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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P., Sadelain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstructing+blood+from+induced+pluripotent+stem+cells.">Reconstructing blood from induced pluripotent stem cells.</a> F1000 Med Rep. 2, (2010).</li><li>Szabo, E., 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+conversion+of+human+fibroblasts+to+multilineage+blood+progenitors.">Direct conversion of human fibroblasts to multilineage blood progenitors.</a> Nature. 468 (7323), 521-526 (2010).</li><li>Mitchell, R., 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=Molecular+evidence+for+OCT4-induced+plasticity+in+adult+human+fibroblasts+required+for+direct+cell+fate+conversion+to+lineage+specific+progenitors.">Molecular evidence for OCT4-induced plasticity in adult human fibroblasts required for direct cell fate conversion to lineage specific progenitors.</a> Stem Cells. 32 (8), 2178-2187 (2014).</li><li>Takahashi, K., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+mouse+embryonic+and+adult+fibroblast+cultures+by+defined+factors.">Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.</a> Cell. 126 (4), 663-676 (2006).</li><li>Takahashi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+adult+human+fibroblasts+by+defined+factors.">Induction of pluripotent stem cells from adult human fibroblasts by defined factors.</a> Cell. 131 (5), 861-872 (2007).</li><li>Qiu, J., Papatsenko, D., Niu, X., Schaniel, C., Moore, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Divisional+history+and+hematopoietic+stem+cell+function+during+homeostasis.">Divisional history and hematopoietic stem cell function during homeostasis.</a> Stem Cell Reports. 2 (4), 473-490 (2014).</li><li>Riddell, J., 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=Reprogramming+committed+murine+blood+cells+to+induced+hematopoietic+stem+cells+with+defined+factors.">Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors.</a> Cell. 157 (3), 549-564 (2014).</li><li>Sandler, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+human+endothelial+cells+to+haematopoietic+cells+requires+vascular+induction.">Reprogramming human endothelial cells to haematopoietic cells requires vascular induction.</a> Nature. 511 (7509), 312-318 (2014).</li><li>Daniel, M. G., Pereira, C. F., Lemischka, I. R., Moore, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+a+Hematopoietic+Stem+Cell.">Making a Hematopoietic Stem Cell.</a> Trends Cell Biol. , (2015).</li><li>Pereira, C. F., 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=Induction+of+a+hemogenic+program+in+mouse+fibroblasts.">Induction of a hemogenic program in mouse fibroblasts.</a> Cell Stem Cell. 13 (2), 205-218 (2013).</li><li>Mader, S. L., Libal, N. L., Pritchett-Corning, K., Yang, R., Murphy, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refining+timed+pregnancies+in+two+strains+of+genetically+engineered+mice.">Refining timed pregnancies in two strains of genetically engineered mice.</a> Lab Anim (NY. 38 (9), 305-310 (2009).</li><li>Jain, K., Verma, P. J., Liu, J. <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+handling+of+mouse+embryonic+fibroblasts).">Isolation and handling of mouse embryonic fibroblasts).</a> Methods Mol Biol. 1194, 247-252 (2014).</li><li>Bryja, V., Bonilla, S., Arenas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivation+of+mouse+embryonic+stem+cells.">Derivation of mouse embryonic stem cells.</a> Nat Protoc. 1 (4), 2082-2087 (2006).</li><li>Carey, B. W., 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=Reprogramming+of+murine+and+human+somatic+cells+using+a+single+polycistronic+vector.">Reprogramming of murine and human somatic cells using a single polycistronic vector.</a> Proc Natl Acad Sci U S A. 106 (1), 157-162 (2009).</li><li>Hockemeyer, 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=A+drug-inducible+system+for+direct+reprogramming+of+human+somatic+cells+to+pluripotency.">A drug-inducible system for direct reprogramming of human somatic cells to pluripotency.</a> Cell Stem Cell. 3 (3), 346-353 (2008).</li><li>Taoudi, S., 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=Extensive+hematopoietic+stem+cell+generation+in+the+AGM+region+via+maturation+of+VE-cadherin+CD45++pre-definitive+HSCs.">Extensive hematopoietic stem cell generation in the AGM region via maturation of VE-cadherin+CD45+ pre-definitive HSCs.</a> Cell Stem Cell. 3 (1), 99-108 (2008).</li><li>Gekas, C., K, E. 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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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 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>
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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...

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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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7.1K Görüntüleme.  Icahn School of Medicine at Mount Sinai. 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 advantag...