Towards tissue-specific vascularization of bio-engineered skeletal muscle constructs using autologous skeletal muscle microvascular endothelial cells

Jun 30, 2022, 4:40 PM
Room: S1

Room: S1


Terrie, Lisanne (Tissue Engineering Lab, Department of Development and Regeneration, KU Leuven)


Prevascularization of tissue-engineered constructs before implantation is used to accelerate anastomosis with the host vasculature and thus to enhance successful implantation. To induce prevascularization, the co-culture of tissue-specific cells with endothelial cells is explored. To date, most co-culture studies are still conducted with human umbilical vein endothelial cells (HUVECs). However, this ultimately restricts clinical applications and thus ideally autologous cells are used. Furthermore, the mounting evidence of the endothelium as a regulator of regenerative processes in an organ/tissue-specific manner warrants the use of tissue-specific cells for tissue engineering. Skeletal muscle microvascular endothelial cells (SkMVECs) are an interesting candidate for prevascularizing tissue-engineered skeletal muscle as they are both autologous and tissue-specific. Here, we compare SkMVECs to HUVECs, both in 2D as well as in 3D bio-artificial muscles.
Primary human SkMVECs were obtained from 3 different donors. These cells were thoroughly characterized in comparison to the current standard, HUVECs, through immunostaining and bulk RNA sequencing. Next, in vitro sprouting capacity of the SkMVECs was compared to the HUVECs using a conventional spheroid assay and tube formation assay. In addition, endothelial network formation in a fibrin hydrogel was evaluated over 14 days. And finally, direct and indirect co-cultures with human primary myoblasts were set up to evaluate the interaction between the SkMVECs and myoblasts.
SkMVECs were extensively compared to HUVECs through bulk RNA sequencing to evaluate differentially expressed genes and these were annotated to the related pathways. Next, the angiogenic potential was evaluated and SkMVECs were found to sprout less compared to HUVECs in terms of sprout number but sprout length was found to be similar. Also, from the tube formation assay, a similar extent of tube formation was found but HUVECs were found to form tubes more rapidly. However, when both endothelial cell types were embedded in a fibrin hydrogel over a longer period, which is similar to our tissue engineering system, SkMVEC constructs were found to result in more branched endothelial networks compared to HUVECs. In addition, the more rapid proliferation of HUVECs compared to SkMVECs seemed to interfere with stable endothelial network formation after 10 days. Conditioned medium of both cell types was used to dissect the cross-talk between myoblasts and endothelial cells and effect. Finally, to explore the potential of SkMVECs for skeletal muscle tissue engineering, 3D co-culture experiments with autologous myoblasts were performed to evaluate the interaction between the SkMVECs and myoblasts.
Taken together, SkMVECs are capable of forming stable, extensive endothelial networks in a relevant model for tissue-engineering applications. In contrast, the currently used standard endothelial cell type, HUVECs, was found to be highly angiogenic in short-term assays but less suited for long-term endothelial network formation. Furthermore, SkMVECs interact with autologous myoblasts and vice-versa, which further underscores their potential as a suitable endothelial cell source for the prevascularization of engineered skeletal muscle tissue."

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