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 mice via CO2 inhalation followed by cervical dislocation.</li>
	<li>Soak pregnant female abdomen with 70% ethanol, dissect the abdominal cavity with sterile scissors and forceps down the midline, and surgically remove the uterine horns containing the embryos14. Take out the embryos gently while cutting off the remaining abdominal tissue. 
	NOTE: Recover about 8 embryos with 4 on each side.</li>
	<li>Place the uterine horns containing the embryos in a 10 cm dish with 5-10 ml of sterile chilled phosphate buffered saline (PBS).</li>
	<li>With sterile scissors and forceps, make an incision along the uterine horns to isolate the sacs carrying the embryos. Transfer the sacs to a new 10 cm dish with 5-10 ml of sterile chilled PBS and place on ice.</li>
	<li>Place a few of the sacs into a new 10 cm dish with 5-10 ml of sterile chilled PBS. Using sterile scissors and forceps gently cut open the sacs (without cutting too deep) in order to avoid piercing the embryos.</li>
	<li>Separate the embryos from the placenta by cutting the umbilical cord.</li>
	<li>Transfer the embryos to a new 10 cm dish containing 5-10 ml of chilled PBS and leave on ice while completing the remaining dissections.</li>
	<li>Decapitate the embryos using sterile forceps. Carefully cut down the midline of the embryo using sterile scissors and forceps, starting from the decapitated area to open up the abdomen. Position forceps underneath the visceral organs (including the heart, liver, and lungs) and scrape them out15.</li>
	<li>Cut the remaining tissue into small pieces using scissors and forceps and transfer to a 50 ml conical tube containing 10 ml of trypsin.</li>
	<li>Pipet up and down with a 10 ml pipet 20-30 times to mix and dissociate the tissue.</li>
	<li>Incubate the tissue and trypsin mix in a 37 &#176;C water bath for 45 min, swirling occasionally.</li>
	<li>Pipet up and down with a 10 ml pipet for about 5 minutes to mix the contents.</li>
	<li>Add trypsinized cells to 3x volume of standard Dulbecco&#39;s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS), 10 &#181;g/ml Penicillin/Streptomycin (P/S), and 1 mM L-Glutamine (L-GLUT) in 10 cm dishes (~10 ml) and culture at 37 &#176;C until confluent (about 2-4 days). Culture about 1-2 embryos per 10 cm dish. 
	NOTE: Freeze cells once plates are confluent. Frozen cells can be thawed and passaged 2 more times. Cells can alternatively be split 2 times prior to freezing, and then once more after being thawed.</li>
</ol>
2. Viral Production
<ol>
	<li>Expand 293T cells in 10 cm plates with 10 ml DMEM with 10% FBS, 1% P/S, and 1% L-GLUT for transfection.
	<ol>
		<li>Trypsinize 293T cells with 2 ml trypsin, incubate at 37 &#176;C for 5 min, collect the cells into 15 ml tubes, spin at 300 x g, and plate appropriately (10 ml per 10 cm dish).</li>
	</ol>
	</li>
	<li>24 hr prior to transfection, split confluent 10 cm plate of 293T cells 1:6 and seed in 10 cm gelatin coated (0.1% gelatin in PBS) plates. 
	NOTE: 4 plates per virus will be required.
	<ol>
		<li>To coat plates in gelatin, add at least 2 ml of the 0.1% gelatin solution to coat the bottom of 10 cm plates. Incubate at 37 &#176;C for at least 20 min. Aspirate off gelatin prior to adding cells to the plates.</li>
	</ol>
	</li>
	<li>In a 15 ml tube, add 84 &#181;g plasmid (e.g. Gata2, Gfi1b, cFos, or Etv6 (sub-cloned into the pFUW-TetO vector16) or FUW-M2rtTA17), 84 &#181;g psPAX2, and 42 &#181;g pMD2.G. Add water (H2O) to make up the volume to 2 ml. Prepare a tube for each factor (e.g. Gata2, Gfi1b, cFos, Etv6 and FUW-M2rtTA).</li>
	<li>Add 250 &#181;l of 2M CaCl2 to each tube containing the 2 ml mixture composed of 84 &#181;g of the chosen gene (Gata2, Gfi1b, cFos, Etv6 or FUW-M2rtTA) + 84 &#181;g psPAX2 + 42 &#181;g pMD2.G + H2O.</li>
	<li>Using a Pasteur pipet inserted into the pipet-aid, release bubbles into the 2.25 ml DNA + H2O + 2 M CaCl2 mixture. As the bubbles are being produced, place the tip of a P1000 against the glass pipet and slowly eject 2 ml of 100 mM N,N-bis (2-hydroxyethyl)-2-aminoethane sulfonic acid (BES) saline down the Pasteur pipet 1 ml at a time.</li>
	<li>Incubate all tubes in the culture hood at room temperature for at least 15 min. 
	NOTE: The 4.25 ml mixture (now DNA + H2O + 2M CaCl2 + BES saline) will look slightly cloudy.</li>
	<li>As the mixture incubates, aspirate media off 293T plates and add 10 ml DMEM + 10% FBS + 1 mM L-Glutamine + 25 nM chloroquine. 
	NOTE: This media does not contain P/S.</li>
	<li>After at least 15 min of incubation (or until changing media on 293T cells), add the 4.25 ml DNA + H2O + 2 M CaCl2 + BES saline mixture drop wise to the 293T cells, distributing evenly across 4 cell plates per tube (i.e. slightly more than 1 ml of the mixture per plate).</li>
	<li>Incubate plates overnight (O/N) at 37 &#176;C.</li>
	<li>24 hr post incubation, replace media on all plates with 4 ml standard DMEM (see step 1.15).</li>
	<li>Keep cells in a 32 &#176;C incubator from now on.</li>
	<li>24 hr after step 2.10, collect media into separate 50 ml tubes. Replace the collected media from each plate with 4 ml of standard DMEM. 
	&#8203;NOTE: 4 plates per initial tube mixture results in 16 ml of collected media.
	<ol>
		<li>After 12 hr of incubation, collect media again into the same 50 ml tubes and replace with another 4 ml of standard DMEM.</li>
		<li>After 12 hr of incubation, collect the media and add to the same 50 ml tubes. 
		NOTE: each tube should now contain about 48 ml of media containing virus.</li>
	</ol>
	</li>
	<li>To concentrate the collected virus, first filter the collected media through 0.45 &#181;M low-protein binding filters.</li>
	<li>Pour the filtered media containing virus into viral concentration centrifuge tubes (about 16 ml at a time).</li>
	<li>Spin at 4,000 x g at 4 &#176;C for 25 min and remove flow-through. 
	NOTE: A small volume of concentrated media and virus will be visible in the filter.</li>
	<li>Add another 16 ml of filtered virus-containing media to the concentrated media + virus volume remaining in the filter and mix by inverting the tube.</li>
	<li>Spin tube again at 4,000 x g at 4 &#176;C for 25 min and discard flow-through.</li>
	<li>Add remaining filtered collection to the concentrated media + virus volume left in the filter and mix by inverting the tube.</li>
	<li>Spin once more at 4,000 x g at 4 &#176;C for 10-20 min (depending on the volume in the filter) and ensure that the remaining volume of concentrated media + virus left in the filter is about 1 ml. If greater than 1 ml of concentrated media + virus remains in the filter, spin again at 4,000 x g for 1 min and repeat until 1 ml is left in the filter.</li>
	<li>Aliquot 200 &#181;l of the concentrated virus into 1.5 ml tubes and freeze at -80 &#176;C or use immediately.</li>
</ol>
3. MEF Reprogramming
<ol>
	<li>Pre-coat 6 well-plates with 0.5 ml of 0.1% gelatin in PBS per well, ensuring that the gelatin covers the entire area of the well. Place in a 37 &#176;C incubator and incubate for at least 20 min. After incubation, remove the plates and aspirate the gelatin.</li>
	<li>Plate MEFs with standard DMEM at a density of 25,000 cells per well (150,000 cells per 6-well plate) on the gelatinized 6-well plate(s) from step 3.1 and allow to settle O/N in a 37 &#176;C incubator.</li>
	<li>Prepare a 100 &#181;l virus cocktail by mixing 50 &#181;l M2rtTA and the remaining 50 &#181;l a mix of Gata2, Gfi1b, cFos, and Etv6 (12.5 &#181;l each).</li>
	<li>Aspirate media from MEFs and add 2 mL standard DMEM with 8 &#181;g/ml hexadimethrine bromide.</li>
	<li>Transduce each well of the MEF plates with 15 &#181;l of the virus cocktail and incubate O/N at 37 &#176;C. 
	NOTE: This is Day 0 of the reprogramming process.</li>
	<li>On Day 1, after 16-20 hr of incubation, replace media with 2 ml fresh standard DMEM supplemented with 1 &#181;g/ml DOX per well.</li>
	<li>Prepare hematopoietic culture media supplemented with hydrocortisone (10-6 M), 100 ng/ml stem cell factor (SCF), 100 ng/ml Fms-related tyrosine kinase 3 ligand (Flt3L), 20 ng/ml interleukin-3 (IL-3), 20 ng/ml interleukin-6 (IL-6), and 1 &#181;g/ml DOX.</li>
	<li>On Day 4 aspirate media from each well and wash cells with 1 ml of PBS per well.</li>
	<li>Aspirate PBS, dissociate cells using 1 ml of 0.05% trypsin per well and collect the cells.</li>
	<li>Count cells with a hemocytometer, spin at 300 x g for 5 min, resuspend in 12 ml of hematopoietic media described in step 3.7 and plate 60,000 cells per 6-well plate on 0.1% gelatin coated plates (2 ml per well, 10,000 cells per well).</li>
	<li>Replace media with fresh supplemented hematopoietic culture media every 6 days for the duration of culture (35 to 36 days).</li>
	<li>Analyze either intermediate cells or emerging hematopoietic-like cells via immuno-staining12, FACS analysis8,12, qRT-PCR12, and mRNA sequencing12 to confirm the acquisition of hemogenic endothelial or HSC-like markers and gene expression.</li>
</ol>
4. Placental Aggregation and Colony Forming Unit (CFU) Assays in Methylcellulose
<ol>
	<li>Dissect placentas from pregnant C57BL/6 mice as previously described18 at E12.5 and keep tissue on ice.</li>
	<li>To start placenta dissociation19, wash placental tissue in 2-5 ml of 0.2% collagenase type I in PBS with 20% FBS and mechanically dissociate with an 18G needle fitted into a 5 ml syringe by passaging the placenta and PBS through the needle at least 3 times.</li>
	<li>After mechanical dissociation, keep placental cells in 2-5 ml of collagenase solution at 37 &#176;C for 1.5 hr.</li>
	<li>Passage cells through 20-25 G needles fitted onto 5 ml syringes several times to further mechanically dissociate the placental cells.</li>
	<li>Filter single cell suspensions through 70 &#181;m cell strainers.</li>
	<li>Count cells with a hemocytometer and irradiate at 2,000 cGy for mitotic inactivation20.</li>
	<li>Add 10 ml of hematopoietic culture media supplemented with 100 ng/ml SCF, 100 ng/ml Flt3L, 20 ng/ml IL-3, 20 ng/ml IL-6, and 10 ng/ml thrombopoietin (TPO) to a 10 cm dish. 
	NOTE: DOX is not required at this stage.</li>
	<li>Place a 0.65 &#181;m filter directly on hematopoietic culture media in the dish, allowing it to float to create a liquid-gas interface and set the dish aside.</li>
	<li>Dissociate day 25 transduced MEFs (derived from step 3.11) as described in steps 3.8-3.10 and mix 33,000 of the transduced MEFs with 167,000 cells of the placental aggregates from step 4.6 (1:6 ratio).</li>
	<li>To generate an aggregate18,21, first spin down the MEF and placental cell mix from step 4.9 for 5 min at 300 x g. Aspirate the media and resuspend the pelleted cells in 30-50 &#181;l of standard DMEM using a P200 pipette, drawing the single cell suspension into the 200 &#181;l nonbevelled pipette tip.</li>
	<li>Occlude the pipette tip with paraffin film. Place the blocked tip into a 15 ml centrifuge tube and spin down for 5 min at 300 x g so a pellet forms at the covered end of the tip.</li>
	<li>Remove the tip from the 15 ml tube carefully and place the tip onto the end of the P200 pipette. Discard the paraffin film and extrude the cell pellet onto the floating filter from step 4.8. 
	NOTE: Plate at most 3 aggregates per filter.</li>
	<li>Culture the aggregates on the floating 0.65 &#181;m filters for 4-5 days in 37 &#176;C with 5% CO2.</li>
	<li>Collect the aggregates (at most 3 per filter) with a cell scraper, combine them, and digest with 0.2% collagenase, following the same protocol as done with the whole placentas to dissociate the cells (steps 4.2-4.5).</li>
	<li>After dissociation, wash the aggregates with 2-5 ml of PBS supplemented with 5% FBS and spin down cells at 300 x g for 5 min.</li>
	<li>Resuspend the aggregates in 500 &#181;l of DMEM without FBS, P/S, or L-GLUT. Take this 500 &#181;l resuspension and add it to 3 ml 1% methylcellulose media supplemented with 10 ng/ml of TPO, carefully avoiding the formation of bubbles. Plate equal volumes of this mixture in 3 35 x 10 mm non-treated culture dishes for CFU assays12.</li>
	<li>As a control, culture irradiated placental aggregates without mixing in reprogrammed MEFs 4-5 days and place in CFU assays as described above in steps 4.7-4.16. 
	NOTE: These aggregates alone do not form colonies.</li>
	<li>Count hematopoietic colonies manually under the microscope after 10-14 days of culture at 37 &#176;C with 5% CO2.</li>
	<li>Cytospin22 picked colonies to show morphological phenotype of single cells.</li>
</ol>

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A., Stekleneva, A. E., Medvedev, S. P., Zakian, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Epigenetic+memory:+phenomenon+in+induced+pluripotent+stem+cells.">"Epigenetic memory: phenomenon in induced pluripotent stem cells.</a> Acta Naturae. 5 (4), 15-21 (2013).</li><li>Elcheva, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+induction+of+haematoendothelial+programs+in+human+pluripotent+stem+cells+by+transcriptional+regulators.">Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators.</a> Nat Commun. 5, 4372 (2014).</li><li>Vereide, D. T., 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=An+expandable,+inducible+hemangioblast+state+regulated+by+fibroblast+growth+factor.">An expandable, inducible hemangioblast state regulated by fibroblast growth factor.</a> Stem Cell Reports. 3 (6), 1043-1057 (2014).</li><li>Batta, K., Florkowska, M., Kouskoff, V., Lacaud, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+murine+fibroblasts+to+hematopoietic+progenitor+cells.">Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells.</a> Cell Rep. 9 (5), 1871-1884 (2014).</li></ol>

The authors have no conflict of interest to disclose.

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><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>0.45 &amp;mu;M 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&#039;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>18 G needles</td><td>BD</td><td>305195</td><td></td></tr><tr><td>20 G needles</td><td>BD</td><td>305175</td><td></td></tr><tr><td>25 G 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 &amp;amp; D Systems</td><td>455-MC</td><td></td></tr><tr><td>Flt3L</td><td>R &amp;amp; D Systems</td><td>427-FL</td><td></td></tr><tr><td>IL-3</td><td>R &amp;amp; D Systems</td><td>403-ML</td><td></td></tr><tr><td>IL-6</td><td>R &amp;amp; D Systems</td><td>406-ML</td><td></td></tr><tr><td>TPO</td><td>R &amp;amp; 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 0.65 &amp;mu;M 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&amp;nbsp;</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>CaCl&lt;sub&gt;2&lt;/sub&gt;</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 mm 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 ...

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Research

JoVE Journal

Developmental Biology

7.1K Views.  Icahn School of Medicine at Mount Sinai. 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.

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

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 ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

Biology

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 ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

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 ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery 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 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 ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

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

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