Name: reprogramming mouse embryonic fibroblasts with transcription factors to induce a hemogenic program
Uploaded: 2016-12-16T12:30:00
Description: Watch this Scientific Journal Video about Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program at JoVE.com

This work was supported by an NHLBI grant to K.M. and I.R.L. (1RO1HL119404). Carlos-Filipe Pereira was the recipient of a Revson Senior Biomedical Fellowship. We gratefully acknowledge the Mount Sinai hESC/iPSC Shared Resource Facility and S. D'Souza for assistance with materials and protocols. We also thank the Mount Sinai Flow Cytometry, Genomics, and Mouse facilities.

Ethics statement: Mouse cell lines are derived following the animal care guidelines of the Icahn School of Medicine at Mount Sinai, and should be done in compliance with any host institution.
1. Mouse Embryonic Fibroblast (MEF) Isolation of C57BL/6 Mice
<ol>
	<li>Set up timed mating13. Once a vaginal plug is visualized, consider this Day 0.5.</li>
	<li>Separate the plugged females on the plug date and check on them on Day 10-11 to confirm pregnancy.</li>
	<li>On Day 13.5-14.5 euthanize pregnant female...

<ol>
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R., H, K. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+analysis+of+hematopoietic+stem+cells+from+the+placenta.">Isolation and analysis of hematopoietic stem cells from the placenta.</a> J Vis Exp. (16), (2008).</li><li>Lewis, I. D., 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=Umbilical+cord+blood+cells+capable+of+engrafting+in+primary,+secondary,+and+tertiary+xenogeneic+hosts+are+preserved+after+ex+vivo+culture+in+a+noncontact+system.">Umbilical cord blood cells capable of engrafting in primary, secondary, and tertiary xenogeneic hosts are preserved after ex vivo culture in a noncontact system.</a> Blood. 97 (11), 3441-3449 (2001).</li><li>Sheridan, J. M., Taoudi, S., Medvinsky, A., Blackburn, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+the+generation+of+reaggregated+organotypic+cultures+that+permits+juxtaposition+of+defined+cell+populations.">A novel method for the generation of reaggregated organotypic cultures that permits juxtaposition of defined cell populations.</a> Genesis. 47 (5), 346-351 (2009).</li><li>Koh, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cells+for+microscopy+using+cytospin.">Preparation of cells for microscopy using cytospin.</a> Methods Enzymol. 533, 235-240 (2013).</li><li>Orkin, S. H., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoiesis:+an+evolving+paradigm+for+stem+cell+biology.">Hematopoiesis: an evolving paradigm for stem cell biology.</a> Cell. 132 (4), 631-644 (2008).</li><li>Martinez-Agosto, J. A., Mikkola, H. K., Hartenstein, V., Banerjee, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hematopoietic+stem+cell+and+its+niche:+a+comparative+view.">The hematopoietic stem cell and its niche: a comparative view.</a> Genes Dev. 21 (23), 3044-3060 (2007).</li><li>Butler, J. 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Generating HSPCs de novo from easily attainable somatic cells offers a unique method to study hematopoiesis in vitro, and the opportunity to potentially apply this technology to the human system. This translation would generate a new tool to study human hematologic disease in a dish, as well as provide drug testing platforms and gene targeting opportunities to treat numerous disorders with novel therapeutics or HSC transplants. In the field, recent studies have expanded on the ability to generate HSPCs ...

Hematopoiesis is a complex developmental process where hematopoietic stem cells (HSCs) bud off hemogenic endothelium present in a variety of embryonic hematopoietic sites such as the Aorta-Gonad-Mesonephros and the placenta1,2. The inability to culture HSCs in vitro prevents the in depth analysis of this process as well as the clinical application of these studies. To circumvent this limitation, previous studies have attempted to derive HSCs de novo either via differentiation of pluripotent stem cells (PSCs)3, or induced plasticity in somatic cells and directed differentiation using reprogramming media4,5. These studies, however, do not generate clinically safe engraftable cells or allow study of definitive developmental hematopoiesis &#34;in a dish.&#34;
The novel work established by Yamanaka and colleagues to generate induced pluripotent stem cells (iPSCs) from somatic fibroblasts provides a framework for transcription factor (TF) based overexpression strategies in reprogramming cell fate6,7. This work has prompted investigators in several fields to generate cell types of choice via TF reprogramming of easily obtainable somatic cells. The goal of the reprogramming strategy described here is to induce a hemogenic process from mouse somatic cells using a TF based reprogramming approach with the goal of translating these findings to the human system to reprogram patient-specific fibroblasts in order to study human hematopoiesis in vitro and generate patient-specific blood products for disease modeling, drug testing, and stem cell transplant.
The first step to ensure proper reprogramming in this mouse system was to develop a reporter line that served as a read-out for CD34 expression, a known marker in endothelial progenitor cells and HSCs. To do this, the huCD34-tTA and TetO-H2BGFP transgenic mouse lines were used to generate double transgenic mouse embryonic fibroblasts (MEFs), now denoted 34/H2BGFP, that fluoresce green upon activation of the CD34 promoter8. This allowed screening of a variety of TFs known to be required at different points during hematopoietic specification and development. Beginning with 18 TFs in pMX retrovial vectors (determined through literature mining and profiling of GFP label retaining HSCs from the previously described 34/H2BGFP mice), 34/H2BGFP MEFs were transduced with all factors and cultured on AFT024 HSC-supporting stromal cells. After detection of 34/H2BGFP activation, TFs were subsequently removed from the reprogramming cocktail until the optimal set of TFs for reporter activation was identified. After this initial screen, the factors were transferred to a DOX inducible pFUW vector system to allow controllable expression of the TFs. Since these two DOX controllable systems are incompatible (the 34/H2BGFP cells and the pFUW inducible vectors), MEFs from wild-type C57BL/6 mice were required. It was also necessary to provide an appropriate microenvironment to allow hemogenesis to proceed and create multilineage clonogenic progenitors.
Current studies attempting to reprogram somatic cells into hematopoietic stem and progenitor cells (HSPCs) have met varied levels of success9-11. To date, the generation of both mouse and human transplantable HSPCs with long term and self-renewing repopulating ability has not been achieved using the same set of TFs. In this protocol, we provide a detailed description of the previously established strategy to reproducibly induce hemogenesis in MEFs. We demonstrate that introduction of a minimal set of TFs (Gata2, Gfi1b, cFos, and Etv6) is capable of instigating a complex developmental program in vitro that provides a platform by which developmental hematopoiesis and clinical application of hematopoietic reprogramming can be further studied12.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45uM filters</td>
			<td>Corning</td>
			<td>430625</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra centrifugal filters</td>
			<td>Millipore</td>
			<td>UFC900324</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini&#39;s Benchmark</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>18G needles</td>
			<td>BD</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>20G needles</td>
			<td>BD</td>
			<td>305175</td>
			<td></td>
		</tr>
		<tr>
			<td>25G needles</td>
			<td>BD</td>
			<td>305125</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type I</td>
			<td>Sigma</td>
			<td>C0130-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Invitrogen</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Myelocult media</td>
			<td>Stem Cell Technologies</td>
			<td>M5300</td>
			<td></td>
		</tr>
		<tr>
			<td>SCF</td>
			<td>R &#38; D Systems</td>
			<td>455-MC</td>
			<td></td>
		</tr>
		<tr>
			<td>Flt3L</td>
			<td>R &#38; D Systems</td>
			<td>427-FL</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-3</td>
			<td>R &#38; D Systems</td>
			<td>403-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6</td>
			<td>R &#38; D Systems</td>
			<td>406-ML</td>
			<td></td>
		</tr>
		<tr>
			<td>TPO</td>
			<td>R &#38; D Systems</td>
			<td>488-TO</td>
			<td></td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D9891-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene (hexadimethrine bromide)</td>
			<td>Sigma</td>
			<td>AL-118</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore 0.65uM membrane filters</td>
			<td>Millipore</td>
			<td>DVPP14250</td>
			<td></td>
		</tr>
		<tr>
			<td>Methylcellulose media</td>
			<td>Stem Cell Technologies</td>
			<td>Methocult M3434</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Stem Cell Technologies</td>
			<td>07904</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>The Jackson Laboratory</td>
			<td>000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma</td>
			<td>G-1890 100g</td>
			<td></td>
		</tr>
		<tr>
			<td>pFUW-tetO</td>
			<td>Addgene</td>
			<td>Plasmid #20321</td>
			<td></td>
		</tr>
		<tr>
			<td>Gata2</td>
			<td>Origene</td>
			<td>MR226728</td>
			<td></td>
		</tr>
		<tr>
			<td>Gfi1b</td>
			<td>Origene</td>
			<td>MR204861</td>
			<td></td>
		</tr>
		<tr>
			<td>cFos&#160;</td>
			<td>Addgene</td>
			<td>Plasmid #19259</td>
			<td></td>
		</tr>
		<tr>
			<td>Etv6</td>
			<td>Origene</td>
			<td>MR207053</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>Plasmid #12260</td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>Plasmid #12259</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C5670-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>FUW-M2rtTA</td>
			<td>Addgene</td>
			<td>Plasmid #20342</td>
			<td></td>
		</tr>
		<tr>
			<td>35 x 10 mm culture dishes</td>
			<td>Thermo Scientific</td>
			<td>171099</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Hematopoiesis is a complex developmental process that begins in various embryonic sites. Hemogenic endothelial cells reside in these sites and give rise to HSCs via cell budding23. This process currently cannot be reproduced by placing HSCs or hematopoietic precursors in culture, necessitating a methodology to somehow obtain these cells in vitro, either by HSPC expansion ex vivo or generation de novo. This protocol demonstrates our novel technology th...

Watch this Scientific Journal Video about Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program at JoVE.com

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

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.

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

The overall goal of this hemogenic induction protocol is to instigate a developmental process in mouse fibroblasts through transcription factor overexpression to generate hematopoietic precursor cells in vitro that product hematopoietic cells upon prolonged culture. This method can help answer key questions in the hematopoietic programming field such as whether or not a protocol can be developed to obtain bonafide HSCs de novo that are capable of multi-lineage and graftment. The main advantage of this technique is that through overexpression of a minimal set of transcription factors, the developmental process can be initiated in reprogrammed cells that does not require travel through a pluripotent intermediate.After culturing mouse embryonic fibroblasts or MEFS, and producing virus according to the text protocol, use 0.1 percent gelatin in PBS to pre-coat 6-well plates. Ensuring that the gelatin covers the entire area of the well. Place the plates in the 37 degrees celsius incubator for at least 20 minutes.Then, remove the plates and aspirate the gelatin. Plate MEFs with standard DMEM at a density of 25, 000 cells per well and allow the cells to settle at 37 degrees celsius overnight. Prepare a 100 microliter virus cocktail by mixing 50 microliters of M2RTTA and 12.5 microliters each of GATA2, GFI1B, CFOS and ETV6.Aspirate the medium from the MEFS and add two milliliters of standard DMEM with 8 micrograms per milliliters of hexadimethrine bromide. Then, transduce each well of MEFs with 15 microliters of the virus cocktail and incubate at 37 degrees celsius overnight. Following 16 to 20 hours of incubation, replace the medium in each well with two milliliters of fresh standard DMEM, supplemented with one microgram per milliliter of DOX.Incubate at 37 degrees Celsius for three days. On day four, prepare hematopoietic culture medium supplemented with hydrocortisone, stem cell factor, MFS-related tyrosine kinase three ligand, interleukin three, interleukin six and DOX. Next, aspirate the medium from the MEFs and use one milliliter of PBS to wash the cells.Then, after aspirating the PBS, add one milliliter of 0.05 percent trypsin per well to dissociate and collect the cells. Using the hemocytometer, count the cells, then spin at 300 times g for five minutes and use 12 milliliters of the hematopoietic medium just prepared to resuspend the cells. Then, plate 10, 000 cells per well on gelatin-coated six-well plates.Every six days, replace the medium with fresh supplemented hematopoietic culture medium for the duration of the culture. Analyze the cells according to the text protocol. After dissecting placentas from pregnant mice according to the text protocol, use two to five milliliters of 0.2 percent collagenase type one in PBS with 20 percent FBS to wash the tissue and using an 18 gauge needle fitted on a five milliliter syringe, mechanically dissociate the tissue in the PBS solution.Incubate the placental cells in two to five milliliters of collagenase solution at 37 degrees Celsius for 1.5 hours. Then, passage the cells several times through 20 to 25 gauge needles fitted on 5 milliliter syringes to further mechanically dissociate them. Next, filter single cell suspensions through 70 micron cell strainers.Then, using a hemocytometer, count the cells and irradiate at 2, 000 centigray volts for mitotic inactivation. Add ten milliliters of hematopoietic culture medium supplemented with the following to a ten centimeter dish. Place the 0.65 micron filter directly on the culture medium in the dish, allowing it to float to create a liquid-gas interface and set the dish aside.Dissociate day 25 transduced MEFs as demonstrated earlier in this video and mix 33, 000 of the transduced MEFs with 167, 000 cells of the mitotically inactivated placental aggregates. To generate an aggregate, spin down the MEF and placental cell mix at 300 times g for five minutes. Aspirate the medium and use 30 to 50 microliters of standard DMEM to resuspend the pelleted cells, drawing the single cell suspension into a 200 microliter non-beveled pipette tip.Next, with paraffin film, occlude the pipette tip. Then, place the blocked tip into a 15 milliliter centrifuge tube and spin it down at 300 times g for five minutes so that a pellet forms at the covered end of the tip. Carefully remove the tip from the 15 milliliter tube and place it onto the end of a P200 pipette.Discard the paraffin film and extrude the cell pellet onto the filter floating in the dish of culture medium. Culture the aggregates on the floating filters at 37 degrees Celsius and five percent carbon dioxide for four to five days. Using a cell scraper, collect the aggregates then combine them and use 0.5 percent collagenase to digest them.After dissociation, use two to five milliliters of PBS with five percent FBS to wash the aggregates and spin down at 300 times g for five minutes. Resuspend the aggregates in 500 microliters of DMEM without FBS, Pen-Strep or L-glutamine. Add the entire suspension to three milliliters of one percent of methylcellulose medium supplemented with ten nanograms per milliliter of Tepo, carefully avoiding the formation of bubbles.Plate equal volumes of this mixture in three, 35 by ten millimeter non-treated culture dishes for CFU assays. As a control, culture irradiated placental aggregates. Finally, count hematopoietic colonies manually under a microscope after ten to 14 days of culture at 37 degrees Celsius with 5 percent carbon dioxide.In the process of reprogramming MEFs to HSCs, cells are transduced with transcription factors as shown here. After three days of expansion and exposure to DOX to begin transgene activation, cells are dissociated, split and grown on gelatin-coated plates and supplemented hematopoietic medium. At day 20 in culture, post-transduction with reprogramming medium, 34/H2BGFP MEFs adopt clear morphological changes including endothelial morphology distinct from MEFs.Further culture to day 35 results in several round hematopoietic-like cells emerging from the endothelial-like intermediates that are GFP positive and stain positive for the hematopoietic markers Sca1 and CD45. Further culture results in cells expressing CD45 while maintaining expression of the human CD34 reporter. Upon placental aggregation, culture cells adopt clonogenic potential and generate colonies in methylcellulose containing various hematopoietic morphologies and blast-like cells.While attempting this procedure, it's important to remember to perform each section slowly so as to not get mixed up. Any mistakes will be hard to notice until weeks after you start the reprogramming. Following this procedure, other methods such as cobblestone-forming cell assays and long-term culture initiating cell assays can be performed in order to answer additional questions such as if the derived cells truly possess hematopoietic function as demonstrated in the functional assays.After its development, this technique paved the way for researchers in the field of hematopoietic stem cell programming to explore other reprogramming strategies and optimization protocols in blood, endothelial and other cell types. After watching this video, you should have a good understanding of how to induce a hemogenic program in mouse fibroblasts, the overexpression of transcription factors and how to functionally assay the derived cell. Thank you for watching and good luck with your experiments.

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Research

JoVE Journal

Developmental Biology

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

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.

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

Collection of Serum- and Feeder-free Mouse Embryonic Stem Cell-conditioned Medium for a Cell-free Approach

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field of regenerative medicine. However, despite their benefits, the tumorigenic potential of ESCs and iPSCs has long been a barrier for clinical applications. Interestingly, it has been shown that ESCs produce several soluble factors that can promote tissue regeneration and delay cellular aging, suggesting that ESCs and iPSCs can also be utilized as a cell-free intervention method. Therefore, the method for harvesting mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) with minimal contamination of serum components (fetal bovine serum, FBS) and feeder cells (mouse embryonic fibroblasts, MEFs) has been highly demanded. Here, the present study demonstrates an optimized method for the collection of mESC-CM under serum- and feeder-free conditions and for the characterization of mESC-CM using senescence-associated multiple readouts. This protocol will provide a method to collect pure mESC-specific secretory factors without serum and feeder contamination.

This protocol provides a method for the collection of mouse embryonic stem cell (mESC)-conditioned medium (mESC-CM) derived from serum (fetal bovine serum, FBS)- and feeder (mouse embryonic fibroblasts, MEFs)-free conditions for a cell-free approach. It may be applicable for the treatment of aging and aging-associated diseases.

The capacity of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to generate various cell types has opened new avenues in the field ...

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.

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.

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

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.

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).

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

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...