Name: Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells (Video) | JoVE Journal
Uploaded: 2015-12-03T10:30:00.000+00:00
Duration: 8 min 14 s
Description: Learn to generate erythroid and myeloid blood cells from human pluripotent stem cells using transcription factor overexpression. This method enables efficient hemogenic endothelium induction for studying hematopoietic development.

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5 minutes

We thank Matt Raymond for editorial assistance. This work was supported by funds from the National Institute of Health (U01HL099773, R01HL116221, and P51 RR000167) and The Charlotte Geyer Foundation.

1. Virus Preparations and Transcription Factor Combinations
<ol>
	<li>Prepare solutions with pSIN-EF1&#945; lentiviral expression plasmid containing protein-coding DNA for ETV2, GATA1, GATA2, TAL1 and LMO2 (Table 1).</li>
	<li>Measure the concentration and purity of plasmid preparations used for lentiviral production by recording UV absorption with a spectrophotometer at 230 nm, 260 nm, and 280 nm. Note: DNA preparations demonstrating A260/280 and A260/230 values greater than 1.8 are generally considered good quality. Lower A260/280 values may indicate protein contamination, while lower A260/230&#160;values indicate impurities with salts or some solvent such as phenol. Recommended plasmid concentration is 1-3 &#181;g/&#181;l.</li>
	<li>Produce lentiviruses for transductions as described in previously published protocols11. Induction of HE with pan-myeloid potential requires co-expression of ETV2 and GATA1, or ETV2 and GATA2. Induction of HE with erythro- and megakaryocytic potential requires GATA2 and TAL1. The addition of LMO2 significantly improves the yield of erythro-megakaryocytic cells from hPSCs in the presence of GATA2 and TAL110.</li>
</ol>
2. hPSCs Culture Protocol
<ol>
	<li>Grow pluripotent stem cells in tight colonies with sharp edges to 70-80% confluency before subculturing. Mark and remove spontaneously differentiated colonies before passaging cells.</li>
	<li>Dilute commercial extracellular matrix gel (see Table 2) according to the manufacturer&#8217;s instructions and coat 6-well plates overnight at 4&#160;&#176;C, or for at least one hour at 37&#160;&#176;C prior to use. Before passaging cells, aspirate the matrix, add 2 ml of fresh commercial complete culturemedium (see Table 2) and keep plates at 37 &#176;C, 5% CO2 until the cells are ready for seeding.</li>
	<li>Passage hPSCs
	<ol>
		<li>Aspirate medium from one confluent well of a 6-well plate with hPSCs and add 1.5 ml of pre-warmed Dispase II solution (2 mg/ml in DMEM/F12, sterilized by filtration through 0.22 &#181;m filters). Incubate for 5-7 min at 37 &#176;C, 5% CO2 until the edges of colonies begin to lift from the surface.</li>
		<li>Aspirate Dispase II solution and carefully wash the colonies twice by adding and then aspirating 2 ml of fresh DMEM/F12 medium. Then, using a 5 ml serological glass pipette, dissociate hPSCs with 3 ml of fresh commercial complete culture medium (see Table 2) by pipetting cell suspension up and down several times.</li>
		<li>Add 0.5 ml of cell suspension to each well of a 6-well plate containing 1.5 ml of commercial complete culture medium (see Table 2). Agitate plate back and forth and right to left several times to ensure a uniform cell distribution when situating the plate into the incubator. Keep cells at 37 &#176;C, 5% CO2 for 18-24 hr.</li>
		<li>The next day change medium to fresh commercial complete culture medium (see Table 2) and examine hPSCs morphology. Successful passaging results in small, well-attached colonies retaining hPSC morphology. hPSCs must be fed with 2.5-3 ml freshmedium per well on a daily basis and passaged every 4-5 days at no more than 70-80% confluence. 
		NOTE: If hiPSCs were maintained on mouse embryonic fibroblasts (MEFs), they need to be transferred to feeder-free conditions and passaged 2-3 times before transduction to ensure efficient differentiation.</li>
	</ol>
	</li>
</ol>
3. Induction of Hematoendothelial Pprecursors from hPSCs (day 0, Transduction of hPSCs)
<ol>
	<li>To prepare reaction medium combine 1.3 ml of commercial complete serum-free medium (see Table 2), virus, polybrene, and Y27632 ROCK inhibitor (Table 1): add 1.3 &#181;l of 10 mM ROCK inhibitor to 1.3 ml medium to a final concentration of 10 &#956;M, and polybrene to a final concentration 6 &#181;g/ml.
	<ol>
		<li>Add an appropriate amount of viral concentrate(s) at a final concentration of 0.5-1.0 MOI (multiplicity of infection) of each virus per cell. Keep reaction medium on ice or at 4 &#176;C until the single cell suspension is ready. 
		NOTE: The same amount of MOI for each virus in the GATA1/ETV2, GATA2/TAL1, and GATA2/TAL1/LMO2 reaction mixtures is optimal for induction of differentiation. However, in ETV2/GATA2 transduced cultures doubling MOI for GATA2, relative to ETV2, enhances differentiation. Therefore, for these cultures, ratio 1:2 for MOI of ETV2 and 1 MOI of GATA2 is recommended (e.g. if viral concentrate is estimated approximately 6.8 x 107 particles/ml, add 10 &#181;l of ETV2 and 20 &#181;l of GATA2 viral concentrates to 0.68 x 106 cells per one ETV2/GATA2 reaction in a 35 mm well of a 6-well plate).</li>
	</ol>
	</li>
	<li>Prepare hPSCs in a single cell suspension.
	<ol>
		<li>Grow hPSCs in feeder-free conditions as described in section 2. On day 4 following the last passage, observe cell density and morphology under an inverted microscope. hPSCs should grow in tight colonies with sharp edges with no spontaneous differentiation and reach ~60-70% confluency on the day of transduction.</li>
		<li>Aspirate medium, add 1.5-2 ml of commercial cell dissociation reagent (Table 1) per well and incubate at 37 &#176;C with 5% CO2 for 5-7 min.</li>
		<li>Collect cells into equal volumes of commercial complete medium (see Table 2) with 10 &#181;M of ROCK inhibitor, count the cells and determine viability. Pellet cells by centrifugation at 200 x g for 5 min and then resuspend cells in commercial complete medium with 10 &#181;M of ROCK inhibitor to a concentration of 3.4-5 x 106 cells per ml. 
		NOTE: Cell viability is critical for successful viral transduction and subsequent differentiation. Typically, cell viability before transduction should exceed 90%.</li>
	</ol>
	</li>
	<li>Add 200 &#181;l of the cell suspension, containing 0.68-1 x106 cells, into 1.3 ml of the reaction medium prepared in 3.1.</li>
	<li>Aspirate the matrix solution from the overnight coated 6-well plate prepared in 2.2., transfer the reaction mixture to one well of a 6-well plate and distribute cells evenly. Incubate at 37&#160;&#176;C with 5% CO2 for 24 hr. After 24 hr at least 80% of the cells should be attached to the matrix.</li>
</ol>
4. Induction of Hematoendothelial Precursors from hPSCs (Day 1-7)
<ol>
	<li>24 hr post-transduction, remove virus-containing medium and wash attached cells with commercial incomplete cell culture medium (see Table 1) and then add 3 ml per well of commercial incomplete cell culture medium containing hematopoietic cytokines: SCF 100 ng/ml, TPO 50 ng/ml, bFGF 20 ng/ml (hereafter referred to as 3F-medium).</li>
	<li>Replace total medium with fresh 3F-medium on days 2, 3 and day 4 to remove dead cells and debris. After day 4, when floating hematopoietic cells are emerging, replace half of the 3F-medium every other day while maintaining the total volume at 4 ml. 
	NOTE: pSIN-EF1&#945; lentiviral expression plasmids incorporate puromycin resistance gene thereby allowing for positive selection of transduced cells. The 1 &#956;g/ml of puromycin can be added to medium during the first 1-2 days of differentiation to eliminate the any residual undifferentiated hPSCs.</li>
	<li>Observe cell morphology on day 4 under an inverted microscope: tight clusters of cells with typical endothelial morphology begin to appear (Figures 2A, 3A). Round blood cells appears from day 5 to day 7 of differentiation, while some areas may retain hPSCs morphology. Analyze HE and hematopoietic differentiation as described below. 
	NOTE: Successful hematoendothelial differentiation results in the production of blood cells that appear as refractile round cells loosely attached to underlying flat endothelial cells. These cells actively proliferate forming small aggregates, and eventually detach and float (Figures 2A and 3A). Live blood cells can be visually distinguished from the dead and dying cells based on their ability to reflect the light and expand in culture conditions.</li>
</ol>
5. Analysis of Hemogenic Endothelium (HE) Stage of Differentiation.
<ol>
	<li>Detect the formation of HE between days 3 and 4 of differentiation by microscopic observation of cells with endothelial morphology. There are no floating blood cells in the culture at this stage of differentiation. The presence of HE can be confirmed by immunofluorescent staining and flow cytometric analysis using VE-cadherin, CD73, CD226 and CD43 antibodies.
	<ol>
		<li>To assess HE formation by flow cytometric analysis use cells on day 3 and day 4 from one experiment. Collect cells by incubating of cultures with commercial cell dissociation reagent (see Table 1) for 5-10 min at 37 &#176;C, 5% CO2 and then use up to 1 x 105 cells per staining reaction (flow cytometry staining procedure described in 12). 
		NOTE: VE-cadherin+ HE cells acquire expression of early hematopoietic marker CD226, but lack the expression of CD73 and CD436. In contrast, non-HE cells express CD73 (Figures 2B, 3B).</li>
	</ol>
	</li>
	<li>Immunofluorescence staining for hPSC-derived HE.
	<ol>
		<li>Wash the attached monolayer of cells with phosphate buffered saline (PBS) and then fix cells in 4% paraformaldehyde from 30 to 60 min at room temperature.</li>
		<li>Wash cells again with PBS and then permeabilize using 0.1% Triton X-100 in PBS for 20 min at room temperature.</li>
		<li>Wash cells in PBS two to three times and then incubate in blocking buffer consisting of PBS with 10% fetal bovine serum (FBS), for 30 to 120 min.</li>
		<li>Prepare staining solution, containing both primary antibodies (1:1,000 dilution) mouse anti-human CD43 and rabbit anti-human VE-cadherin in PBS with 5% FBS, (Table 1).</li>
		<li>Aspirate the blocking buffer from the cells and add 1 ml per well of staining buffer with the added primary antibodies; incubate 3 hr at room temperature, or overnight at 4 &#176;C.</li>
		<li>Wash the cells three times by adding 2 ml of PBS with 5% FBS.</li>
		<li>Prepare a second staining buffer, containing secondary antibodies at 1:500 dilution in PBS with 5% FBS: donkey anti-rabbit conjugated with green fluorescent dye, and anti-mouse conjugated with red fluorescent dye (Table 1). Add 1 ml per well of secondary staining buffer and incubate at room temperature for 1 hr.</li>
		<li>Wash three times with PBS without serum. Stain cells with 300 nM DAPI working solution in dH2O for 10-15 min.</li>
		<li>Wash cells with PBS twice and add 2-3 ml of PBS into well with stained cells. Observe fluorescence with green, red and blue microscope filters.</li>
	</ol>
	</li>
</ol>
6. Analysis of Induced Hematopoietic Precursors
<ol>
	<li>Maintain differentiation cultures until floating cells expand significantly, up to 10-14 days, changing half of the 3F-medium, as described in 3.1, every two days.</li>
	<li>Dissociate and collect differentiating monolayers of cells by treating cells with a commercial cell dissociation reagent (see Table 1) for 5-10 min at 37 &#176;C, 5% CO2.</li>
	<li>Perform flow cytometry using anti-VE-cadherin, CD43 and CD45 antibodies to evaluate hematopoiesis in cultures12. Note that a large fraction of cells, up to 40% of ETV2 and GATA2 transduced cells, co-expresses VE-cad and CD43. The following antibodies can be used to detect specific cell lineages following expansion or differentiation of induced blood progenitors: CD235a (erythroid cells), CD41a (megakaryocytic), CD32 (all types of myeloid cells), CD66b (neutrophils) and CD163 (macrophages).</li>
	<li>Perform CFC-assay using a commercial clonogenic medium (Table 1) according to the manufacturer&#8217;s instructions.
	<ol>
		<li>Transfer 1-2 x 104 cells into 3 ml of commercial clonogenic medium (see Table 3) to assess the number of colony-forming cells and types of hematopoietic colonies. Optimal seeding density allows for the identification and isolation of single colonies that grow separately from each other and do not overlap. Seeding density may vary between experiments depending on differentiation efficiency, but should not exceed 1 x 104 cells per 1 ml of commercial clonogenic medium.</li>
		<li>Mix cells in commercial clonogenic medium by gentle vortexing, and transfer 3 ml of suspension to two 35 mm ultra-low attachment dishes for CFC assay, 1.5 ml of suspension to each dish. Distribute medium evenly and incubate at 37 &#176;C, 5% CO2 for 14 days. Avoid disturbing the dishes; observe colony formation on day 7 and day 10 of assay.</li>
		<li>Identify and count hematopoietic colonies according to their morphologies on day 14.</li>
	</ol>
	</li>
</ol>

<ol><li>Zape, J. P., Zovein, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hemogenic+endothelium%3A+origins%2C+regulation%2C+and+implications+for+vascular+biology%5BTitle%5D)+AND+%22Semin+Cell+Dev+Biol%22%5BJournal%5D)">Hemogenic endothelium: origins, regulation, and implications for vascular biology.</a> Semin Cell Dev Biol. 22, 1036-1047 (2011).</li>
<li>Swiers, G., Rode, C., Azzoni, E., de Bruijn, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+short+history+of+hemogenic+endothelium%5BTitle%5D)+AND+%22Blood+Cells%2C+Mol+%26+Dis%22%5BJournal%5D)">A short history of hemogenic endothelium.</a> Blood Cells, Mol & Dis. 51, 206-212 (2013).</li>
<li>Kennedy, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T+lymphocyte+potential+marks+the+emergence+of+definitive+hematopoietic+progenitors+in+human+pluripotent+stem+cell+differentiation+cultures%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2, 1722-1735 (2012).</li>
<li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endothelial+and+hematopoietic+cell+fate+of+human+embryonic+stem+cells+originates+from+primitive+endothelium+with+hemangioblastic+properties%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties.</a> Immunity. 21, 31-41 (2004).</li>
<li>Rafii, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+ESC-derived+hemogenic+endothelial+cells+undergo+distinct+waves+of+endothelial+to+hematopoietic+transition%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition.</a> Blood. 121 (5), 770-780 (2012).</li>
<li>Choi, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+of+the+hemogenic+endothelial+progenitor+and+its+direct+precursor+in+human+pluripotent+stem+cell+differentiation+cultures%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Identification of the hemogenic endothelial progenitor and its direct precursor in human pluripotent stem cell differentiation cultures.</a> Cell Rep. 2, 553-567 (2012).</li>
<li>Vodyanik, M. A., Thomson, J. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Leukosialin+%28CD43%29+defines+hematopoietic+progenitors+in+human+embryonic+stem+cell+differentiation+cultures%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures.</a> Blood. 108, 2095-2105 (2006).</li>
<li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TGFbeta+inhibition+enhances+the+generation+of+hematopoietic+progenitors+from+human+ES+cell-derived+hemogenic+endothelial+cells+using+a+stepwise+strategy%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">TGFbeta inhibition enhances the generation of hematopoietic progenitors from human ES cell-derived hemogenic endothelial cells using a stepwise strategy.</a> Cell Res. 22, 194-207 (2012).</li>
<li>Uenishi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tenascin+C+promotes+hematoendothelial+development+and+T+lymphoid+commitment+from+human+pluripotent+stem+cells+in+chemically+defined+conditions%5BTitle%5D)+AND+%22Stem+Cell+Rep%22%5BJournal%5D)">Tenascin C promotes hematoendothelial development and T lymphoid commitment from human pluripotent stem cells in chemically defined conditions.</a> Stem Cell Rep. 3, 1073-1084 (2014).</li>
<li>Elcheva, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+induction+of+haematoendothelial+programs+in+human+pluripotent+stem+cells+by+transcriptional+regulators%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators.</a> Nat Commun. 5, 4372(2014).</li>
<li>Tiscornia, G., Singer, O., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+and+purification+of+lentiviral+vectors%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Production and purification of lentiviral vectors.</a> Nat Protoc. 1, 241-245 (2006).</li>
<li>Vodyanik, M. A., Slukvin, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hematoendothelial+differentiation+of+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Curr+Protoc+Cell+Biol%22%5BJournal%5D)">Hematoendothelial differentiation of human embryonic stem cells.</a> Curr Protoc Cell Biol. Chapter 23, Unit 23.6(2007).</li>
<li>Cao, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+gene-transfer+efficiency+in+human+embryonic+stem+cells%5BTitle%5D)+AND+%22Mol+Imgn+and+Biol%22%5BJournal%5D)">Comparison of gene-transfer efficiency in human embryonic stem cells.</a> Mol Imgn and Biol. 12, 15-24 (2010).</li>
</ol>

I.S. is a founding shareholder and consultant for Cynata.

The above-described method for hematopoietic differentiation of hPSCs by overexpression of TFs, represents a rapid and efficient approach for the generation of HE and myeloid and erytho-magakaryocytic progenitors from hESCs and iPSCs, thereby allowing the production of up to 30 millions blood cells from one million pluripotent stem cells10. This method exhibited consistent differentiation in multiple hESC and iPSCs lines10. During differentiation by ETV2 and GATA2, GATA1 factors as well as GATA2 and...

The unique ability of human pluripotent stem cells (hPSCs) to self-renew and to differentiate into cells of the three germ layers, including blood, make them a valuable tool for the mechanistic studies of hematopoietic development, modeling of blood diseases, drug screening, toxicity studies, and the development of cellular therapies. Because blood formation in the embryo proceeds from hemogenic endothelium (HE) through an endothelial hematopoietic transition1,2, the generation of HE in cultures would be essential to study the molecular mechanisms regulating the endothelial to hematopoietic transition and hematopoietic specification. Current methods for studies of HE are based on the induction of hematoendothelial differentiation in aggregates (EBs) with the addition of hematopoietic cytokines3-5, and coculture of hPSCs with hematopoiesis-supportive stromal cells6,7 or in two-dimensional cultures with extracellular matrices and cytokines8,9. These classical differentiation methods are based on the introduction of external signals acting at the cell surface and initiating cascades of molecular pathways that eventually lead to the activation of transcriptional program guiding hematoendothelial development. Thus, the efficiency of hPSCs differentiations in these systems relies on an effective induction of those signals, signal transduction to the nucleus, and the resulting activation of specific transcriptional regulators. In addition, the study of HE in conventional differentiation cultures requires the additional step of isolating HE cells using cell sorting. Here, we describe a simple protocol for the direct induction of HE and blood by overexpression of hematopoietic transcription factors. This method allows for the efficient induction of HE in a dish and direct observation of the endothelial to hematopoietic transition without the need for isolation of HE using a cumbersome cell sorting procedure.
Formation of HE and blood from human pluripotent stem cells can be efficiently induced by overexpressing just a few transcription factors (TFs). The optimal combination of TFs capable of inducing robust pan-myeloid hematopoiesis from hPSCs includes ETV2 and GATA1 or GATA2. In contrast, combination of GATA2 and TAL1 induces erythromegakaryocytopoiesis10. Programming hPSCs through overexpression of these factors differentiates hPSCs directly to the VE-cad+CD43-CD73- HE cells that gradually acquire the hematopoietic phenotype defined by the expression of the early hematopoietic marker CD437. This lentiviral-based method for the direct programming of human pluripotent stem cells method is applicable for the generation of HE and blood cells for mechanistic studies, studies of endothelial to hematopoietic transition, and transcriptional regulation of hematopoietic development and specification. Although the current protocol describes blood production using constitutive expression of the transgenes, similar results could be obtained using modified mRNA10.

<table><tbody><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 1.&lt;/strong&gt; &lt;strong&gt;Induction of hPSCs differentiation with trancription factors and analysis of hemogenic endothelium and blood cells.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>pSIN4-EF1a-ETV2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61061</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-GATA2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61063</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-GATA1-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61062</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-TAL1-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61062</td><td>Lentiviral Vector</td></tr><tr><td>pSIN4-EF1a-LMO2-IRES-Puro&amp;nbsp;</td><td>Addgene</td><td>Plasmid #61064</td><td>Lentiviral Vector</td></tr><tr><td>Hexadimethrine bromide (Polybrene)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>107689-10G</td><td>Cationic polymer used to increase the efficiency of infection</td></tr><tr><td>Y-27632 (Dihydrochloride) ROCK inhibitor</td><td>STEMCELL Technologies</td><td>72302</td><td>RHO/ROCK pathway inhibitor Inhibits ROCK</td></tr><tr><td>StemPro Accutase Cell Dissociation Reagent</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>A11105-01</td><td>Cell Dissociation Reagent</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Incomplete (growth factor- free) culture medium&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; mTeSR1 Custom formulation&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>MCF</td><td>&lt;a name=&quot;RANGE!D12&quot;&gt;Serum-free mTeSR1 medium without bFGF and TGFb&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>human SCF</td><td>Peprotech</td><td>300-07</td><td>Premium grade</td></tr><tr><td>human TPO</td><td>Peprotech</td><td>300-18</td><td>Research grade</td></tr><tr><td>human FGF-basic</td><td>Peprotech</td><td>100-18B</td><td>Premium grade</td></tr><tr><td>CD144 (VE-cad) FITC</td><td>BD Biosciences</td><td>560411</td><td>Endothelial marker (FACS)</td></tr><tr><td>CD226 PE</td><td>BD Biosciences</td><td>338305</td><td>Hematopoietic (FACS)</td></tr><tr><td>CD43 PE</td><td>BD Biosciences</td><td>560199</td><td>Hematopoietic (FACS)</td></tr><tr><td>CD73 APC</td><td>R&amp;amp;D Systems</td><td>FAB5795A</td><td>Endothelial marker (FACS)</td></tr><tr><td>CD45 APC</td><td>BD Biosciences</td><td>555485</td><td>Hematopoietic (FACS)</td></tr><tr><td>7AAD</td><td>Life Technologies</td><td>A1310</td><td>Live/Dead assay (FACS)</td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P6148-500G</td><td>Cell fixation&amp;nbsp;</td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T9284-500ML&amp;nbsp;</td><td>Permeabilization&amp;nbsp;</td></tr><tr><td>FBS</td><td>Fisher Scientific</td><td>SH3007003</td><td>Fetal bovine serum</td></tr><tr><td>Mouse anti-human CD43</td><td>BD Biosciences</td><td>551457</td><td>Pure, primary antibody for Immunofluorescence (IF) staining</td></tr><tr><td>Rabbit anti-human VE-cadherin</td><td>BenderMedSystem</td><td>BMS158</td><td>Primary (IF)</td></tr><tr><td>Anti-rabbit Alexa Fluor 488-conjugated</td><td>JacksonResearch</td><td>715-486-152</td><td>Secondary (IF)</td></tr><tr><td>Anti-mouse Alexa Fluor 594-conjugated</td><td>JacksonResearch</td><td>715-516-150&amp;nbsp;</td><td>Secondary (IF)</td></tr><tr><td>DAPI nucleic acid stain</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>D1306&amp;nbsp;</td><td>Live/Dead assay (IF)</td></tr><tr><td>Clonogenic medium MethoCult H4435 Enriched</td><td>STEMCELL Technologies</td><td>4435</td><td>CFC-assay</td></tr><tr><td>Wright Stain solution</td><td>Sigma -Aldrich</td><td>32857</td><td>Staining cytospins</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 2.&lt;/strong&gt; &lt;strong&gt;hPSCs culture.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>Human Pluripotent Stem Cells (hPSCs)&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>hESCs (WA01, WA09) human Embryonic Stem cells; iPSCs (DF-19-9-7T, DF-4-3-7T) transgene-free induced Pluripotent Stem Cells</td><td>hPSCs are able to self-renew and to differentiate into cells of three germ layers</td></tr><tr><td>Complete serum-free medium for culture of hPSCs&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; mTeSR1 media&amp;nbsp;&amp;nbsp;</td><td>WiCell Research Institute (Madison, WI)</td><td>M500&amp;nbsp;</td><td>Serum-free&amp;nbsp; medium with growth factors for feeder free culture of ESC/iPSCs</td></tr><tr><td>Matrigel</td><td>BD Biosciences/ Corning&amp;nbsp;</td><td>356234</td><td>Matrix for maintenance of human ESC/iPSCs</td></tr><tr><td>DMEM/F-12, powder&amp;nbsp;</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>12500-062</td><td>Basal Medium&amp;nbsp;</td></tr><tr><td>HyClone Dulbecco&#039;s PBS powder</td><td>Fisher Scientific</td><td>dSH30013.04</td><td>PBS</td></tr><tr><td>Dispase II, powder</td><td>&lt;a href=&quot;http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/stem-cell-culture/stem-cell-research-misc/stempro-accutase.html&quot;&gt;Life Technologies&lt;/a&gt;</td><td>17105-041</td><td>Neutral protease, Cell dissociation</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot; fo:keep-together.within-page=&quot;1&quot; fo:keep-with-previous.within-page=&quot;always&quot;&gt;&lt;strong&gt;Table 3.&lt;/strong&gt; &lt;strong&gt;Primers for detection of virus genomic integration.&lt;/strong&gt;&lt;/p&gt;</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primer&#039;s Name&lt;/strong&gt;</td><td>&lt;strong&gt;Forward (Fwd)&amp;nbsp; 5&amp;rsquo; --&gt;3&amp;rsquo;&lt;/strong&gt;</td><td>&lt;strong&gt;Reverse (Rev) 5&amp;rsquo;--&gt;3&amp;rsquo;&lt;/strong&gt;</td><td>&lt;strong&gt;Discription&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>pSIN EF1a Fwd</td><td>TTC CAT TTC AGG TGT CGT GA</td><td>--</td><td>EF1a promoter sequence</td></tr><tr><td>GATA1 Rev</td><td>--</td><td>TCC CTG TAG TAG GCC AGT GC</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>GATA2 Rev</td><td>--</td><td>GGT TGG CAT AGT AGG GGT TG</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>TAL1 Rev</td><td>--</td><td>AGG CGG AGG ATC TCA TTC TT</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>LMO2 Rev</td><td>--</td><td>GGC CCA GTT TGT AGT AGA GGC</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr><tr><td>ETV2 Rev</td><td>--</td><td>GAA CTT CTG GGT GCA GTA AC&amp;nbsp;</td><td>&amp;nbsp;Coding Region&amp;nbsp;</td></tr></tbody></table>

direct induction of hemogenic endothelium and blood by overexpression of transcription factors in human pluripotent stem cells

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.

The schematic diagram of HE and blood induction from hPSCs by overexpression of transcription factors is shown in Figure 1. ETV2 with GATA1 or GATA2 combination induces pan-myeloid hematopoiesis, while a GATA2, TAL1 +/- LMO2 combination induces predominantly erythro-megakaryocytic hematopoiesis. Both TF combinations directly induced HE cells that subsequently transformed into blood progenitors with a distinct spectrum of hematopoietic differentiation. Differentiation of hPSCs from the pluripotent state t...

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 overall goal of this procedure is to generate erythroid and myeloid blood cells from human pluripotent stem cells by forced expression of transcription factors. This method is applicable for the generation of homogenic endothelium in blood cells for studies of endothelial to hepo transition and transcriptional regulation of hepo development and specification. The main advantage of this technique is that the method provides inefficient and rapid means of homogenic endothelium induction from human preport stem cells, and allows for observation of the endothelial hemmie transition in a cell culture dish.Dr.Bruske, a research assistant in Dr.Lin's laboratory, will demonstrate the procedure Before beginning preco six wall plates with an extracellular matrix such as mat gel by diluting it according to the manufacturer's instructions and pouring it into the wells. Incubate the plates for 30 to 60 minutes at 37 degrees Celsius for the transduction medium. First, prepare lentiviral aliquots for each transduction set such that the estimated multiplicity of infection or MOI of virus to cells is one virus per cell and keep them at four degrees Celsius.Next, combine complete serum free medium for hbsc viruses poly brain to a final concentration of six micrograms per milliliter and rock inhibitor Y 2 7 6 3 2 to a final concentration of 10 micromolar in a total volume of 1.3 milliliters for each transduction set and set the mixes on ice to prepare the cells. Start with a healthy batch of pluripotent stem cells at 70%confluence about four days after the last passage. If human pluripotent stem cells were maintained in maps, they must be transferred to feeder free conditions, two to three passages prior to transduction experiments and represent healthy pluripotent stem cell cultures.Free of spontaneous differentiation. Aspirate the HPSC medium and add two milliliters of salt detachment solutions such as Accutane per well of a six Well plate incubating the plate at 37 degrees Celsius and 5%carbon dioxide for five to seven minutes. Then add two milliliters of complete medium with 10 micromolar rock inhibitor and collect the detached cells.After preparing a homogenous cell suspension, take a small aliquot and count the viable cells by the Trian blue exclusion method. Next, centrifuge the cells at 200 times G for five minutes and resuspend the pellet in complete medium with 10 micromolar rock inhibitor at a final concentration of 3.4 times 10 to the six live cells per milliliter. Then add 200 microliters of cell suspension containing 0.68 times 10 to the six cells to each of the 1.3 milliliter transduction medium prepared earlier.Remove the matrix coated six wall plate from 37 to degrees Celsius. Next, aspirate the matrix solution and transfer each of the transduction mixes into a well and swirl the plate to distribute the cells evenly in the well. 24 hours.After transduction, visualize the wells under the microscope to make sure that at least 80%of the cells are adhered. Discard the virus solution and wash the attached cells with incomplete medium without any growth factors. Next, add three milliliters of the medium supplemented with cytokines referred to as three F.Medium to the cells.Incubate the cells overnight on the following day. Change the medium with fresh three F medium containing one microgram per milliliter of pur mycin. To eliminate the residual nont transduced cells.Replace this medium every 24 hours to remove the dead cells. After 48 hours of the treatment, discontinue pur mycin and use only fresh three F medium on day four. After transduction, observe the cells under the microscope for the appearance of clustered cells with endothelial morphology to determine the formation of hemo endothelial cells.First associate the cells between days three or four from the culture dish with the cell detachment solution. Collect the detached cells in complete media and centrifuge them, resuspend the cells in max buffer at room temperature and count the cells in a hemo cytometer, then incubate one times 10 to the fifth cells per staining reaction with suitable antibodies for the detection of v catrin, CD 2 26, CD 73, and CD 43. Cell surface markers.Perform flow cytometry is described in the text protocol from day four. After transduction, continue differentiation of cells by replacing half of the medium from each well, every 48 hours between five to seven days round blood cells will appear loosely aggregated and refile above the endothelial cell layer. Maintain the cultures for up to 14 days during which time the floating cells should expand significantly.Flow cytometric analysis of ETV two, gata one and ETV two. Gata two transduced cells shows the formation of terin positive hemo endothelium, which can be identified by the expression of CD 2 26 and lack of CD 73 expression at day three to four of differentiation flow cytometric analysis of ETV two, gata one and ETV two. Gata two transduced cells shows induction of CD 43 expressing blood cells at day seven.After transduction flow cytometric analysis of gata two TAL one LMO two induced cells on day 14 after transduction shows formation of blood cells that express CD 2 35 A and CD 41 A right stained cytosine preparations of gata two TAL one lmo two induced hematopoietic cells showed erythroid macrophage and mega cario acidic cell morphologies in colony forming cell assays. The transduction of human pluripotent stem cells can be done in less than an hour while performing the transduction procedure. It is important to consider stem cell viability always to include rock inhibitor in the reaction mixture and to use an optimal amount of virus per cell following the first three days.Post transduction are the methods like immuno staining, colon forming asay, and flow cytometry can be performed in order to answer questions about the efficiency of differentiation and the type of hemato dease is induced. After watching this video, you should have a good understanding of how to ize and infect human pluripotent stem cells and what to expect in differentiating cultures within the first seven to 10 days. After transcription factors were successfully delivered into human purport stem cells, cell lose their per potency and gradually commit to a hematopoietic fate.Thus, researchers will have a convenient in vitro model to study molecular mechanisms of the endothelial to hematopoietic transition. Don't forget that working with lentivirus containing regions can be extremely hazardous and precautions such as wearing personal protection equipment and proper disposal of regions containing viruses should always be taking while performing this procedure.

direct-induction-hemogenic-endothelium-blood-overexpression

Research

JoVE Journal

Developmental Biology

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Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

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.

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

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

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.

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

Learn to generate erythroid and myeloid blood cells from human pluripotent stem cells using transcription factor overexpression. This method enables efficient hemogenic endothelium induction for studying hematopoietic development.

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

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