Nogo-A (Rtn 4A), a member of the reticulon 4 (Rtn4) protein family, is a neurite outgrowth inhibitor protein that is primarily expressed in the central nervous system (CNS). However, previous studies revealed that Nogo-A was upregulated in skeletal muscles of Amyotrophic lateral sclerosis (ALS) patients. Additionally, experiments showed that endoplasmic reticulum (ER) stress marker, C/EBP homologous protein (CHOP), was upregulated in gastrocnemius muscle of a murine model of ALS. We therefore hypothesized that Nogo-A might relate to skeletal muscle diseases. According to our knocking down and overexpression results in muscle cell line (C2C12), we have found that upregulation of Nogo-A resulted in upregulation of CHOP, pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while downregulation of Nogo-A led to downregulation of CHOP, IL-6 and TNF-α. Immunofluorescence results showed that Nogo-A and CHOP were expressed by myofibers as well as tissue macrophages. Since resident macrophages share similar functions as bone marrow-derived macrophages (BMDM), we therefore, isolated macrophages from bone marrow to study the role of Nogo-A in activation of these cells. Lipopolysaccharide (LPS)-stimulated BMDM in Nogo-KO mice showed low mRNA expression of CHOP, IL-6 and TNF-α compared to BMDM in wild type (WT) mice. Interestingly, Nogo knockout (KO) BMDM exhibited lower migratory activity and phagocytic ability compared with WT BMDM after LPS treatment. In addition, mice experiments data revealed that upregulation of Nogo-A in notexin- and tunicamycin-treated muscles was associated with upregulation of CHOP, IL-6 and TNF-α in WT group, while in Nogo-KO group resulted in low expression level of CHOP, IL-6 and TNF-α. Furthermore, upregulation of Nogo-A in dystrophin-deficient (mdx) murine model, myopathy and Duchenne muscle dystrophy (DMD) clinical biopsies was associated with upregulation of CHOP, IL-6 and TNF-α. To the best of our knowledge, this is the first study to demonstrate Nogo-A as a regulator of inflammation in diseased muscle and bone marrow macrophages and that deletion of Nogo-A alleviates muscle inflammation and it can be utilized as a therapeutic target for improving muscle diseases.

Life-long regeneration of healthy muscle by cell transplantation is an ideal therapy for patients with degenerative muscle diseases. Yet, obtaining muscle stem cells from patients is very limited due to their exhaustion in disease condition. Thus, development of a method to obtain healthy myogenic stem cells is required. Here, we showed that the four transcription factors, Six1, Eya1, Esrrb, and Pax3, converts fibroblasts into induced myogenic stem cells (iMSCs). The iMSCs showed effective differentiation into multinucleated myotubes and also higher proliferation capacity than muscle derived stem cells both in vitro and in vivo. The iMSCs do not lose their proliferation capacity though the passaging number is increased. We further isolated CD106-negative and α7-integrin-positive iMSCs (sort-iMSCs) showing higher myogenic differentiation capacity than iMSCs. Moreover, genome-wide transcriptomic analysis of iMSCs and sort-iMSCs, followed by network analysis, revealed the genes and signaling pathways associated with enhanced proliferation and differentiation capacity of iMSCs and sort-iMSCs, respectively. The stably expandable iMSCs provide a new source for drug screening and muscle regenerative therapy for muscle wasting disease.

In previous work, we established an equine induced pluripotent stem cell line (E-iPSCs) from equine adipose-derived stem cells (ASCs) using a lentiviral vector encoding four transcription factors: Oct4, Sox2, Klf4, and c-Myc. In the current study, we attempted to differentiate these established E-iPSCs into mesenchymal stem cells (MSCs) by serial passaging using MSC-defined media for stem cell expansion. Differentiation of the MSCs was confirmed by analyzing expression levels of the MSC surface markers CD44 and CD29, and the pluripotency markers Nanog and Oct4. Results indicated that the E-iPSC-derived MSCs (E-iPSC-MSCs) retained the characteristics of MSCs, including the ability to differentiate into chondrogenic, osteogenic, or myogenic lineages. E-iPSC-MSCs were rendered suitable for therapeutic use by inhibiting immune rejection through exposure to transforming growth factor beta 2 (TGF-β2) in culture, which down-regulated the expression of major histocompatibility complex class I (MHC class I) proteins that cause immune rejection if they are incompatible with the MHC antigen of the recipient. We reported 16 cases of E-iPSC-MSC transplantations into injured horses with generally positive effects, such as reduced lameness and fraction lines. Our findings indicate that E-iPSC-MSCs can demonstrate MSC characteristics and be safely and practically used in the treatment of musculoskeletal injuries in horses.

Adult articular chondrocytes retain the ability to repopulate the matrix, an ability which is demonstrated with this specific cryoinjury. This may be an initial stage of cartilage regeneration.

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