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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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Co-transplanted hESC-ECs form functional vessels in the host myocardium. a Small clusters of GFP-expressing cells scattered through the host myocardium 24 weeks after transplantation, b–d Human endothelial cells forming capillaries in mouse myocardium; black dots GFP, e erythrocyte, l leukocyte, n nucleus, EC endothelial cell, hu human, ms mouse. e Overview of endothelial structures in a beating area from hES3-GFP. f, g Ki-67-positive endothelial cells expressing human PECAM and human von Willebrand Factor. h Ki-67-positive hESC-derived endothelial cells in a graft 12 weeks after transplantation. i,j Undifferentiated hESCs; immunogold labeling for Tra-1-60 at the apical side (i) and intracellular (j); black dots Tra-1-60; n nucleus. Scale bars (a,e) 100 μm, (b,c) 1 μm (d) 500 nm (f,g) 20 μm (h,i) 200 nm
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Fig6: Co-transplanted hESC-ECs form functional vessels in the host myocardium. a Small clusters of GFP-expressing cells scattered through the host myocardium 24 weeks after transplantation, b–d Human endothelial cells forming capillaries in mouse myocardium; black dots GFP, e erythrocyte, l leukocyte, n nucleus, EC endothelial cell, hu human, ms mouse. e Overview of endothelial structures in a beating area from hES3-GFP. f, g Ki-67-positive endothelial cells expressing human PECAM and human von Willebrand Factor. h Ki-67-positive hESC-derived endothelial cells in a graft 12 weeks after transplantation. i,j Undifferentiated hESCs; immunogold labeling for Tra-1-60 at the apical side (i) and intracellular (j); black dots Tra-1-60; n nucleus. Scale bars (a,e) 100 μm, (b,c) 1 μm (d) 500 nm (f,g) 20 μm (h,i) 200 nm

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)

Co-transplanted hESC-ECs form functional vessels in the host myocardium. a Small clusters of GFP-expressing cells scattered through the host myocardium 24 weeks after transplantation, b–d Human endothelial cells forming capillaries in mouse myocardium; black dots GFP, e erythrocyte, l leukocyte, n nucleus, EC endothelial cell, hu human, ms mouse. e Overview of endothelial structures in a beating area from hES3-GFP. f, g Ki-67-positive endothelial cells expressing human PECAM and human von Willebrand Factor. h Ki-67-positive hESC-derived endothelial cells in a graft 12 weeks after transplantation. i,j Undifferentiated hESCs; immunogold labeling for Tra-1-60 at the apical side (i) and intracellular (j); black dots Tra-1-60; n nucleus. Scale bars (a,e) 100 μm, (b,c) 1 μm (d) 500 nm (f,g) 20 μm (h,i) 200 nm
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Fig6: Co-transplanted hESC-ECs form functional vessels in the host myocardium. a Small clusters of GFP-expressing cells scattered through the host myocardium 24 weeks after transplantation, b–d Human endothelial cells forming capillaries in mouse myocardium; black dots GFP, e erythrocyte, l leukocyte, n nucleus, EC endothelial cell, hu human, ms mouse. e Overview of endothelial structures in a beating area from hES3-GFP. f, g Ki-67-positive endothelial cells expressing human PECAM and human von Willebrand Factor. h Ki-67-positive hESC-derived endothelial cells in a graft 12 weeks after transplantation. i,j Undifferentiated hESCs; immunogold labeling for Tra-1-60 at the apical side (i) and intracellular (j); black dots Tra-1-60; n nucleus. Scale bars (a,e) 100 μm, (b,c) 1 μm (d) 500 nm (f,g) 20 μm (h,i) 200 nm
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