A.F.-P. was supported by the National Science Foundation Graduate Research Fellowship under Grant No.2015165336. N.V.M was supported by grants from the NIH (HL094699), Burroughs Wellcome Fund (1009838), and the March of Dimes (#5-FY14-203). We acknowledge Young-Jae Nam, Christina Lubczyk, and Minoti Bhakta for their important contributions to protocol development and data analysis. We also thank John Shelton for valuable technical input and members of the Munshi lab for scientific discussion.

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The authors have nothing to disclose.

המחקר הנוכחי מספק אסטרטגיה ישיר תכנות מחדש לגיור של MEFs לתוך קבוצה מגוונת של תת הלב באמצעות ביטוי בתיווך רטרו-וירוס של שעתוק לב גורמי Gata4, Mef2c, Tbx5, ו Hand2 (GHMT). שימוש בגישת immunostaining זמנית בשילוב עם עכבר כתב PM-ספציפי, אנו מסוגלים לזהות IAM, iVMs, ו iPMs ברזולוצית תא בודד. כזה assay מאפש?...

הלב הוא האיבר הפונקציונלי הראשון לפתח בעובר 1, 2. בשיתוף עם מערכת הדם, היא מספקת חמצן, חומרי הזנה, וכן מנגנון סילוק פסולת במהלך פיתוח. שלושה שבועות לאחר ההפריה, הלב האנושי פועם בפעם הראשונה והרגולציה הראויה מתוחזק על ידי cardiomyocytes (CMS). ההפסד בלתי הפיך של תאים מיוחדים אלה היא אפוא הנושא היסודי אותו אי ספיקת לב מתקדמת. בעוד כמה אורגניזמים כגון דג הזברה Xenopus יש פוטנציאל להתחדשות לב, הלב של יונקי הבוגרים מוגבל יותר 3, 5, 6. לפיכך, בהתחשב הפונקציה הקריטית של הלב, אין זה מפתיע כי מחלת לב היא הגורם המוביל למוות בעולם, והיוותה 600,000 מקרי מוות בארצות הברית לבדה 7. הrefore, טיפולים מבוססי תאים כדי לתקן או להחליף את שריר הלב נפגע ביעילות הם עניין קליני גדול. המחקר הזרע של יאמאנאקה ועמיתיו 8 הראה כי ביטוי כפוי של ארבעה גורמי שעתוק מספיק כדי להמיר תאי פיברובלסטים בדיל באופן מלא לתאי גזע פלוריפוטנטיים. עם זאת, יכולת tumorigenic של כל אסטרטגיות תאי גזע פלוריפוטנטיים כבר דאגה קריטית בשימוש שלהם למטרות טיפוליות. זה הניע התחום המדעי לחפש שיטות חלופיות כדי transdifferentiate תאים תוך הימנעות שלב פלוריפוטנטיים. לאחרונה, מספר קבוצות הראו את הכדאיות של אסטרטגיה זו על ידי הצגת המרה ישירה של פיברובלסטים עכבר לתאי cardiomyocyte דמוי המושרה (iCLMs) עם ביטוי אקטופי של שעתוק גורמי Gata4, Mef2c, Tbx5, ובהמשך, Hand2 (GMT ו GHMT 9, בהתאמה), 10. Furthermore, באותה האסטרטגיה ניתן לבצע in vivo ו ברקמות אדם נגזר 9, 11, 12. מחקרים שנעשה לאחרונה הדגישו גורמים נוספים או מסלולי איתות כי יכול להיות מווסת כדי לשפר את יעילות תכנות מחדש של לב עוד 13, 14, 15. יחדיו, מחקרים אלה להדגים את הפוטנציאל של transdifferentiation ביים עבור טיפולים רגנרטיבית. עם זאת, היעילות הנמוכה של תכנות מחדש CM, המנגנונים המולקולריים הידועים, שחזור עולה בקנה אחד בשל הבדלים מתודולוגיים 16, ואת האופי הטרוגני של iCLMs להישאר unaddressed. על מנת ישירות להעריך ההטרוגניות iCLM, עצבנו assay בדידה החזקה תא בודד לזיהוי התפתחות sarcomere ו specificatio שושלת לבn ושניים מאפיינים דרושים של cardiomyocytes הפונקציונלי. יש לפחות שלושה סוגים עיקריים של CM בלב כהגדרתו לפי המיקום שלהם תכונות חשמליות ייחודי: פרוזדורים (PM), חדרית (VM) ו קוצב לב (PM) 17, 18, 19, 20. בשנת מערך מתוזמר, הם לאפשר שאיבה הנכונה של דם. במהלך פגיעה לבבית, אחד או כל תת עלול להיפגע, ואת הסוג של טיפול בתא היה צורך לטפל על בסיס כל מקרה לגופו. כיום, רוב האסטרטגיות להתמקד בדור הכולל של cardiomyocytes, בעוד מעט עבודה נעשתה כדי ללמוד את המנגנונים המולקולריים אשר מסדיר מפרט תת סוג. המחקר הבא מפרט כיצד לכמת כראוי סרקומר מאורגן היטב לזהות קבוצה מגוונת של תת cardiomyocyte. באמצעות קוצב לב (PM) עכבר כתב -specific, אנו מסוגלים להחיל iהגישה mmunocytochemical להבחין מיוציטים המושרה דמוי פרוזדורים (IAM), myocytes חדרית דמוי המושרה (IVM), ו myocytes PM דמוי המושרה (iPMs) 21. בהתבסס על התצפיות שלנו, רק תאים כי תערוכת ארגון sarcomere מסוגלים פועם ספונטני. פלטפורמת תכנות מחדש ייחודית זו מאפשרת להערכת התפקיד פרמטרים מסוימים של בארגון sarcomere, מפרט תת סוג, והיעילות של תכנות מחדש CM ברזולוציה תא בודד.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">DMEM</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">D5796</td>
			<td style="width:347px;">Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Medium 199</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11150059</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Fetal bovine serrum (FBS)</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">F2442</td>
			<td style="width:347px;">Component of iCLM media, Plat-E media, fibroblast, and Transfection media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Insulin-Transferrin-Selenium G</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">41400-045</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">MEM vitamin solution</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11120-052</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">MEM amino acids</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">1601149</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Non-Essential amino acids</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11140-050</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Antibiotic-Antimycotics</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15240062</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">B-27 supplement</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">17504044</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Heat-Inactivated Horse Serum</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">26050-088</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">NaPyruvate</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11360-70</td>
			<td style="width:347px;">Component of iCLM media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Penicillin/Streptomycin</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">1514022</td>
			<td style="width:347px;">Component of Plat-E media and fibroblast media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Puromycin&#160;</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A11139-03</td>
			<td>Component of Plat-E media</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Blasticidin&#160;&#160;</td>
			<td style="width:91px;">Gemini Bio-Products</td>
			<td>400-128P</td>
			<td>Component of Plat-E media</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Glutamax</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>35050-061</td>
			<td>Component of Fibroblast media</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Confocal laser scanning LSM700</td>
			<td style="width:91px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>For confocal analysis</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">FuGENE 6 transfection Reagent</td>
			<td style="width:91px;">Promega</td>
			<td style="width:119px;">E2692</td>
			<td>Transfection reagent&#160;</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Opti-MEM Reduced Serum Medium</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">31985-070</td>
			<td>Transfection reagent&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Polybrene</td>
			<td style="width:91px;">Millipore</td>
			<td style="width:119px;">TR-1003-G</td>
			<td>Induction reagent. Use at a final concentration of 8um/mL</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Platinium-E (PE) Retroviral Packagin Cell Line, Ecotropic</td>
			<td style="width:91px;">CellBiolabs</td>
			<td style="width:119px;">RV-101</td>
			<td>Retroviral pacaking cell line</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Trypsin 0.25% EDTA</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;"></td>
			<td>For MEFs and Plat-E dissociation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti &#945;-Actinin (Clone EA-53)</td>
			<td style="width:91px;">Sigma</td>
			<td>A7811</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Chicken anti-GFP IgY</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A10262</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit Pab anti-NPPA&#160;</td>
			<td style="width:91px;">Abgent</td>
			<td>AP8534A</td>
			<td>Antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit Pab anti Myl2 IgG&#160;</td>
			<td style="width:91px;">ProteinTech</td>
			<td>10906-1-AP</td>
			<td>Antibody for confocal analysis. Use at 1:200</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Vectashield solution with DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:91px;">Vector Labs</td>
			<td style="width:119px;">H-1500</td>
			<td>Dye for confocal analysis</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:319px;">Superfrost Plus Microscope slides</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12-550-15</td>
			<td>25 x 75 x 1.0 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">BioCoat Fibronectin 12mm coverslips</td>
			<td style="width:91px;">NeuVitro Corp</td>
			<td>GG-12-1.5</td>
			<td>Coverslips for confocal analysis</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">100um cell strainer</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">0.45um Syringes filters SFCA 25MM</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">09-740-106</td>
			<td>For virus filtration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">6ml Syringes</td>
			<td style="width:91px;">Covidien</td>
			<td>8881516937</td>
			<td>For virus filtration</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-Chicken IgY (H&#38;L) A488</td>
			<td style="width:91px;">Abcam</td>
			<td>AB150169</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Donkey anti-rabbit A647 IgG(H+L)&#160;</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A31573</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;">Goat anti-mouse IgG(H+L) A555</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>A21422</td>
			<td style="width:347px;">Secondary antibody for confocal analysis. Use at 1:400</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Triton X-100</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">93443-100ml</td>
			<td>For cell permeabilization&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">Dulbecco&#39;s PBS without CaCl2 and MgCl2 (D-PBS)</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Power Block 10X Universal Blocking reagent</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">NC9495720</td>
			<td>Dilute to 1X in H20</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:319px;">16% Paraformaldehyde aqueous solution (PFA)</td>
			<td style="width:91px;">Electro Microscopy Sciences&#160;</td>
			<td style="width:119px;">15710</td>
			<td>Use at 4% diluted in dH20</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">6 cm&#160; plates</td>
			<td style="width:91px;">Olympus</td>
			<td style="width:119px;">25-260</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">6-well plates</td>
			<td style="width:91px;">Genesee Scientific</td>
			<td style="width:119px;">25-105</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">24-well plates</td>
			<td style="width:91px;">Genesee Scientific</td>
			<td style="width:119px;">25-107</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">10 cm Tissue culture dishes</td>
			<td style="width:91px;">Corning</td>
			<td>4239</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">15 cm&#160; Tissue culture dishes</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td>5442</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">15 ml Conical tubes</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">4308</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">50 ml Conical tubes</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">4249</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:319px;">0.4% Trypan blue solution</td>
			<td style="width:91px;">Sigma</td>
			<td style="width:119px;">T8154</td>
			<td>For viability</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Ethyl Alcohol 200 proof</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">7005</td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:319px;">Bleach</td>
			<td style="width:91px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">6009</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cardiomyocyte subtypes following transcription factor-mediated reprogramming of mouse embryonic fibroblasts

Direct reprogramming of one cell type into another has recently emerged as a powerful paradigm for regenerative medicine, disease modeling, and lineage specification. In particular, the conversion of fibroblasts into induced cardiomyocyte-like myocytes (iCLMs) by Gata4, Hand2, Mef2c, and Tbx5 (GHMT) represents an important avenue for generating de novo cardiac myocytes in vitro and in vivo. Recent evidence suggests that GHMT generates a greater diversity of cardiac subtypes than previously appreciated, thus underscoring the need for a systematic approach to conducting additional studies. Before direct reprogramming can be used as a therapeutic strategy, however, the mechanistic underpinnings of lineage conversion must be understood in detail to generate specific cardiac subtypes. Here we present a detailed protocol for generating iCLMs by GHMT-mediated reprogramming of mouse embryonic fibroblasts (MEFs). 
We outline methods for MEF isolation, retroviral production, and MEF infection to accomplish efficient reprogramming. To determine the subtype identity of reprogrammed cells, we detail a step-by-step approach for performing immunocytochemistry on iCLMs using a defined set of compatible antibodies. Methods for confocal microscopy, identification, and quantification of iCLMs and individual atrial (iAM), ventricular (iVM), and pacemaker (iPM) subtypes are also presented. Finally, we discuss representative results of prototypical direct reprogramming experiments and highlight important technical aspects of our protocol to ensure efficient lineage conversion. Taken together, our optimized protocol should provide a stepwise approach for investigators to conduct meaningful cardiac reprogramming experiments that require identification of individual CM subtypes.

ניצול של העכבר הכתב PM-ספציפי, פיתחנו אסטרטגיה immunostaining זמנית לזהות מיוציטים אנדוגני מגוונים כמו מתואר באיור 1. בעקבות צעדי תכנות מחדש שמוצגים באיור 2, אינדוקציה של CMS ספציפי תת סוג ניתן לאתר מוקדם ככל יום 4 21, אם כי שיעור ?...

Watch this Scientific Journal Video about Assessing Cardiomyocyte Subtypes Following Transcription Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts at JoVE.com

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

This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5.

הערכה תת סוגי Cardiomyocyte בעקבות תמלול פקטור בתיווך תכנות מחדש של פיברובלסטים עכבר עובריים

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

assessing-cardiomyocyte-subtypes-following-transcription-factor

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

7.4K Views.  UT Southwestern Medical Center. This manuscript describes a step-by-step protocol for the generation and quantification of diverse reprogrammed cardiac subtypes using a retrovirus-mediated delivery of Gata4, Hand2, Mef2c, and Tbx5. 

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

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

תרבות של עוברי Explants שבלול העכבר וג&#39;ין העברה על ידי Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

ניתוח של Cardiomyocyte פיתוח באמצעות Immunofluorescence בלב העכבר עוברי

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

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

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

הנדסה גנטית יציבה ויעילה של תאי V-SVZ העכבר למבוגרים עבור הניתוח של הגזע עצבי נייד אוטונומי והשפעות חד אוטונומיות

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

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptome Profiling של<em&gt; In-Vivo</em&gt; עוברי אדם שור הופק באמצעות פלטפורמת microarray שני צבעים

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4&#8242;,6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Quantification of cardiomyocyte turnover is challenging. The protocol described here makes an important contribution to this challenge by enabling accurate and sensitive quantification of neo-cardiomyocyte nuclei generation and nuclei ploidy.

הערכה סימולטני של DNA Cardiomyocyte סינתזה ploidy: שיטה Assist כימות Cardiomyocyte רגנרציה מחזור

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover ...

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

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

פרוטוקול עבור ההמרה הישירה של Murine העוברי פיברובלסטים לתאי גזע Trophoblast

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

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

תכנות מחדש עכבר עוברי פיברובלסטים עם גורמי שעתוק כדי לגרום תכנית Hemogenic

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

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

דיספציה ותרבות של כליה עובריים עכבר

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

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

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

דיכוי של Pro-fibrotic איתות Potentiates בתיווך גורם התכנות של העכבר מתחלקים Fibroblasts לתוך Cardiomyocytes המושרה

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

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

ישיר לשושלת התכנות של העכבר למבוגרים פיברובלסט Erythroid אבות

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

הערכה תת סוגי Cardiomyocyte בעקבות תמלול פקטור בתיווך תכנות מחדש של פיברובלסטים עכבר עובריים

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תרבות של עוברי Explants שבלול העכבר וג'ין העברה על ידי Electroporation