152
6.4 Multicellular Strategies
Several strategies have been used to develop vascularized contractile grafts that
recapitulate key characteristics of the native myocardium. A common feature of
these approaches is the culture of the three essential cell types: (1) contractile, electrically excitable cardiomyocytes; (2) endothelial cells, to self-assemble into vascular networks; and (3) fibroblasts-like cells, to stabilize cardiac and vascular function
(see Table 6.2). The first such reported tri-culture system utilized hESC-CMs,
hESC-ECs, or HUVECs and mouse embryonic fibroblasts (mFBs) combined in a
cell ratio of 1:1:1 (hCM-mFB-hEC) and seeded into 1:1 mixed poly-L-lactic acid
(PLLA)/polylactic-glycolic acid (PLGA) scaffolds via Matrigel [13, 63] (Fig. 6.3).
This study demonstrated that vascular structures were stabilized in the presence of
fibroblasts and developed increased lumens and vessel density. Without the presence of fibroblasts, vessel structures were limited in development. In this tri-culture
system, cardiomyocytes displayed chronotropic responses to compounds like isoproterenol and carbamylcholine [13, 63]. While this approach was a landmark in the
development of vascularized cardiac tissues, there was limited quantitative characterization of cardiomyocyte interconnectivity or electrophysiology.
Fibrin hydrogels have also been used extensively as a scaffold for cardiac grafts
[101, 117, 134]. Ye et al. co-cultured cardiomyocytes, smooth muscle cells, and
endothelial cells, all derived from hiPSCs at a ratio of 1:1:1. These cells were directly
injected into the infarct or directly injected into a fibrin hydrogel loaded with insulin-like growth factor (IGF). Groups that were tested included direct cell injection,
fibrin patch only, and hEC plus hSMC encapsulated within the fibrin hydrogel. After
4 weeks of in vivo implantation, the cardiac patch resulted in increased ejection fraction which was near that of the sham rat. In addition, the injection of all three cell
types into fibrin hydrogels resulted in the smallest infarct size compared to MI with
no treatment and patch only groups after 4 weeks. The tri-culture group injected into
the fibrin hydrogel had the greatest thickening fraction comparable to that of the
sham. In this study, cells were not cultivated in hydrogels before injection; therefore,
characterization of the cardiomyocyte contractility or vessel development was not
completed in vitro before implantation. Schaefer et al. co-cultured hiPSC-CMs,
human pericytes, and human blood outgrowth endothelial cells (BOEC). In this
study, the presence of vasculature structures improved the force of contraction over
time and increased the presence of the mature form of cardiac troponin (cTnI) compared to the immature form (ssTnI) normally observed in immature tissues. There
was nearly 1 mN of force generated in these constructs by day 14. When the patch
was implanted in vivo, the vasculature anastomosed with the host vasculature and
also integrated with cardiomyocytes. There were nearly 150 vessels/mm
2
in the triculture system. One limitation of these approaches is that hydrogel systems are isotropic, with cells growing in several directions. The native myocardium is anisotropic.
In addition, hydrogels do not allow for the highest level of homotypic cell-cell connections among cardiomyocytes, when multiple cell types are encapsulated.
Scaffold and hydrogel studies typically require all cells to be seeded simultaneously. Subsequent studies demonstrate advantages associated with a sequential
J. Morrissette-McAlmon et al.
6.4 Multicellular Strategies
Several strategies have been used to develop vascularized contractile grafts that
recapitulate key characteristics of the native myocardium. A common feature of
these approaches is the culture of the three essential cell types: (1) contractile, electrically excitable cardiomyocytes; (2) endothelial cells, to self-assemble into vascular networks; and (3) fibroblasts-like cells, to stabilize cardiac and vascular function
(see Table 6.2). The first such reported tri-culture system utilized hESC-CMs,
hESC-ECs, or HUVECs and mouse embryonic fibroblasts (mFBs) combined in a
cell ratio of 1:1:1 (hCM-mFB-hEC) and seeded into 1:1 mixed poly-L-lactic acid
(PLLA)/polylactic-glycolic acid (PLGA) scaffolds via Matrigel [13, 63] (Fig. 6.3).
This study demonstrated that vascular structures were stabilized in the presence of
fibroblasts and developed increased lumens and vessel density. Without the presence of fibroblasts, vessel structures were limited in development. In this tri-culture
system, cardiomyocytes displayed chronotropic responses to compounds like isoproterenol and carbamylcholine [13, 63]. While this approach was a landmark in the
development of vascularized cardiac tissues, there was limited quantitative characterization of cardiomyocyte interconnectivity or electrophysiology.
Fibrin hydrogels have also been used extensively as a scaffold for cardiac grafts
[101, 117, 134]. Ye et al. co-cultured cardiomyocytes, smooth muscle cells, and
endothelial cells, all derived from hiPSCs at a ratio of 1:1:1. These cells were directly
injected into the infarct or directly injected into a fibrin hydrogel loaded with insulin-like growth factor (IGF). Groups that were tested included direct cell injection,
fibrin patch only, and hEC plus hSMC encapsulated within the fibrin hydrogel. After
4 weeks of in vivo implantation, the cardiac patch resulted in increased ejection fraction which was near that of the sham rat. In addition, the injection of all three cell
types into fibrin hydrogels resulted in the smallest infarct size compared to MI with
no treatment and patch only groups after 4 weeks. The tri-culture group injected into
the fibrin hydrogel had the greatest thickening fraction comparable to that of the
sham. In this study, cells were not cultivated in hydrogels before injection; therefore,
characterization of the cardiomyocyte contractility or vessel development was not
completed in vitro before implantation. Schaefer et al. co-cultured hiPSC-CMs,
human pericytes, and human blood outgrowth endothelial cells (BOEC). In this
study, the presence of vasculature structures improved the force of contraction over
time and increased the presence of the mature form of cardiac troponin (cTnI) compared to the immature form (ssTnI) normally observed in immature tissues. There
was nearly 1 mN of force generated in these constructs by day 14. When the patch
was implanted in vivo, the vasculature anastomosed with the host vasculature and
also integrated with cardiomyocytes. There were nearly 150 vessels/mm
2
in the triculture system. One limitation of these approaches is that hydrogel systems are isotropic, with cells growing in several directions. The native myocardium is anisotropic.
In addition, hydrogels do not allow for the highest level of homotypic cell-cell connections among cardiomyocytes, when multiple cell types are encapsulated.
Scaffold and hydrogel studies typically require all cells to be seeded simultaneously. Subsequent studies demonstrate advantages associated with a sequential
J. Morrissette-McAlmon et al.