Speaker
Description
Symposium: Cell-rich constructs for tissue engineering.
Introduction: The development of in-vitro model of skeletal muscle regeneration is extremely challenging. Human Bone Marrow-derived Mesenchymal Stem Cell (hBM-MSCs) and human Myoblast co-culture with growth factor supplementation has been described for in vitro myogenic commitment [1-2]; among them, b-Fibroblast Growth Factor (b-FGF) was largely used at concentrations of 10 ng/mL [2]. On the other hand, human Peripheral blood mononuclear cells (hPBMCs) were also described as significantly involved in myogenesis [3]; however, their contribution is still under debate. Thus, to better understand their role, in our study, hPBMCs were added to an in vitro 3D co-culture of BM-MSCs and myoblasts (hSkMs) to investigate their potential contribution on hBM-MSC myogenic commitment.
Materials and methods: In vitro 3D scaffold-based systems were assembled using fibrin hydrogel cultured in perfusion conditions, and three cell types were seeded together. 2D in vitro culture of human BM-MSCs and hSkMs in the presence of PBMNCs placed in an upper chamber of transwell insert was adopted as control.
Results and Discussion: hBM-MSC commitment towards a myogenic phenotype was confirmed by qRT-PCR showing upregulation of myogenic-related genes, such as Pax3, MyoD1, Myf5, Myf6, Desmin and Myosin Heavy Chain II (MYH2). In particular, Desmin expression showed an increasing trend in the presence of PBMNCs, suggesting a synergist paracrine effect of peripheral blood cells on myogenic commitment. The presence of Desmin and MYH2 at protein level was also confirmed by immunofluorescence assay. Finally, the 3D environment in dynamic culture was an extremely favorable biomimetic environment allowing the study of all myogenic marker expression at both gene and protein levels.
References:
1.Witt R. et al. BMC Cell Biol. 18 (2017) 15.
2.Scala P. et al. Artificial cells, Nanomedicine and Biotechonology, 2021, in press.
3.Scala P. et al. Int J Mol Sci. 2021 Oct 8;22(19):10867.
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