Name: suppression of pro-fibrotic signaling potentiates factor-mediated reprogramming of mouse embryonic fibroblasts into induced cardiomyocytes
Uploaded: 2018-06-03T14:00:00
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

This research was supported by funds from the Boettcher Foundation&#39;s Webb-Waring Biomedical Research Program, American Heart Association Scientist Development Grant (13SDG17400031), University of Colorado Department of Medicine Outstanding Early Career Scholar Program, University of Colorado Division of Cardiology Barlow Nyle endowment, and NIH R01HL133230 (to K.S). A.S.R was supported by NIH/NCATS Colorado CTSA Grant Number TL1TR001081 and a pre-doctoral fellowship from the University of Colorado Consortium for Fibrosis Research &#38; Translation (CFReT). This research was also supported by the Cancer Center Support Grant (P30CA046934), the Skin Diseases Research Cores Grant (P30AR057212), and the Flow Cytometry Core at the University of Colorado Anschutz Medical Campus.

All experiments requiring animals were approved by the Institutional Animal Care and Use Committee at the UC Denver Anschutz Medical Campus.
1. Isolation of MEFs
<ol>
	<li>Purchase C57BL/6 pregnant mice at E13. Ship overnight.</li>
	<li>Euthanize the mother according to approved IACUC protocols (ex: ~1.3 L/min CO2 until animal appears dead followed by cervical dislocation)</li>
	<li>Spray the mother with 70% ethanol and open abdominal cavity. Remove the uterine horn containing emb...

<ol>
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The present study outlines a high-efficiency strategy to directly reprogram fibroblasts into functional iCMs via delivery of GHMT2m reprogramming factors combined with suppression of pro-fibrotic signaling pathways. Using flow cytometry, immunofluorescent imaging, calcium imaging, and beating cell counts, we show the majority of cells in this protocol undergo successful reprogramming and adopt CM lineage fate. We have previously shown that the addition of anti-fibrotic compounds including the TGF-&#946; type I receptor i...

Ischemic heart disease is a leading cause of death in the United States1. Approximately 800,000 Americans experience a first or recurrent myocardial infarction (MI) per year1. Following MI, the death of cardiomyocytes (CMs) and cardiac fibrosis, deposited by activated cardiac fibroblasts, impair heart function2,3. Progression of heart failure following MI is largely irreversible due to the poor regenerative capacity of adult CMs4,5. While current clinical therapies slow disease progression and decrease risk of future cardiac events6,7,8,9, no therapies reverse disease progression due to the inability to regenerate CMs post-infarction10. Novel cell therapies are emerging to treat patients following MI. Disappointingly, clinical trials delivering stem cells to the heart following MI thus far have shown inconclusive regenerative potential11,12,13,14,15,16,17,18.
The generation of human-derived induced pluripotent stem cells (hiPSCs) from fibroblasts by overexpression of four transcription factors, first demonstrated by Takahashi &#38; Yamanaka, opened the door to new breakthroughs in cell therapy19. These cells can differentiate into all three germ layers19, and several highly efficient methods for generating large numbers of CMs have been previously shown20,21. HiPSC-derived CMs (hiPS-CMs) offers a powerful platform to study cardiomyogenesis and may have important implications for repairing the heart following injury. However, hiPS-CMs currently face translational hurdles due to concerns of teratoma formation22, and their immature nature may be pro-arrhythmogenic23. Reprogramming fibroblasts into hiPSCs sparked interest in directly reprogramming fibroblasts into other cell types. Ieda et al. demonstrated that overexpression of GATA4, Mef2c, and Tbx5 (GMT) in fibroblasts results in direct reprogramming to cardiac lineage, albeit at low efficiency24. Reprogramming efficiency was improved with the addition of Hand2 (GHMT)25. Since these early studies, many publications have demonstrated that altering the reprogramming factor cocktail with additional transcription factors26,27,28,29, chromatin modifiers30,31, microRNAs32,33, or small molecules34 leads to improved reprogramming efficiency and/or maturation of induced cardiomyocyte-like cells (iCMs).
Here we provide a detailed protocol to generate iCMs from mouse embryonic fibroblasts (MEFs) with high efficiency. We previously showed that the GHMT cocktail is significantly improved with the addition of miR-1 and miR-133 (GHMT2m) and is further improved when pro-fibrotic signaling pathways including transforming growth factor &#946; (TGF-&#946;) signaling or Rho-associated protein kinase (ROCK) signaling pathways are inhibited35. Using this protocol, we show that approximately 60% of cells express cardiac Troponin T (cTnT), approximately 50% express &#945;-actinin, and a high number of beating cells can be observed as early as Day 11 following transduction of reprogramming factors and treatment with the TGF-&#946; type I receptor inhibitor A-83-01. Furthermore, these iCMs express gap junction proteins including connexin 43 and exhibit spontaneous contraction and calcium transients. This marked improvement in reprogramming efficiency compared to earlier studies demonstrates the potential to regenerate CMs from endogenous cell populations that remain in the heart post-infarction.

<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-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.

Using the reprogramming strategy outlined above and in Figure 1B, we generated iCMs with approximately 70% of cells expressing cardiac Troponin T and approximately 55% of cells expressing cardiac &#945;-actinin, quantified by flow cytometry at Day 9 following transduction of GHMT2m (Figure 2A and B). Additionally, the majority of cells express cardiac Troponin T, Troponin I, and cardiac &#945;-actinin as well as ...

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

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.

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

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

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

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 ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

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

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

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

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

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

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