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Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.

ABSTRACT: The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts with HUVECs were determined in a fusion assay. Endothelial growth medium proved to be the best compromise for the coculture, without affecting the myoblast fusion index. Second, both cell types were cocultured in a BAM maintained under tension to stimulate myofiber alignment. We then tested different total cell numbers containing 50% HUVECs and found that BAMs with a total cell number of 2?×?10(6) resulted in well-aligned and densely packed myofibers while allowing for improved interspersed endothelial network formation. Third, we compared different myoblast-HUVEC ratios. Including higher numbers of myoblasts improved endothelial network formation at lower total cell density; however, improvement of network characteristics reached a plateau when 1?×?10(6) or more myoblasts were present. Finally, addition of Matrigel to the fibrin ECM did not enhance overall myofiber and endothelial network formation. Therefore, in our BAM model, we suggest the use of a fibrin extracellular matrix containing 2?×?10(6) cells of which 50-70% are muscle cells. Optimizing these coculture conditions allows for a physiologically more relevant muscle model and paves the way toward engineering of larger in vitro muscle constructs.

SUBMITTER: Gholobova D

PROVIDER: S-EPMC4605445 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.

Gholobova Dacha D Decroix Lieselot L Van Muylder Vicky V Desender Linda L Gerard Melanie M Carpentier Gilles G Vandenburgh Herman H Thorrez Lieven L

Tissue engineering. Part A 20150901 19-20

The size of in vitro engineered skeletal muscle tissue is limited due to the lack of a vascular network in vitro. In this article, we report tissue-engineered skeletal muscle consisting of human aligned myofibers with interspersed endothelial networks. We extend our bioartificial muscle (BAM) model by coculturing human muscle progenitor cells with human umbilical vein endothelial cells (HUVECs) in a fibrin extracellular matrix (ECM). First, the optimal medium conditions for coculturing myoblasts ...[more]

PMID: 26177063

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Project description:UnlabelledTransplantation of human muscle precursor cells (hMPCs) is envisioned for the treatment of various muscle diseases. However, a feasible noninvasive tool to monitor cell survival, migration, and integration into the host tissue is still missing.MethodsIn this study, we designed an adenoviral delivery system to genetically modify hMPCs to express a signaling-deficient form of human dopamine D2 receptor (hD2R). The gene expression levels of the receptor were evaluated by reverse transcriptase polymerase chain reaction, and infection efficiency was evaluated by fluorescent microscopy. The viability, proliferation, and differentiation capacity of the transduced cells, as well as their myogenic phenotype, were determined by flow cytometry analysis and fluorescent microscopy. (18)F-fallypride and (18)F-fluoromisonidazole, two well-established PET radioligands, were assessed for their potential to image engineered hMPCs in a mouse model and their uptakes were evaluated at different time points after cell inoculation in vivo. Biodistribution studies, autoradiography, and PET experiments were performed to determine the extent of signal specificity. To address feasibility for tracking hMPCs in an in vivo model, the safety of the adenoviral gene delivery was evaluated. Finally, the harvested tissues were histologically examined to determine whether survival of the transplanted cells was sustained at different time points.ResultsAdenoviral gene delivery was shown to be safe, with no detrimental effects on the primary human cells. The viability, proliferation, and differentiation capacity of the transduced cells were confirmed, and flow cytometry analysis and fluorescent microscopy showed that their myogenic phenotype was sustained. (18)F-fallypride and (18)F-fluoromisonidazole were successfully synthesized. Specific binding of (18)F-fallypride to hD2R hMPCs was demonstrated in vitro and in vivo. Furthermore, the (18)F-fluoromisonidazole signal was high at the early stages. Finally, sustained survival of the transplanted cells at different time points was confirmed histologically, with formation of muscle tissue at the site of injection.ConclusionOur proposed use of a signaling-deficient hD2R as a potent reporter for in vivo hMPC PET tracking by (18)F-fallypride is a significant step toward potential noninvasive tracking of hD2R hMPCs and bioengineered muscle tissues in the clinic.

Dataset Information

Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.

Publications

Endothelial Network Formation Within Human Tissue-Engineered Skeletal Muscle.

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