Generation of allogenic and xenogeneic functional muscle stem cells for intramuscular transplantation.

Satellite cells, the stem cells of skeletal muscle tissue, hold a remarkable regeneration capacity and therapeutic potential in regenerative medicine. However, low satellite cell yield from autologous or donor-derived muscles hinders the adoption of satellite cell transplantation for the treatment of muscle diseases, including Duchenne muscular dystrophy (DMD). To address this limitation, here we investigated whether satellite cells can be derived in allogeneic or xenogeneic animal hosts. First, injection of CRISPR/Cas9-corrected mouse DMD-induced pluripotent stem cells (iPSCs) into mouse blastocysts carrying an ablation system of host satellite cells gave rise to intraspecies chimeras exclusively carrying iPSC-derived satellite cells. Furthermore, injection of genetically corrected DMD-iPSCs into rat blastocysts resulted in the formation of interspecies rat-mouse chimeras harboring mouse satellite cells. Remarkably, iPSC-derived satellite cells or derivative myoblasts produced in intraspecies or interspecies chimeras restored dystrophin expression in DMD mice following intramuscular transplantation, and contributed to the satellite cell pool. Collectively, this study demonstrates the feasibility of producing therapeutically competent stem cells across divergent animal species, raising the possibility of generating human muscle stem cells in large animals for regenerative medicine purposes.

Exclusive generation of rat spermatozoa in sterile mice utilizing blastocyst complementation with pluripotent stem cells.

Blastocyst complementation denotes a technique that aims to generate organs, tissues, or cell types in animal chimeras via injection of pluripotent stem cells (PSCs) into genetically compromised blastocyst-stage embryos. Here, we report on successful complementation of the male germline in adult chimeras following injection of mouse or rat PSCs into mouse blastocysts carrying a mutation in Tsc22d3, an essential gene for spermatozoa production. Injection of mouse PSCs into Tsc22d3-Knockout (KO) blastocysts gave rise to intraspecies chimeras exclusively embodying PSC-derived functional spermatozoa. In addition, injection of rat embryonic stem cells (rESCs) into Tsc22d3-KO embryos produced interspecies mouse-rat chimeras solely harboring rat spermatids and spermatozoa capable of fertilizing oocytes. Furthermore, using single-cell RNA sequencing, we deconstructed rat spermatogenesis occurring in a mouse-rat chimera testis. Collectively, this study details a method for exclusive xenogeneic germ cell production in vivo, with implications that may extend to rat transgenesis, or endangered animal species conservation efforts.

Screening method to identify hydrogel formulations that facilitate myotube formation from encapsulated primary myoblasts.

Hydrogel-based three-dimensional (3D) cellular models are attractive for bioengineering and pharmaceutical development as they can more closely resemble the cellular function of native tissue outside of the body. In general, these models are composed of tissue specific cells embedded within a support material, such as a hydrogel. As hydrogel properties directly affect cell function, hydrogel composition is often tailored to the cell type(s) of interest and the functional objective of the model. Here, we develop a parametric analysis and screening method to identify suitable encapsulation conditions for the formation of myotubes from primary murine myoblasts in methacryloyl gelatin (GelMA) hydrogels. The effect of the matrix properties on the myotube formation was investigated by varying GelMA weight percent (wt%, which controls gel modulus), cell density, and Matrigel concentration. Contractile myotubes form via myoblast fusion and are characterized by myosin heavy chain (MyHC) expression. To efficiently screen the gel formulations, we developed a fluorescence-based plate reader assay to quantify MyHC staining in the gel samples, as a metric of myotube formation. We observed that lower GelMA wt% resulted in increased MyHC staining (myotube formation). The cell density did not significantly affect MyHC staining, while the inclusion of Matrigel increased MyHC staining, however, a concentration dependent effect was not observed. These findings were supported by the observation of spontaneously contracting myotubes in samples selected in the initial screen. This work provides a method to rapidly screen hydrogel formulations for the development of 3D cellular models and provides specific guidance on the formulation of gels for myotube formation from primary murine myoblasts in 3D.

Exercise promotes satellite cell contribution to myofibers in a load-dependent manner.

BACKGROUND: Satellite cells (SCs) are required for muscle repair following injury and are involved in muscle remodeling upon muscular contractions. Exercise stimulates SC accumulation and myonuclear accretion. To what extent exercise training at different mechanical loads drive SC contribution to myonuclei however is unknown. RESULTS: By performing SC fate tracing experiments, we show that 8 weeks of voluntary wheel running increased SC contribution to myofibers in mouse plantar flexor muscles in a load-dependent, but fiber type-independent manner. Increased SC fusion however was not exclusively linked to muscle hypertrophy as wheel running without external load substantially increased SC fusion in the absence of fiber hypertrophy. Due to nuclear propagation, nuclear fluorescent fate tracing mouse models were inadequate to quantify SC contribution to myonuclei. Ultimately, by performing fate tracing at the DNA level, we show that SC contribution mirrors myonuclear accretion during exercise. CONCLUSIONS: Collectively, mechanical load during exercise independently promotes SC contribution to existing myofibers. Also, due to propagation of nuclear fluorescent reporter proteins, our data warrant caution for the use of existing reporter mouse models for the quantitative evaluation of satellite cell contribution to myonuclei.

Expression and Activity of TRPA1 and TRPV1 in the Intervertebral Disc: Association with Inflammation and Matrix Remodeling.

Transient receptor potential (TRP) channels have emerged as potential sensors and transducers of inflammatory pain. The aims of this study were to investigate (1) the expression of TRP channels in intervertebral disc (IVD) cells in normal and inflammatory conditions and (2) the function of Transient receptor potential ankyrin 1 (TRPA1) and Transient receptor potential vanilloid 1 (TRPV1) in IVD inflammation and matrix homeostasis. RT-qPCR was used to analyze human fetal, healthy, and degenerated IVD tissues for the gene expression of TRPA1 and TRPV1. The primary IVD cell cultures were stimulated with either interleukin-1 beta (IL-1ß) or tumor necrosis factor alpha (TNF-α) alone or in combination with TRPA1/V1 agonist allyl isothiocyanate (AITC, 3 and 10 µM), followed by analysis of calcium flux and the expression of inflammation mediators (RT-qPCR/ELISA) and matrix constituents (RT-qPCR). The matrix structure and composition in caudal motion segments from TRPA1 and TRPV1 wild-type (WT) and knock-out (KO) mice was visualized by FAST staining. Gene expression of other TRP channels (A1, C1, C3, C6, V1, V2, V4, V6, M2, M7, M8) was also tested in cytokine-treated cells. TRPA1 was expressed in fetal IVD cells, 20% of degenerated IVDs, but not in healthy mature IVDs. TRPA1 expression was not detectable in untreated cells and it increased upon cytokine treatment, while TRPV1 was expressed and concomitantly reduced. In inflamed IVD cells, 10 µM AITC activated calcium flux, induced gene expression of IL-8, and reduced disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and collagen 1A1, possibly via upregulated TRPA1. TRPA1 KO in mice was associated with signs of degeneration in the nucleus pulposus and the vertebral growth plate, whereas TRPV1 KO did not show profound changes. Cytokine treatment also affected the gene expression of TRPV2 (increase), TRPV4 (increase), and TRPC6 (decrease). TRPA1 might be expressed in developing IVD, downregulated during its maturation, and upregulated again in degenerative disc disease, participating in matrix homeostasis. However, follow-up studies with larger sample sizes are needed to fully elucidate the role of TRPA1 and other TRP channels in degenerative disc disease.

An Inflammatory Nucleus Pulposus Tissue Culture Model to Test Molecular Regenerative Therapies: Validation with Epigallocatechin 3-Gallate.

Organ cultures are practical tools to investigate regenerative strategies for the intervertebral disc. However, most existing organ culture systems induce severe tissue degradation with only limited representation of the in vivo processes. The objective of this study was to develop a space- and cost-efficient tissue culture model, which represents degenerative processes of the nucleus pulposus (NP). Intact bovine NPs were cultured in a previously developed system using Dyneema jackets. Degenerative changes in the NP tissue were induced either by the direct injection of chondroitinase ABC (1-20 U/mL) or by the diffusion of interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α) (both 100 ng/mL) from the culture media. Extracellular matrix composition (collagens, proteoglycans, water, and DNA) and the expression of inflammatory and catabolic genes were analyzed. The anti-inflammatory and anti-catabolic compound epigallocatechin 3-gallate (EGCG, 10 µM) was employed to assess the relevance of the degenerative NP model. Although a single injection of chondroitinase ABC reduced the proteoglycan content in the NPs, it did not activate cellular responses. On the other hand, IL-1ß and TNF-α significantly increased the mRNA expression of inflammatory mediators IL-6, IL-8, inducible nitric oxide synthase (iNOS), prostaglandin-endoperoxide synthase 2 (PTGS2) and matrix metalloproteinases (MMP1, MMP3, and MMP13). The cytokine-induced gene expression in the NPs was ameliorated with EGCG. This study provides a proof of concept that inflammatory NP cultures, with appropriate containment, can be useful for the discovery and evaluation of molecular therapeutic strategies against early degenerative disc disease.