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Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes.

van Laake LW, van Donselaar EG, Monshouwer-Kloots J, Schreurs C, Passier R, Humbel BM, Doevendans PA, Sonnenberg A, Verkleij AJ, Mummery CL - Cell. Mol. Life Sci. (2009)

Bottom Line: Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling.Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks.These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Heart Lung Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling. Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks. These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

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Survival and continuing maturation of transplanted hESC-CM for at least 24 weeks. a,b Immature hESC-CM in vitro containing poorly organized myofibrils and keratin threads; black dots cytokeratin 8. c–f hESC-CM grafts; black dots GFP; d desmosome, n nucleus, m myofibrils, hu human, ms mouse, ECM extracellular matrix. c Increased myofibril content and organization and desmosome formation in hESC-CM 12 weeks after transplantation. d Further increased myofibril content and organization and desmosome formation in hESC-CM 24 weeks after transplantation. e Extracellular matrix between donor and host cardiomyocytes 24 weeks after transplantation. f Overview of hESC-CM with flanking hESC-derived endothelial cells (ECs) 24 weeks after transplantation. Scale bars (a–e) 200 nm, (f) 1 μm
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Fig5: Survival and continuing maturation of transplanted hESC-CM for at least 24 weeks. a,b Immature hESC-CM in vitro containing poorly organized myofibrils and keratin threads; black dots cytokeratin 8. c–f hESC-CM grafts; black dots GFP; d desmosome, n nucleus, m myofibrils, hu human, ms mouse, ECM extracellular matrix. c Increased myofibril content and organization and desmosome formation in hESC-CM 12 weeks after transplantation. d Further increased myofibril content and organization and desmosome formation in hESC-CM 24 weeks after transplantation. e Extracellular matrix between donor and host cardiomyocytes 24 weeks after transplantation. f Overview of hESC-CM with flanking hESC-derived endothelial cells (ECs) 24 weeks after transplantation. Scale bars (a–e) 200 nm, (f) 1 μm

Mentions: Since fibrosis was present at early time-points and was not degraded 12 weeks post-grafting, the question arose whether transplanted hESC-CM could survive at all for longer periods. We therefore increased follow-up to 24 weeks and found viable grafts that appeared similar to those at 12 weeks in terms of graft size and GFP-fluorescence. At both time-points, the hESC-CM showed signs of maturation in vivo, including loss of punctuate staining for cytokeratin 8 which was found in intracellular keratin threads and desmosomes in immature hESC-CM in vitro (Fig. 5a, b), as previously reported for human fetal cardiomyocytes [29]. However, EM analysis of the grafted cells 24 weeks after transplantation revealed that the hESC-CM had matured further, evidenced by increased myofibril content and improved sarcomeric organization compared to 12 weeks in vivo maturation (Fig. 5c, d). Paradoxically, a fibrotic layer surrounding the graft still largely separated human from mouse myocardium, preventing desmosome formation between human and mouse cells (Fig. 5e) even though desmosomes between human cardiomyocytes were abundant (Fig. 5d). It was thus likely that the grafts received oxygen and nutrients through infiltrating vessels. We observed previously that some mouse-derived vessels were present in the grafts [10]. Surprisingly, a subset of 12- and 24-week hESC-CM grafts (4 out of 16 hearts) were surrounded by small clusters of GFP-expressing cells which formed a mosaic-like pattern with the native mouse cardiomyocytes (Figs. 5f and 6a). The morphology of these hESC-derived cells was typical of endothelial cells: long flat cells aligning into capillaries (Fig. 6d) with the typical appearance of caveolae and Weibel-palade bodies. The capillaries consisted uniquely of GFP-expressing human endothelial cells but were connected to the mouse vasculature, as evidenced by the presence of mouse leukocytes and erythrocytes in their lumen (Fig. 6b, c), and were associated with an increased vascular density in the area around the grafts.Fig. 5


Extracellular matrix formation after transplantation of human embryonic stem cell-derived cardiomyocytes.

van Laake LW, van Donselaar EG, Monshouwer-Kloots J, Schreurs C, Passier R, Humbel BM, Doevendans PA, Sonnenberg A, Verkleij AJ, Mummery CL - Cell. Mol. Life Sci. (2009)

Survival and continuing maturation of transplanted hESC-CM for at least 24 weeks. a,b Immature hESC-CM in vitro containing poorly organized myofibrils and keratin threads; black dots cytokeratin 8. c–f hESC-CM grafts; black dots GFP; d desmosome, n nucleus, m myofibrils, hu human, ms mouse, ECM extracellular matrix. c Increased myofibril content and organization and desmosome formation in hESC-CM 12 weeks after transplantation. d Further increased myofibril content and organization and desmosome formation in hESC-CM 24 weeks after transplantation. e Extracellular matrix between donor and host cardiomyocytes 24 weeks after transplantation. f Overview of hESC-CM with flanking hESC-derived endothelial cells (ECs) 24 weeks after transplantation. Scale bars (a–e) 200 nm, (f) 1 μm
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Fig5: Survival and continuing maturation of transplanted hESC-CM for at least 24 weeks. a,b Immature hESC-CM in vitro containing poorly organized myofibrils and keratin threads; black dots cytokeratin 8. c–f hESC-CM grafts; black dots GFP; d desmosome, n nucleus, m myofibrils, hu human, ms mouse, ECM extracellular matrix. c Increased myofibril content and organization and desmosome formation in hESC-CM 12 weeks after transplantation. d Further increased myofibril content and organization and desmosome formation in hESC-CM 24 weeks after transplantation. e Extracellular matrix between donor and host cardiomyocytes 24 weeks after transplantation. f Overview of hESC-CM with flanking hESC-derived endothelial cells (ECs) 24 weeks after transplantation. Scale bars (a–e) 200 nm, (f) 1 μm
Mentions: Since fibrosis was present at early time-points and was not degraded 12 weeks post-grafting, the question arose whether transplanted hESC-CM could survive at all for longer periods. We therefore increased follow-up to 24 weeks and found viable grafts that appeared similar to those at 12 weeks in terms of graft size and GFP-fluorescence. At both time-points, the hESC-CM showed signs of maturation in vivo, including loss of punctuate staining for cytokeratin 8 which was found in intracellular keratin threads and desmosomes in immature hESC-CM in vitro (Fig. 5a, b), as previously reported for human fetal cardiomyocytes [29]. However, EM analysis of the grafted cells 24 weeks after transplantation revealed that the hESC-CM had matured further, evidenced by increased myofibril content and improved sarcomeric organization compared to 12 weeks in vivo maturation (Fig. 5c, d). Paradoxically, a fibrotic layer surrounding the graft still largely separated human from mouse myocardium, preventing desmosome formation between human and mouse cells (Fig. 5e) even though desmosomes between human cardiomyocytes were abundant (Fig. 5d). It was thus likely that the grafts received oxygen and nutrients through infiltrating vessels. We observed previously that some mouse-derived vessels were present in the grafts [10]. Surprisingly, a subset of 12- and 24-week hESC-CM grafts (4 out of 16 hearts) were surrounded by small clusters of GFP-expressing cells which formed a mosaic-like pattern with the native mouse cardiomyocytes (Figs. 5f and 6a). The morphology of these hESC-derived cells was typical of endothelial cells: long flat cells aligning into capillaries (Fig. 6d) with the typical appearance of caveolae and Weibel-palade bodies. The capillaries consisted uniquely of GFP-expressing human endothelial cells but were connected to the mouse vasculature, as evidenced by the presence of mouse leukocytes and erythrocytes in their lumen (Fig. 6b, c), and were associated with an increased vascular density in the area around the grafts.Fig. 5

Bottom Line: Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling.Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks.These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Heart Lung Center Utrecht, Utrecht, The Netherlands.

ABSTRACT
Transplantation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) for cardiac regeneration is hampered by the formation of fibrotic tissue around the grafts, preventing electrophysiological coupling. Investigating this process, we found that: (1) beating hESC-CM in vitro are embedded in collagens, laminin and fibronectin, which they bind via appropriate integrins; (2) after transplantation into the mouse heart, hESC-CM continue to secrete collagen IV, XVIII and fibronectin; (3) integrin expression on hESC-CM largely matches the matrix type they encounter or secrete in vivo; (4) co-transplantation of hESC-derived endothelial cells and/or cardiac progenitors with hESC-CM results in the formation of functional capillaries; and (5) transplanted hESC-CM survive and mature in vivo for at least 24 weeks. These results form the basis of future developments aiming to reduce the adverse fibrotic reaction that currently complicates cell-based therapies for cardiac disease, and to provide an additional clue towards successful engraftment of cardiomyocytes by co-transplanting endothelial cells.

Show MeSH
Related in: MedlinePlus