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Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

Christoforou N, Oskouei BN, Esteso P, Hill CM, Zimmet JM, Bian W, Bursac N, Leong KW, Hare JM, Gearhart JD - PLoS ONE (2010)

Bottom Line: Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances.Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function.Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, North Carolina, United States of America. nc28@duke.edu

ABSTRACT
Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

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Structural and functional assessment of a NRVM monolayer with a central island comprised of differentiated CPCs.A GFP(+) CPC island (green) within the cardiac network (red) (a–b). Composite image and separate fluorescence channels showing the border between NRVMs and CPCs (c). Note that significant number of GFP positive cells are also α-Actinin positive (cardiomyocytes). Composite image and separate channels showing the Connexin 43 staining within the CPC island. While NRVMs were connected via relatively long Connexin 43(+) gap-junction plaques (data not shown), the gap-junctions in CPC differentiated cardiomyocytes appear small and irregular (d). Electrical propagation initiated inside the CPC island propagated into the surrounding cardiac network (e). Individual hexagonal frames denote 19.5 mm diameter recording area with membrane voltage snapshots shown at given times. Electrical stimulus (pulse sign) was applied in the center of the CPC island at time 0 ms. Membrane voltage is color coded from rest (blue) to peak (red). Circles denote 504 recordings sites. Dashed white line denotes the CPC island. Membrane potentials at selected sites within (1) and outside (2&3) the CPC island are also shown. Electrical stimulus (yellow triangle) yielded action potential propagation that was significantly slower within the island than in the surrounding cardiomyocytes. Isochrones of electrical propagation (white lines) are shown at the bottom right of. Note central isochrone crowding due to slow propagation within the CPC island (black dashed line).
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pone-0011536-g002: Structural and functional assessment of a NRVM monolayer with a central island comprised of differentiated CPCs.A GFP(+) CPC island (green) within the cardiac network (red) (a–b). Composite image and separate fluorescence channels showing the border between NRVMs and CPCs (c). Note that significant number of GFP positive cells are also α-Actinin positive (cardiomyocytes). Composite image and separate channels showing the Connexin 43 staining within the CPC island. While NRVMs were connected via relatively long Connexin 43(+) gap-junction plaques (data not shown), the gap-junctions in CPC differentiated cardiomyocytes appear small and irregular (d). Electrical propagation initiated inside the CPC island propagated into the surrounding cardiac network (e). Individual hexagonal frames denote 19.5 mm diameter recording area with membrane voltage snapshots shown at given times. Electrical stimulus (pulse sign) was applied in the center of the CPC island at time 0 ms. Membrane voltage is color coded from rest (blue) to peak (red). Circles denote 504 recordings sites. Dashed white line denotes the CPC island. Membrane potentials at selected sites within (1) and outside (2&3) the CPC island are also shown. Electrical stimulus (yellow triangle) yielded action potential propagation that was significantly slower within the island than in the surrounding cardiomyocytes. Isochrones of electrical propagation (white lines) are shown at the bottom right of. Note central isochrone crowding due to slow propagation within the CPC island (black dashed line).

Mentions: The potential of CPC derived cardiomyocytes to form functional gap junctions was determined by utilizing a previously described in vitro NRVM co-culture assay [17], [18] which allows the observation of action potential propagation by the means of optical mapping over a macroscopic (3 cm2) culture area [18]. In particular, the CPCs were selectively seeded at high density in the central region of pre-masked coverslips with the NRVMs surrounding them (Fig. 2a). The co-cultures exhibited distinct border between the CPCs and the NRVMs as assayed by GFP and α-Actinin expression (Figs. 2a, b). Immunostaining for cardiomyocyte, smooth muscle and endothelial markers after 14 days of co-culture demonstrated that the majority of the CPCs differentiated into cardiomyocytes (Fig. 2c) with limited smooth muscle or endothelial cell differentiation (data not shown). On the other hand, CPCs plated at low density in the central region of the coverslips formed few patches of cardiomyocytes with the majority of cells differentiating into smooth muscle cells (Fig. S1). Gap junctions indicated by punctuate Connexin 43 localization at the site of cell-cell contacts were detected in the differentiated cardiomyocytes (Fig. 2d). However, unlike the NRVMs that expressed Connexin 43 in relatively long plaques at the sites of cell contacts, the CPC-derived cardiomyocytes expressed Connexin 43 sporadically and irregularly.


Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.

Christoforou N, Oskouei BN, Esteso P, Hill CM, Zimmet JM, Bian W, Bursac N, Leong KW, Hare JM, Gearhart JD - PLoS ONE (2010)

Structural and functional assessment of a NRVM monolayer with a central island comprised of differentiated CPCs.A GFP(+) CPC island (green) within the cardiac network (red) (a–b). Composite image and separate fluorescence channels showing the border between NRVMs and CPCs (c). Note that significant number of GFP positive cells are also α-Actinin positive (cardiomyocytes). Composite image and separate channels showing the Connexin 43 staining within the CPC island. While NRVMs were connected via relatively long Connexin 43(+) gap-junction plaques (data not shown), the gap-junctions in CPC differentiated cardiomyocytes appear small and irregular (d). Electrical propagation initiated inside the CPC island propagated into the surrounding cardiac network (e). Individual hexagonal frames denote 19.5 mm diameter recording area with membrane voltage snapshots shown at given times. Electrical stimulus (pulse sign) was applied in the center of the CPC island at time 0 ms. Membrane voltage is color coded from rest (blue) to peak (red). Circles denote 504 recordings sites. Dashed white line denotes the CPC island. Membrane potentials at selected sites within (1) and outside (2&3) the CPC island are also shown. Electrical stimulus (yellow triangle) yielded action potential propagation that was significantly slower within the island than in the surrounding cardiomyocytes. Isochrones of electrical propagation (white lines) are shown at the bottom right of. Note central isochrone crowding due to slow propagation within the CPC island (black dashed line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2902505&req=5

pone-0011536-g002: Structural and functional assessment of a NRVM monolayer with a central island comprised of differentiated CPCs.A GFP(+) CPC island (green) within the cardiac network (red) (a–b). Composite image and separate fluorescence channels showing the border between NRVMs and CPCs (c). Note that significant number of GFP positive cells are also α-Actinin positive (cardiomyocytes). Composite image and separate channels showing the Connexin 43 staining within the CPC island. While NRVMs were connected via relatively long Connexin 43(+) gap-junction plaques (data not shown), the gap-junctions in CPC differentiated cardiomyocytes appear small and irregular (d). Electrical propagation initiated inside the CPC island propagated into the surrounding cardiac network (e). Individual hexagonal frames denote 19.5 mm diameter recording area with membrane voltage snapshots shown at given times. Electrical stimulus (pulse sign) was applied in the center of the CPC island at time 0 ms. Membrane voltage is color coded from rest (blue) to peak (red). Circles denote 504 recordings sites. Dashed white line denotes the CPC island. Membrane potentials at selected sites within (1) and outside (2&3) the CPC island are also shown. Electrical stimulus (yellow triangle) yielded action potential propagation that was significantly slower within the island than in the surrounding cardiomyocytes. Isochrones of electrical propagation (white lines) are shown at the bottom right of. Note central isochrone crowding due to slow propagation within the CPC island (black dashed line).
Mentions: The potential of CPC derived cardiomyocytes to form functional gap junctions was determined by utilizing a previously described in vitro NRVM co-culture assay [17], [18] which allows the observation of action potential propagation by the means of optical mapping over a macroscopic (3 cm2) culture area [18]. In particular, the CPCs were selectively seeded at high density in the central region of pre-masked coverslips with the NRVMs surrounding them (Fig. 2a). The co-cultures exhibited distinct border between the CPCs and the NRVMs as assayed by GFP and α-Actinin expression (Figs. 2a, b). Immunostaining for cardiomyocyte, smooth muscle and endothelial markers after 14 days of co-culture demonstrated that the majority of the CPCs differentiated into cardiomyocytes (Fig. 2c) with limited smooth muscle or endothelial cell differentiation (data not shown). On the other hand, CPCs plated at low density in the central region of the coverslips formed few patches of cardiomyocytes with the majority of cells differentiating into smooth muscle cells (Fig. S1). Gap junctions indicated by punctuate Connexin 43 localization at the site of cell-cell contacts were detected in the differentiated cardiomyocytes (Fig. 2d). However, unlike the NRVMs that expressed Connexin 43 in relatively long plaques at the sites of cell contacts, the CPC-derived cardiomyocytes expressed Connexin 43 sporadically and irregularly.

Bottom Line: Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances.Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function.Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Pratt School of Engineering, Duke University, Durham, North Carolina, United States of America. nc28@duke.edu

ABSTRACT
Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.

Show MeSH
Related in: MedlinePlus