Topography-Mediated Myotube and Endothelial Alignment, Differentiation, and Extracellular Matrix Organization for Skeletal Muscle Engineering

Understanding the response of endothelial cells to aligned myotubes is important to create an appropriate environment for tissue-engineered vascularized skeletal muscle. Part of the native tissue environment is the extracellular matrix (ECM). The ECM is a supportive scaffold for cells and allows cellular processes such as proliferation, differentiation, and migration. Interstitial matrix and basal membrane both comprise proteinaceous and polysaccharide components for strength, architecture, and volume retention. Virtually all cells are anchored to their basal lamina. One of the physical factors that affects cell behavior is topography, which plays an important role on cell alignment. We tested the hypothesis that topography-driven aligned human myotubes promote and support vascular network formation as a prelude to in vitro engineered vascularized skeletal muscle. Therefore, we used a PDMS-based topography substrate to investigate the influence of pre-aligned myotubes on the network formation of microvascular endothelial cells. The aligned myotubes produced a network of collagen fibers and laminin. This network supported early stages of endothelial network formation.


SI
. The complex plasmids-Endofectin was left for 15 minutes. Next, the complex was added to the HEK cells culture dropwise while stirring gently. Next day, the HEK cell medium was refreshed with either myoblasts cell medium or ECs cell medium. At the third day, the virus-containing medium was centrifuged at 300 xg, filtered through a 0.45 µm filter, and then, polybrene (6 µg/ml) was added to the virus-containing medium which was then added to the myoblasts or ECs cell culture. Fresh medium was put into the virus producing HEK cell culture. Finally, at the fourth day, virus-containing medium was collected and treated in the same way as previously described and added to the myoblasts or ECs. After a week in culture, FACsVerse SH800S Sony Cell Sorter (Copyright ©2019 Sony Biotechnology Inc.) was used for cell sorting. Then, individual cells, previously sorted for either green or red, were cultured on a 96 well plate. Clones with high proliferation rate were selected and expanded for cell culture and experiments.

Gene expression analyses
Cells were washed with PBS and then lysed after two and five days of co-culture using TRIzol™ Reagent ©(Thermo Fisher, USA) according to the manufacturer's protocol. An UV-Vis Spectrophotometer Nanodrop (1000, Thermo Scientific) was used to measure RNA concentration.
ΔCt value was calculated as the fold difference between the gene of interest and the reference gene HPRT.  1: Early steps of vasculogenesis observed by endothelial tube formation. a. Life image of co-culture after five days on pre-formed myotubes. Bottom, cross-section (Z stack) shows primitive tube-like structure of ECs. GFP+ myotubes, DAPI, and dTom+ ECs b. Fibronectin production was increased on the interface between ECs and myotubes. Left micrograph corresponds to the co-culture of myotubes (green) and ECs (red), nuclei (blue) and FN (yellow). Right micrograph shows ECs (red) and FN (yellow).

SI 2: pericytic phenotype of myotubes
Supplementary information figure 2: Myotubes did not harbor a pericytic phenotype in monoculture. a. Life image of the co-culture after five days. GFP+ myptubes, nuclei (DAPI, blue), and dTom+ ECs. b,c,d, e RT-qPCR of two pericytic genes (CSPG4 and PDGFRB) and one endothelial specific intercellular adhesion gene (CDH5).

SI 3: Basement membrane protein deposition by myoblasts and myotubes
Supplementary information figure 3: Two-day-old Myoblasts and three-day differentiated myotubes had similar basement membrane protein deposition on TCP. a. EGFP+ myoblasts (green), DAPI nuclei (blue), red color corresponds to the protein of interest (collagen I, III, and IV, fibronectin, and laminin). Zoom-in micrographs left to right correspond to DAPI, myoblast EGFP+ and the merge picture. b. Non-tag three-day old differentiated myotubes stained for myosin heavy chain (green), nuclei (blue, DAPI) and red color corresponds to the protein of interest (collagen I, III, and IV, fibronectin, and laminin). Zoom-in micrographs left to right correspond to DAPI, MHY1 and the merge picture. Large micrographs depict an area of 1 mm by 1 mm. Scale bars are 200 µm and for the zoom-in, scale bars are 50 µm.

SI 4: Myotube maturity in co-cultures
Supplementary information figure 4: Myotube maturity was maintained in the co-cultures with and endothelial cells showed low expresion of MHC2. a. Three-day-old myotubes in TCP (top) and wrinkled PDMS (bottom). Nuclei (DAPI), Myosin heavy chain 1 (green) and collagen IV (red) were immunofluorescent labelled. Micrographs are 2 by 2 mm. Scale bars are 500 µm. Scale bar is 50 um b. RT-qPCR data of expression of myosin heavy chain 2 in myotubes, ECs and co-culture after two and five days on TCP, flat and wrinkled PDMS.

SI 5: Protein expression by Myotubes on different substrates
Supplementary information figure 5: The directional topography positively influences cell attachment and organization of the myotubes' protein deposition. a. Two-day co-culture on flat PDMS. b. Two-day co-culture on the TCP. c. Two-day co-culture on the directional topography. ECs were dTom+ (red), myotubes were EGFP+ (green), nuclei were stained for DAPI (blue), protein of interested was stained with Alexa Fluor 647 and visualized with Cy5 filter (yellow). Scale bars are 200 µm.