (p < 0.05) is indicated with asterisks (***)
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