Human Skeletal Muscle Progenitor Cells (Myoblasts)

ZIKV Replicates in Human Skeletal Muscle Progenitor Cells, Causing Cell Death

Zika virus (ZIKV) infections have raised global concerns due to severe neurological outcomes. While its neurotropic nature is well-known, the role of peripheral tissues in viral amplification is less explored. Gavino-Leooldino et al. aim to uncover the potential of skeletal muscle as an early ZIKV replication site, hypothesizing that muscle replication contributes to increased peripheral viral loads and later neural tissue invasion.

To assess whether muscle could be a ZIKV replication site, primary human skeletal muscle myoblasts (HSMM) were infected at the myoblast stage and as differentiated myotubes. Quantification of infectious particles (Fig. 1A) showed a rapid increase in ZIKV particles in myoblast cultures for at least 48 hours post-infection (hpi). Myotubes showed no increase until 16 hpi, but by 36 hpi, viral titers matched those in myoblasts. Immunostaining confirmed the presence of ZIKV E and NS2B proteins in myoblasts and myotubes at 36 hpi (Fig. 1B-E). No viral protein staining was seen in mock cultures. More ZIKV-positive cells were found in myoblast than myotube cultures at 36 and 48 hpi (Fig. 1H). Then, they assessed ZIKV replication's impact on skeletal muscle cell viability. At 48 hours post-infection (hpi), myoblast viability reduced by about 50%, whereas myotube viability remained unchanged (Fig. 1I). Consistently, myosin heavy chain (MF20) labeling showed no structural changes in myofibers of ZIKV-infected compared to mock-treated myotubes (Fig. 1F and 1G). Quantification confirmed unchanged fiber count and area (Fig. 1J), indicating ZIKV does not disrupt existing myofiber integrity. These findings suggest ZIKV replicates in myogenic cells, causing significant damage.

Fig. 1. ZIKV replicates and promotes death of human skeletal muscle progenitor cells.

USC^IGF1 was Fused with Human and Mouse Skeletal Muscle Progenitor Cells in vitro.

Skeletal muscles, crucial for body function, often suffer injuries affecting health and mobility. Traditional muscle regeneration relies on satellite cells, but their scarcity after severe injuries limits recovery. Stem cell therapy emerges as a promising alternative. Urine-derived stem cells (USC), easily obtained non-invasively, have shown myogenic potential.

Yi's team used USC^IGF1, created by lentivirus to overexpress ^IGF1, assessing its effects on muscle regeneration in a rodent model. Myofusion assays were conducted between mCherry-labeled USC^IGF1 and hSMPC^GFP (human skeletal muscle progenitor cells labeled with a GFP-containing plasmid). The number of fused cells increased significantly, reaching 23 fused myotubes per 2×10⁵ USC^IGF1 at a 1:2 ratio after 7 days of differentiation (Fig. 2A and B). Fusion was first seen 3 days after switching to differentiation media and persisted until Day 14. Human USC^IGF1 fused distinctly with mSMPC^GFP, confirmed by immunostaining for human mitochondria, lamin, and dystrophin 3 (DYS3) in myotubes (Fig. 2C). In contrast, only one myotube was detectable from USC and USC^mCherry controls per 2×10⁵ cells. Cytoplasm and mitochondria were observed in myotubes with mCherry-labeled USC^IGF and hSMPCGFP (Fig. 2D). These results indicated ^IGF1 overexpression increased the ability of USC to fuse with skeletal myocytes to form myotubes.

Fig. 2. USC^IGF1 was fused with human skeletal muscle progenitor cells (hSMPC) and mouse skeletal muscle progenitor cells (mSMPC) in vitro (Yi H, Chen G, et al., 2024).