Production of Proliferation- and Differentiation-Competent Porcine Myoblasts for Preclinical Studies in a Porcine Large Animal Model of Muscular Insufficiency

Muscular insufficiency is observed in many conditions after injury, chronic inflammation, and especially in elderly populations. Causative cell therapies for muscle deficiencies are not state of the art. Animal models to study the therapy efficacy are, therefore, needed. We developed an improved protocol to produce myoblasts suitable for pre-clinical muscle therapy studies in a large animal model. Myoblasts were isolated from the striated muscle, expanded by employing five different protocols, and characterized on transcript and protein expression levels to determine procedures that yielded optimized regeneration-competent myoblasts and multi-nucleated myotubes. We report that swine skeletal myoblasts proliferated well under improved conditions without signs of cellular senescence, and expressed significant levels of myogenic markers including Pax7, MyoD1, Myf5, MyoG, Des, Myf6, CD56 (p ≤ 0.05 each). Upon terminal differentiation, myoblasts ceased proliferation and generated multi-nucleated myotubes. Injection of such myoblasts into the urethral sphincter complex of pigs with sphincter muscle insufficiency yielded an enhanced functional regeneration of this muscle (81.54% of initial level) when compared to the spontaneous regeneration in the sham controls without myoblast injection (67.03% of initial level). We conclude that the optimized production of porcine myoblasts yields cells that seem suitable for preclinical studies of cell therapy in a porcine large animal model of muscle insufficiency

Expression of CD146 and Regenerative Cytokines by Human Placenta-Derived Mesenchymal Stromal Cells upon Expansion in Different GMP-Compliant Media

Mesenchymal stromal cells (MSCs) have been successfully employed in clinical applications. In most studies, autologous MSCs from the bone marrow (bmMSCs) were used, and others employed autologous adipose tissue-derived stromal cells (ADSCs). Recently, clinical feasibility studies provided evidence that MSCs from human term placenta (pMSCs) can be used for homologous therapy facilitating access to regenerative cells in emergency situations, when autologous cells are not available or not suitable. We therefore investigated the expression of MSC stemness marker CD146 and the expression of neuro- and myoregenerative cytokines by human pMSCs after expansion in three different media compliant with good manufacturing protocols (GMP) in comparison to pMSCs expanded in a commercial MSC expansion media. To replace xenobiotic serum in the GMP-compliant media employed in this study, either human serum, human serum plus platelet lysate (PLL), or human plasma plus PLL was used. We report that enrichment of media with PLL accelerates pMSC proliferation but reduces the expression of the stemness marker CD146 significantly, while PLL deprivation enhanced the CD146 expression. In contrast, the reduced expression of CD146 by PLL deprivation was not observed on bmMSCs. The expression of the cytokines investigated was not modulated significantly by PLL. We conclude that accelerated expansion of pMSCs in GMP-compliant media enriched by PLL reduces the expression of stemness marker CD146, but does not influence the expression of neuro- and myoregenerative cytokines

Elevated Expression of the Immune Checkpoint Ligand CD276 (B7-H3) in Urothelial Carcinoma Cell Lines Correlates Negatively with the Cell Proliferation

The cell surface molecule CD276 (B7-H3) is an immune checkpoint antigen. The elevated expression of CD276 on tumors contributes to the suppression of anti-tumor T-cell responses and correlates with poor prognosis. Methods: The expression of CD276 was explored in vitro on eight urothelial carcinoma cell lines (UM-UC) in comparison to eight normal urothelial cells (NUCs) by RT-qPCR, Western blotting, and flow cytometry. Cell proliferation was enumerated over consecutive passages. The expression of cancer stem cell markers CD24 and CD44, cytokeratins, and vimentin was investigated by immunofluorescence. The expression of CD276 in bladder tumor samples and metastases was explored by immunohistochemistry. Results: Expression of CD276 on cell surfaces was elevated on UM-UCs when compared to NUCs. In UM-UCs, CD276 transcripts correlated moderately positive with CD276 protein expression (ρ = 0.660) and strongly positive with CD276 surface-expression (ρ = 0.810). CD276 mRNA expression (ρ = −0.475) and CD276 protein expression (ρ = −0.417) had a significant negative correlation with proliferation, while a significant correlation between proliferation and cell surface expression was not observed in UM-UCs. Conclusion: The expression of CD276 on UM-UC bladder tumor cell surfaces is elevated. Slow proliferating UM-UC cells express more CD276 mRNA and protein than fast proliferating cells. In patients, slow proliferating CD276(high) tumor (stem) cells may evade immune surveillance. However, cancer therapy targeting CD276 may be effective in the treatment of slow proliferating tumor cells