Mesenchymal Progenitor Cells (MPCs) Involvement In Ectopic Fat Formation In Muscular Dystrophy

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1 Mesenchymal Progenitor Cells (MPCs) Involvement In Ectopic Fat Formation In Muscular Dystrophy Jihee Sohn, MS 1, Ying Tang 2, Bing Wang, MD,PhD 3, Aiping Lu 4, Johnny Huard, Ph.D 5. 1 university of pittsburgh, Pittsburgh, PA, USA, 2 Univeristy of Pittsburgh, Pittsburgh, PA, USA, 3 University of Pittsburgh, Pittsburgh, PA, USA, 4 University of pittsburgh, Pittsburgh, PA, USA, 5 university of pittsburgh, Pittsbburgh, PA, USA. Disclosures: J. Sohn: None. Y. Tang: None. B. Wang: None. A. Lu: None. J. Huard: None. Introduction: Ectopic fat formation/accumulation and infiltration in skeletal muscle is closely associated with several genetic disorders including Duchenne muscular dystrophy (DMD), a degenerative muscle disorder caused by point mutations in dystrophin gene. However, neither the origin of ectopic adipocytes, nor the mechanism of their formation in disease, is still unclear. Based on a previously published preplate technique, we isolated two types of muscle derived cells; rapidly adhering cells (RACs) and slowly adhering cells (SACs) from skeletal muscle utrophin/dystrophin double knockout (dys-/- utro-/-, dko) mice, which is a severe animal model of DMD. Previously, we have shown that SACs are muscle derived stem cells (MDSCs) since they are myogenic progenitor cells, which are mainly involved in muscle fiber regeneration. We also have characterized RACs as mysenchymal progenitor cells (MPCs) and showed that dko-mpcs display increased adipogenic, osteogenic, and chondrogenic differentiation potentials compared to wt-mpcs in vitro. In current study, we show that compared to other muscle-derived cell populations from dko mice, MPCs displayed significantly increased proliferation and adipogenic potentials in vivo. We tested the developmental potentials of MDSCs and MPCs in vivo by transplantation. Our data demonstrated that dko-mdscs engrafted by fusing to myofibers in both mdx, which a milder animal model of DMD, and dko mice muscle. In contrast, almost no donor dko-mpcs were found in mdx muscle, yet dko- MPCs engrafted and yielded donor-derived adipocytes in dko muscle. In addition, in vitro co-culture experiment showed that dko-mpcs inhibited myogenic differentiation potential of dko-mdscs. Overall, our data suggest that MPCs and MDSCs have distinct developmental potentials and furthermore, MPCs engraftment and differentiation into adipocytes suggest that they involve in pseudohypertrophy in DMD. Methods: Cell Isolation: Cells were isolated from dko (dys-/-utro-/-) mice at 6 weeks of age, as previously described via a modified preplate technique [1]. After 24hrs, the MPCs were obtained while the MDSCs were obtained after 7 days. Both cells were cultured in proliferation medium (PM, DMEM supplemented with 10% fetal bovine serum, 10% horse serum, 0.5% chicken embryo extract and 1% penicillin-streptomycin). Retrovirus transduction of MPCs and MDSCs to express GFP: Retroviral vector containing GFP had been previously developed under CL promoter and used to transduce MPCs and MDSCs. Cells were transduced with retrovirus at a Multiplicity of Infection (MOI) of 1x10 6 and successful gene transfer was determined by GFP staining. After transduction, GFP expressing cells were sorted using FACS sorting. Transplantation assay: 4.5x10 5 GFP + dko-mpcs and dko-mdscs were injected into the gastrocnemius of 4wk old mdx or dko mice. Both mice were sacrificed 2 wks post cells transplantation and the muscle tissues were frozen in 2-methylbutane precooled in liquid nitrogen and sectioned for IHC staining and histology. Sections were stained with GFP (1:1000) and FABP4 (1:200), a marker for adipocytes, antibodies with DAPI counterstaining. Co-culture assay: 10,000 dko-mdscs were plated in the lower compartment of Transwell Permeable Supports and 10,000 dko- MPCs were seeded into 6.5 mm transwell membrane inserts in PM media. To measure the differentiation potential of dko-mdscs 48 hours after co-culture, PM media was switched to differentiation media (DMEM medium supplemented with 2% FBS) for 3 days. Results: dko muscle may favor stem/progenitor cells proliferation, survival, and differentiation potentials. 14 days post injection, dko-mdscs generated

2 larger engraftment in gastrocnemius of mdx than in dko mice. Donor dko-mdscs formed newly regenerated muscle fibers in both mdx and dko muscle. We observed that GFP + dko-mpcs differentiated into FABP4 + adipocytes when they were transplanted into dko muscle. However, barely any dko-racs survived and engrafted in mdx muscle after transplantation (Figure 1). MPCs may inhibit the regeneration capacity of MDSCs in dystrophic muscle. Co-culture assay using transwell inserts indicated that limited myogenic differentiation potential of dko-mdscs was further reduced by co-culturing them with dko-mpcs (Figure 2). Discussion: The predominant symptoms seen in advanced cases of DMD are sarcopenia and pseudohypertrophy with fatty infiltration in skeletal muscle; however, the origin of ectopic adipoctyes or the mechanism of their formation in disease is unknown. In dko mice, an animal model of DMD, we observed severe peri-muscular adipose tissue on the surface of the gastrocneminus as well as lipid accumulation inside of skeletal muscle myofibers. In this study, we showed that MPCs form adipocytes while MDSCs regenerate muscle fibers in dystrophic muscle. Furthermore, we suggest that the muscle microenvironment may have an important role in determining fate of stem/progenitor cells. Our data demonstrated that dko-mpcs engrafted and differentiated into adipocytes more readily in dko than in mdx muscle which supports that the severity of the disease influences the fate of the cells. Significance: This study suggests that MPCs may involve in forming adipocytes while MDSCs involve in regenerating muscle fibers in dystrophic muscle. We also provide evidence that disease state may affect stem cell niche, impacting their fate. In a dystrophic dko mouse, the muscle tissue is severely affected by disease and the muscle milieu may be altered to favor adipogenic potentials of MPCs. Results from this project could provide insight into new approaches to alleviate muscle weakness and wasting in DMD patients through the inhibition of ectopic adipose cells in skeletal muscle by blocking the adipogenic differentiation of the MPCs. Acknowledgments: References: [1] Gharaibeh, et al. Nat Protoc. 2008; 3: [2] Joe, et al. Nat Cell biology. 2010; 2:

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4 ORS 2014 Annual Meeting Poster No: 0523

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