Name: suppression of pro-fibrotic signaling potentiates factor-mediated reprogramming of mouse embryonic fibroblasts into induced cardiomyocytes
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Description: Watch this Scientific Journal Video about Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes at JoVE.com

Esta investigación fue apoyada por fondos de la Fundación Boettcher Webb Waring biomédica investigación programa, americano corazón Asociación científico desarrollo Grant (13SDG17400031), Departamento de la Universidad de Colorado de destacada carrera medicina Programa, de la Universidad de Colorado División de Cardiología Barlow Nyle dotación y R01HL133230 de NIH (a K.S). ASR fue apoyado por NIH/NCATS Colorado CTSA concesión número TL1TR001081 y una beca predoctoral del consorcio Universidad de Colorado para la investigación de la Fibrosis y traducción (CFReT). Esta investigación fue apoyada también por la concesión de apoyo Centro de cáncer (P30CA046934), beca de núcleos de investigación de enfermedades de piel (P30AR057212) y la base de Cytometry del flujo en el Universidad de Colorado Anschutz Medical Campus.

Todos los experimentos que requieren animales fueron aprobados por el cuidado institucional de animales y uso en la UC Denver Anschutz Medical Campus.
1. aislamiento de MEFs
<ol><li>Comprar ratones C57BL/6 embarazadas E13. Nave durante la noche.</li>
	<li>Eutanasia a la madre según protocolos aprobados de IACUC (ex: ~1.3 CO L/min2 hasta que el animal aparece muerto seguido por dislocación cervical)</li>
	<li>La madre del aerosol con etanol al 70% y abrir la cavidad abdominal. El...

<ol>
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Med. 355 (12), 1199-1209 (2006).</li><li>Hirsch, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracoronary+infusion+of+autologous+mononuclear+bone+marrow+cells+or+peripheral+mononuclear+blood+cells+after+primary+percutaneous+coronary+intervention:+rationale+and+design+of+the+HEBE+trial+-+a+prospective,+multicenter,+randomized+trial.">Intracoronary infusion of autologous mononuclear bone marrow cells or peripheral mononuclear blood cells after primary percutaneous coronary intervention: rationale and design of the HEBE trial - a prospective, multicenter, randomized trial.</a> Am. 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Cardiol. 50 (2), 327-332 (2011).</li><li>Shiba, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+transplantation+of+iPS+cell-derived+cardiomyocytes+regenerates+primate+hearts.">Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts.</a> Nature. 538, 388-391 (2016).</li><li>Ieda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+fibroblasts+into+functional+cardiomyocytes+by+defined+factors.">Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors.</a> Cell. 142 (3), 357-386 (2010).</li><li>Song, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+repair+by+reprogramming+non-myocytes+with+cardiac+transcription+factors.">Heart repair by reprogramming non-myocytes with cardiac transcription factors.</a> Nature. 485, 599-604 (2012).</li><li>Hirai, H., Katoku-Kikyo, N., Keirstead, S. 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El presente estudio esboza una estrategia de alta eficiencia para directamente reprogramar fibroblastos en iCMs funcional vía entrega de GHMT2m reprogramación factores combinados con la supresión de vías de señalización pro-fibróticos. Mediante citometría de flujo, la proyección de imagen inmunofluorescente, calcio imaging y superando a un recuento, nos muestran la mayoría de las células en este protocolo se someten a reprogramación exitosa y adoptar sino del linaje de CM. Previamente hemos demostrado que la ...

Cardiopatía isquémica es la principal causa de muerte en los Estados Unidos1. Aproximadamente 800.000 estadounidenses experimentan un primera o recurrente infarto de miocardio (IM) por año1. Después de MI, la muerte de los cardiomiocitos (CMs) y la fibrosis cardiaca, depositado por los fibroblastos cardiacos activados, afectar corazón función2,3. Progresión de la insuficiencia cardíaca después de MI es en gran medida irreversible debido a la pobre capacidad de regeneración de adultos CMs4,5. Mientras que los tratamientos clínicos actuales lenta progresión de la enfermedad y disminuyen el riesgo de eventos cardíacos futuros6,7,8,9, no hay terapias revertir la progresión de la enfermedad debido a la incapacidad de regenerar el CMs postinfarto10. Terapias celulares nuevos están emergiendo para tratar pacientes con mi desgracia, entrega de células madre al corazón después de MI hasta ahora los ensayos clínicos han demostrado concluyente regenerativo potencial11,12, 13 , 14 , 15 , 16 , 17 , 18.
La generación de células pluripotentes inducidas derivadas de humanos (hiPSCs) de fibroblastos por la sobreexpresión de cuatro factores de transcripción, demostrada por primera vez por Takahashi & Yamanaka, abrió la puerta a nuevos avances en la terapia de la célula19. Estas células pueden distinguir en tres capas germinales todos19y varios métodos eficientes para la generación de grandes cantidades de CMs han demostrado previamente20,21. Derivados de HiPSC CMs (CMs-cadera) ofrece una poderosa plataforma para estudiar cardiomyogenesis y puede tener implicaciones importantes para reparar el corazón después de lesión. Sin embargo, CMs de caderas enfrentan actualmente aplicadas vallas debido a las preocupaciones de teratoma formación22, y su naturaleza inmadura puede ser pro-arritmogénico23. Reprogramación de fibroblastos en hiPSCs despertó interés en directamente reprogramar fibroblastos en otros tipos celulares. Ieda et al demostraron que la sobreexpresión de Mef2c y GATA4 y Tbx5 (GMT) en los resultados de fibroblastos en reprogramación directa al linaje cardíaco, aunque a bajo rendimiento24. Eficiencia de reprogramación fue mejorada con la adición de Hand2 (GHMT)25. Desde estos primeros estudios, numerosas publicaciones han demostrado que alterando el reprogramación factor cóctel con transcripción adicional factores26,27,28,29, modificadores de la cromatina30,31, microRNAs32,33o34 conduce a mejorar las moléculas pequeñas reprogramación eficiencia y maduración de células cardiomiocito inducidas (iCMs ).
Aquí proporcionamos un protocolo detallado para generar iCMs de fibroblastos embrionarios de ratón (MEFs) con alta eficiencia. Demostramos previamente que la GHMT cóctel se mejoró significativamente con la adición de los miR 1 y miR-133 (GHMT2m) y es mejorado cuando pro-fibróticos como transformación de factor de crecimiento β (TGF-β) señalización de rutas de señalización o Rho-associated vías de señalización de la proteína quinasa (ROCK) son inhibidos35. Mediante este protocolo, nos muestran que aproximadamente el 60% de las células express troponina T cardiaca (cTnT), aproximadamente el 50% expresan α-actinina, y un alto número de células de golpeo puede observarse tan pronto como el día 11 después de transducción de reprogramación factores y tratamiento con el TGF-β del tipo I inhibidor del receptor de A-83-01. Además, estos iCMs expresar proteínas de ensambladura de gap incluyendo connexin 43 y exhiben oscilaciones de calcio y la contracción espontánea. Esta marcada mejora en la eficiencia en comparación con estudios anteriores de reprogramación demuestra el potencial para regenerar CMs de poblaciones celulares endógenos que permanecen en el postinfarto de corazón.

<table border="0" cellpadding="0" cellspacing="0" width="1326">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">C57BL/6 Mice</td>
			<td style="width:197px;">Charles River&#39;s Laboratory</td>
			<td style="width:111px;">027</td>
			<td style="width:667px;">For MEF isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Platinum E (PE) Cells</td>
			<td>Cell Biolabs, INC</td>
			<td>RV-101</td>
			<td>For retrovirus production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM High Glucose</td>
			<td>Gibco</td>
			<td>SH30022.FS</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medium 199</td>
			<td>Life Technologies</td>
			<td>11150-059</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td>Gemini</td>
			<td>100106</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donor Horse Serum</td>
			<td>Gemini</td>
			<td>100508 500</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Essential Amino Acids, 50X</td>
			<td>Life Technologies</td>
			<td>11130051</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Sodium Pyruvate Solution, 100X</td>
			<td>Life Technologies</td>
			<td>11360070</td>
			<td>Component of iCM media and for calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Non-Essential Amino Acids, 100X</td>
			<td>Life Technologies</td>
			<td>11140050</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEM Vitamin Solution, 100X</td>
			<td>Life Technologies</td>
			<td>11120-052</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Insulin-Transferrin-Selenium</td>
			<td>Gibco</td>
			<td>41400045</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>Component of iCM media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Penicilin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">GlutaMAX (L-Glutamine Supplement)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>Component of iCM, PE, and Growth media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blasticidin-HCl</td>
			<td>Life Technologies</td>
			<td>A11139-03</td>
			<td>Component of PE media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Puromycin dihydrochloride</td>
			<td>Life Technologies</td>
			<td>A11138-03</td>
			<td>Component of PE media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">0.25% Trypsin/EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>For detaching cells from culture dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A-83-01</td>
			<td>R&#38;D Systems - Tocris</td>
			<td>2939/10</td>
			<td>Treat cells to inhibit TGF-&#946; signaling - promotes high efficiecy reprogramming. Use at 0.5 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Thermo Scientific</td>
			<td>85190</td>
			<td>For dilution and storage of A-83-01 and component of Freeze Medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SureCoat</td>
			<td>Cellutron</td>
			<td>SC-9035</td>
			<td>For coating dishes to plate MEFs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FuGENE 6 Transfection Reagent</td>
			<td>Promega</td>
			<td>E2692</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Opti-MEM Reduced Serum Media</td>
			<td>Gibco</td>
			<td>11058-021</td>
			<td>Transfection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-GATA4</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Hand2</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Mef2c</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X Myc-Tbx5</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X miR-1</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X miR-133</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pBabe-X GFP</td>
			<td></td>
			<td></td>
			<td>Plasmid containing reprogramming factor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polybrene (Hexadimethrine bromide)</td>
			<td>Sigma</td>
			<td>H9268-5G</td>
			<td>For viral induction. Use at a concentration of 6 &#181;g/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Vacuum Filter + bottles (0.22 &#181;m pores)</td>
			<td>Nalgene</td>
			<td>&#160;569-0020&#160;</td>
			<td>For filtering media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringes</td>
			<td>Bd Vacutainer Labware&#160;</td>
			<td>309654</td>
			<td>For viral filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.45 &#181;m Filters</td>
			<td>Celltreat</td>
			<td>229749</td>
			<td>For viral filtration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70 &#181;m cell strainers</td>
			<td>Falcon&#160;</td>
			<td>352350</td>
			<td>For MEF isolation and Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Cytofix/Cytoperm Solution</td>
			<td>BD</td>
			<td>554722</td>
			<td>For fixation and permeabilization of cells for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">perm/wash buffer&#160;</td>
			<td>BD</td>
			<td>554723</td>
			<td>For washing cells for flow cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPBS 1X</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td>For washing cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td>VWR</td>
			<td>0332-100g</td>
			<td>For flow cytometry and calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Serum</td>
			<td>Sigma&#160;</td>
			<td>G9023</td>
			<td>For blocking cells for Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Donkey Serum</td>
			<td>Sigma</td>
			<td>D9663-10mg</td>
			<td>For blocking cells for Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Troponin T</td>
			<td>Thermo Scientific</td>
			<td>ms-295-p</td>
			<td>1:400 IF, 1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse &#945;-actinin</td>
			<td>Sigma</td>
			<td>A7811L</td>
			<td>1:400 IF, 1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit Connexin 43</td>
			<td>Sigma&#160;</td>
			<td>C6219</td>
			<td>1:400 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit Troponin I</td>
			<td>PhosphoSolutions</td>
			<td>2010-TNI</td>
			<td>1:400 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst</td>
			<td>Life Technologies</td>
			<td>62249</td>
			<td>1:10000 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 488, rabbit</td>
			<td>Life Technologies</td>
			<td>A-11034</td>
			<td>1:800 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 555, mouse</td>
			<td>Life Technologies</td>
			<td>A-21422</td>
			<td>1:800 IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa 647, mouse</td>
			<td>Life Technologies</td>
			<td>A-31571</td>
			<td>1:200 Flow Cytometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">27-color ZE5 Flow Cytometer&#160;</td>
			<td>Bio-RAD</td>
			<td></td>
			<td>For FACS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>sigma</td>
			<td>P6148-500mg</td>
			<td>For fixing cells for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td>For permeabilization of cells for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOS&#8482; FL Color Imaging System</td>
			<td>Thermo Scientific</td>
			<td>AMEFC4300</td>
			<td>For IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>RPI</td>
			<td>S23020-5000</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR</td>
			<td>395</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">CaCl2</td>
			<td>Fisher</td>
			<td>C614-500</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td>VWR</td>
			<td>97061-352</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">glucose</td>
			<td>sigma</td>
			<td>G7528-250g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>sigma</td>
			<td>H4034-500g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fura-2 AM</td>
			<td>Life Technologies</td>
			<td>F1221</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluronic F-127</td>
			<td>Sigma</td>
			<td>P2443-250g</td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nifedipine</td>
			<td>Sigma</td>
			<td>N7634-1G</td>
			<td>For disruption calcium transients in iCMs - use at 10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isoproterenol</td>
			<td>sigma</td>
			<td>I6504-1g</td>
			<td>For increasing number of calcium transients in iCMs - use at 1-2 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Marianas Spinning Disk Confocal microscope</td>
			<td>3i</td>
			<td></td>
			<td>For calcium imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol</td>
			<td>Decon Laboratories</td>
			<td>2801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">bleach</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mL conical tubes</td>
			<td>GREINER BIO-ONE</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL conical tubes</td>
			<td>GREINER BIO-ONE</td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm cell culture dishes</td>
			<td>Falcon</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 cm cell culture dishes</td>
			<td>Falcon</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">60 mm cell culture dishes</td>
			<td>GREINER BIO-ONE</td>
			<td>628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>657160&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">12 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>665180&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well cell culture plates</td>
			<td>GREINER BIO-ONE</td>
			<td>662160&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

suppression of pro-fibrotic signaling potentiates factor-mediated reprogramming of mouse embryonic fibroblasts into induced cardiomyocytes

Trans-diferenciación del tipo de una célula somática en otro tiene un enorme potencial para modelar y tratar enfermedades humanas. Estudios anteriores han demostrado que ratón embrionario, fibroblastos cutáneos y cardíacos pueden ser reprogramados en células similares de cardiomiocitos inducida funcionales (iCMs) a través de la sobreexpresión de factores de transcripción cardiogénico como GATA4, Hand2, Mef2c, y TBX5 in vitro e in vivo. Sin embargo, estos estudios previos han demostrado la eficacia relativamente baja. Para restablecer la función del corazón después de lesiones, mecanismos que regulan la reprogramación cardiaca deben ser aclados para aumentar la eficiencia y la maduración del iCMs.
Previamente demostramos que la inhibición de la señalización pro-fibróticos dramáticamente aumenta la eficiencia de reprogramación. Aquí, se detallan métodos para alcanzar una eficiencia de reprogramación de hasta un 60%. Además, se describen varios métodos incluyendo la citometría de flujo, imagen inmunofluorescente y proyección de imagen de calcio para cuantificar la eficiencia de reprogramación y la maduración de reprogramado fibroblastos. Utilizando el protocolo detallado aquí, pueden realizarse estudios mecanísticos para determinar los reguladores positivos y negativos de reprogramación cardiaca. Estos estudios pueden identificar vías de señalización que pueden ser dirigidas para promover la eficiencia de reprogramación y la maduración, que podría conducir a terapias celulares nuevos para el tratamiento de enfermedades del corazón humano.

Usando la estrategia de reprogramación descrita anteriormente y en la figura 1B, generamos iCMs con aproximadamente 70% de las células cardiacas de troponina T y aproximadamente 55% de las células cardiaca α-actinina, cuantificado por citometría de flujo en el día 9 después de transducción de GHMT2m (figura 2A y B). Además, la mayoría de las células expresa cardiaca troponina T, troponina I y α-actini...

Watch this Scientific Journal Video about Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes at JoVE.com

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Aquí presentamos un método robusto para reprogramar fibroblastos embrionarios primarios en cardiomiocitos funcionales a través de la sobreexpresión de GATA4, Hand2, Mef2c, Tbx5, miR-1 y miR-133 (GHMT2m) junto con la inhibición de la señalización de TGF-β. Nuestro protocolo genera cardiomiocitos paliza tan pronto como transducción después de 7 días con hasta un 60% eficiencia.

Supresión de la señalización Pro-fibróticos potencia Factor-mediada la reprogramación de fibroblastos embrionarios de ratón en cardiomiocitos inducida

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Research

JoVE Journal

Developmental Biology

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Estable y eficiente modificación genética de células en el ratón adulto V-SVZ para el análisis de neural de células madre Autónoma y los efectos no autónomos

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Investigación

Biología del Desarrollo

Improved Generation of Induced Cardiomyocytes Using a Polycistronic Construct Expressing Optimal Ratio of Gata4, Mef2c and Tbx5

Mejora de la generación de cardiomiocitos inducida uso de un constructo Polycistronic Expresando relación óptima de Gata4, MEF2C y Tbx5

Direct conversion of cardiac fibroblasts (CFs) into induced cardiomyocytes (iCMs) holds great potential for regenerative medicine by offering alternative ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Protocolo para la conversión directa de murinos fibroblastos embrionarios en células madre Trofoblasto

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

La reprogramación de fibroblastos embrionarios de ratón con factores de transcripción para inducir un programa Hemogenic

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

La estimulación de la señalización de Notch en el ratón osteoclastos precursores

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts

La evaluación de cardiomiocitos subtipos A raíz de reprogramación mediada por el factor de transcripción de fibroblastos embrionarios de ratón

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage ...

Analysis of Retinoic Acid-induced Neural Differentiation of Mouse Embryonic Stem Cells in Two and Three-dimensional Embryoid Bodies

Análisis de inducida por ácido retinoico Neural diferenciación de células madre embrionarias de ratón en cuerpos embrioides dos y tres dimensiones

Mouse embryonic stem cells (ESCs) isolated from the inner mass of the blastocyst (typically at day E3.5), can be used as in vitro model system for ...

Dissection and Culture of Mouse Embryonic Kidney

Disección y cultivo de riñón embrionario de ratón

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Evaluación de la senescencia inducida por la lesión y en Vivo de reprogramación en el músculo esquelético

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Directo linaje reprogramación de fibroblastos de ratón adulto a progenitores eritroides

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...