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1.
Nat Methods ; 8(12): 1037-40, 2011 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-22020065

RESUMEN

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5(eGFP/w) hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP(+) cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.


Asunto(s)
Separación Celular/métodos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Mioblastos Cardíacos/citología , Miocitos Cardíacos/citología , Factores de Transcripción/metabolismo , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Biomarcadores/análisis , Diferenciación Celular , Perfilación de la Expresión Génica , Proteína Homeótica Nkx-2.5 , Proteínas de Homeodominio/genética , Humanos , Mioblastos Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Reacción en Cadena de la Polimerasa , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Factores de Transcripción/genética , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismo
2.
Nat Cardiovasc Res ; 3(2): 145-165, 2024 02.
Artículo en Inglés | MEDLINE | ID: mdl-39196193

RESUMEN

Preclinical data have confirmed that human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can remuscularize the injured or diseased heart, with several clinical trials now in planning or recruitment stages. However, because ventricular arrhythmias represent a complication following engraftment of intramyocardially injected PSC-CMs, it is necessary to provide treatment strategies to control or prevent engraftment arrhythmias (EAs). Here, we show in a porcine model of myocardial infarction and PSC-CM transplantation that EAs are mechanistically linked to cellular heterogeneity in the input PSC-CM and resultant graft. Specifically, we identify atrial and pacemaker-like cardiomyocytes as culprit arrhythmogenic subpopulations. Two unique surface marker signatures, signal regulatory protein α (SIRPA)+CD90-CD200+ and SIRPA+CD90-CD200-, identify arrhythmogenic and non-arrhythmogenic cardiomyocytes, respectively. Our data suggest that modifications to current PSC-CM-production and/or PSC-CM-selection protocols could potentially prevent EAs. We further show that pharmacologic and interventional anti-arrhythmic strategies can control and potentially abolish these arrhythmias.


Asunto(s)
Arritmias Cardíacas , Miocitos Cardíacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/trasplante , Animales , Arritmias Cardíacas/terapia , Humanos , Modelos Animales de Enfermedad , Infarto del Miocardio/terapia , Porcinos , Células Cultivadas , Diferenciación Celular , Células Madre Pluripotentes Inducidas/trasplante , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de los fármacos , Fenotipo , Biomarcadores/metabolismo , Células Madre Pluripotentes/trasplante , Trasplante de Células Madre/métodos , Antiarrítmicos/uso terapéutico , Antiarrítmicos/farmacología , Frecuencia Cardíaca/fisiología
3.
Cardiovasc Res ; 118(3): 828-843, 2022 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-33744937

RESUMEN

AIMS: We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. METHODS AND RESULTS: Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively. CONCLUSIONS: We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects.


Asunto(s)
Células Madre Embrionarias Humanas , Células Madre Pluripotentes Inducidas , Potenciales de Acción/fisiología , Diferenciación Celular , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo
4.
Trends Mol Med ; 23(7): 651-668, 2017 07.
Artículo en Inglés | MEDLINE | ID: mdl-28576602

RESUMEN

Human pluripotent stem cells (hPSCs) offer a practical source for the de novo generation of cardiac tissues and a unique opportunity to investigate cardiovascular lineage commitment. Numerous strategies have focused on the in vitro production of cardiomyocytes, smooth muscle, and endothelium from hPSCs. However, these differentiation protocols often yield undesired cell types. Thus, establishing a set of stage-specific markers for pure cardiac subpopulations will assist in defining the hierarchy of cardiac differentiation, aid in the development of cellular therapy, and facilitate drug screening and disease modeling. The recent characterization of many such markers is enabling the isolation of major cardiac lineages and subpopulations from differentiating hPSCs. We provide here a comprehensive review detailing the suite of biomarkers used to differentiate cardiac lineages from mixed hPSC-derived populations.


Asunto(s)
Antígenos de Diferenciación/metabolismo , Diferenciación Celular , Miocardio/metabolismo , Células Madre Pluripotentes/metabolismo , Células Endoteliales/citología , Células Endoteliales/metabolismo , Humanos , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Células Madre Pluripotentes/citología
5.
Cell Rep ; 18(1): 54-67, 2017 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-27989715

RESUMEN

Applications of embryonic stem cells (ESCs) require faithful chromatin changes during differentiation, but the fate of the X chromosome state in differentiating ESCs is unclear. Female human ESC lines either carry two active X chromosomes (XaXa), an Xa and inactive X chromosome with or without XIST RNA coating (XiXIST+Xa;XiXa), or an Xa and an eroded Xi (XeXa) where the Xi no longer expresses XIST RNA and has partially reactivated. Here, we established XiXa, XeXa, and XaXa ESC lines and followed their X chromosome state during differentiation. Surprisingly, we found that the X state pre-existing in primed ESCs is maintained in differentiated cells. Consequently, differentiated XeXa and XaXa cells lacked XIST, did not induce X inactivation, and displayed higher X-linked gene expression than XiXa cells. These results demonstrate that X chromosome dosage compensation is not required for ESC differentiation. Our data imply that XiXIST+Xa ESCs are most suited for downstream applications and show that all other X states are abnormal byproducts of our ESC derivation and propagation method.


Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias Humanas/metabolismo , Inactivación del Cromosoma X/genética , Diferenciación Celular/efectos de los fármacos , Línea Celular , Metilación de ADN/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Células Madre Embrionarias Humanas/efectos de los fármacos , Humanos , Hibridación Fluorescente in Situ , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Análisis de Secuencia de ARN , Tretinoina/farmacología
6.
Stem Cell Reports ; 6(1): 95-108, 2016 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-26771355

RESUMEN

The generation of tissue-specific cell types from human embryonic stem cells (hESCs) is critical for the development of future stem cell-based regenerative therapies. Here, we identify CD13 and ROR2 as cell-surface markers capable of selecting early cardiac mesoderm emerging during hESC differentiation. We demonstrate that the CD13+/ROR2+ population encompasses pre-cardiac mesoderm, which efficiently differentiates to all major cardiovascular lineages. We determined the engraftment potential of CD13+/ROR2+ in small (murine) and large (porcine) animal models, and demonstrated that CD13+/ROR2+ progenitors have the capacity to differentiate toward cardiomyocytes, fibroblasts, smooth muscle, and endothelial cells in vivo. Collectively, our data show that CD13 and ROR2 identify a cardiac lineage precursor pool that is capable of successful engraftment into the porcine heart. These markers represent valuable tools for further dissection of early human cardiac differentiation, and will enable a detailed assessment of human pluripotent stem cell-derived cardiac lineage cells for potential clinical applications.


Asunto(s)
Antígenos CD13/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Mesodermo/metabolismo , Miocardio/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/metabolismo , Animales , Antígenos CD13/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Células Endoteliales/citología , Células Endoteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica/métodos , Células Madre Embrionarias Humanas/citología , Humanos , Mesodermo/citología , Ratones , Músculo Liso/citología , Músculo Liso/metabolismo , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trasplante de Células Madre/métodos , Porcinos , Factores de Tiempo , Trasplante Heterólogo
7.
Stem Cells Transl Med ; 5(1): 67-74, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26582908

RESUMEN

UNLABELLED: Given the limited regenerative capacity of the heart, cellular therapy with stem cell-derived cardiac cells could be a potential treatment for patients with heart disease. However, reliable imaging techniques to longitudinally assess engraftment of the transplanted cells are scant. To address this issue, we used ferumoxytol as a labeling agent of human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) to facilitate tracking by magnetic resonance imaging (MRI) in a large animal model. Differentiating hESCs were exposed to ferumoxytol at different time points and varying concentrations. We determined that treatment with ferumoxytol at 300 µg/ml on day 0 of cardiac differentiation offered adequate cell viability and signal intensity for MRI detection without compromising further differentiation into definitive cardiac lineages. Labeled hESC-CPCs were transplanted by open surgical methods into the left ventricular free wall of uninjured pig hearts and imaged both ex vivo and in vivo. Comprehensive T2*-weighted images were obtained immediately after transplantation and 40 days later before termination. The localization and dispersion of labeled cells could be effectively imaged and tracked at days 0 and 40 by MRI. Thus, under the described conditions, ferumoxytol can be used as a long-term, differentiation-neutral cell-labeling agent to track transplanted hESC-CPCs in vivo using MRI. SIGNIFICANCE: The development of a safe and reproducible in vivo imaging technique to track the fate of transplanted human embryonic stem cell-derived cardiac progenitor cells (hESC-CPCs) is a necessary step to clinical translation. An iron oxide nanoparticle (ferumoxytol)-based approach was used for cell labeling and subsequent in vivo magnetic resonance imaging monitoring of hESC-CPCs transplanted into uninjured pig hearts. The present results demonstrate the use of ferumoxytol labeling and imaging techniques in tracking the location and dispersion of cell grafts, highlighting its utility in future cardiac stem cell therapy trials.


Asunto(s)
Rastreo Celular/métodos , Células Madre Embrionarias , Óxido Ferrosoférrico/farmacología , Imagen por Resonancia Magnética , Mioblastos Cardíacos , Trasplante de Células Madre , Células Madre Embrionarias/diagnóstico por imagen , Células Madre Embrionarias/trasplante , Compuestos Férricos/farmacología , Óxido Ferrosoférrico/farmacocinética , Xenoinjertos , Humanos , Mioblastos Cardíacos/diagnóstico por imagen , Mioblastos Cardíacos/trasplante , Radiografía
8.
Stem Cell Res ; 13(1): 172-9, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24968096

RESUMEN

The study of human cardiogenesis would benefit from a detailed cell lineage fate map akin to that established for the haematopoietic lineages. Here we sought to define cell lineage relationships based on the expression of NKX2-5 and the cell surface markers VCAM1, SIRPA and CD34 during human cardiovascular development. Expression of NKX2-5(GFP) was used to identify cardiac progenitors and cardiomyocytes generated during the differentiation of NKX2-5(GFP/w) human embryonic stem cells (hESCs). Cardiovascular cell lineages sub-fractionated on the basis of SIRPA, VCAM1 and CD34 expression were assayed for differentiation potential and gene expression. The NKX2-5(pos)CD34(pos) population gave rise to endothelial cells that rapidly lost NKX2-5 expression in culture. Conversely, NKX2-5 expression was maintained in myocardial committed cells, which progressed from being NKX2-5(pos)SIRPA(pos) to NKX2-5(pos)SIRPA(pos)VCAM1(pos). Up-regulation of VCAM1 was accompanied by the expression of myofilament markers and reduced clonal capacity, implying a restriction of cell fate potential. Combinatorial expression of NKX2-5, SIRPA, VCAM1 and CD34 can be used to define discrete stages of cardiovascular cell lineage differentiation. These markers identify specific stages of cardiomyocyte and endothelial lineage commitment and, thus provide a scaffold for establishing a fate map of early human cardiogenesis.


Asunto(s)
Antígenos CD34/metabolismo , Antígenos de Diferenciación/metabolismo , Sistema Cardiovascular/crecimiento & desarrollo , Miocitos Cardíacos/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores Inmunológicos/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Diferenciación Celular/fisiología , Linaje de la Célula , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Humanos , Miocitos Cardíacos/metabolismo
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