This work was partially supported by NIH grants HL128064 and U2EB017103. Further support was provided by CT Innovations 15-RMB-YALE-04 grant.

1. Reagents and reagent preparation
NOTE: A list of reagents is provided in Table of Materials.
<ol>
	<li>Obtain the human pluripotent stem cell lines: H1-hESC, H9-Fucci-hESC. 
	NOTE: The generation of hemogenic ECs may be more efficient in the H1 cell line.</li>
	<li>Prepare matrix protein stocks: Aliquot the matrix protein into pre-chilled 1.5 mL tubes (on ice) so that each tube contains 1 mg of matrix protein. 1 mg of matrix protein is enough to coat all wells of two...

<ol>
	<li>Giacomelli, 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=Three-dimensional+cardiac+microtissues+composed+of+cardiomyocytes+and+endothelial+cells+co-differentiated+from+human+pluripotent+stem+cells.">Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells.</a> Development. 144 (6), 1008-1017 (2017).</li><li>Wu, J., Wu, Z., Xue, Z., Li, H., 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=PHBV/bioglass+composite+scaffolds+with+co-cultures+of+endothelial+cells+and+bone+marrow+stromal+cells+improve+vascularization+and+osteogenesis+for+bone+tissue+engineering.">PHBV/bioglass composite scaffolds with co-cultures of endothelial cells and bone marrow stromal cells improve vascularization and osteogenesis for bone tissue engineering.</a> RSC Advances. 7 (36), 22197-22207 (2017).</li><li>Lee, H., Chung, M., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvasculature:+An+essential+component+for+organ-on-chip+systems.">Microvasculature: An essential component for organ-on-chip systems.</a> MRS Bulletin. 39 (1), 51-59 (2014).</li><li>Gritz, E., Hirschi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+and+function+of+hemogenic+endothelium+during+embryogenesis.">Specification and function of hemogenic endothelium during embryogenesis.</a> Cellular and Molecular Life Sciences. 73 (8), 1547-1567 (2016).</li><li>Williams, I. M., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+endothelial+cells+from+human+pluripotent+stem+cells:+Methods,+considerations,+and+applications.">Generation of endothelial cells from human pluripotent stem cells: Methods, considerations, and applications.</a> Arteriosclerosis, Thrombosis, and Vascular Biology. 39 (7), 1317-1329 (2019).</li><li>Olmer, 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=Differentiation+of+human+pluripotent+stem+cells+into+functional+endothelial+cells+in+scalable+suspension+culture.">Differentiation of human pluripotent stem cells into functional endothelial cells in scalable suspension culture.</a> Stem Cell Reports. 10 (5), 1657-1672 (2018).</li><li>Cossu, G., 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=Lancet+Commission:+Stem+cells+and+regenerative+medicine.">Lancet Commission: Stem cells and regenerative medicine.</a> The Lancet. 391 (10123), 883-910 (2018).</li><li>Fox, I. 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P., 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=Autologous+induced+pluripotent+stem+cell-derived+four-organ-chip.">Autologous induced pluripotent stem cell-derived four-organ-chip.</a> Future Science OA. 5 (8), 1-12 (2019).</li><li>Ronaldson-Bouchard, K., Vunjak-Novakovic, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organs-on-a-Chip:+A+fast+track+for+engineered+human+tissues+in+drug+development.">Organs-on-a-Chip: A fast track for engineered human tissues in drug development.</a> Cell Stem Cell. 22 (3), 310-324 (2018).</li><li>Kane, N. 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X., 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=Differentiation+of+human+embryonic+stem+cells+to+endothelial+progenitor+cells+on+laminins+in+defined+and+xeno-free+systems.">Differentiation of human embryonic stem cells to endothelial progenitor cells on laminins in defined and xeno-free systems.</a> Stem Cell Reports. 7 (4), 802-816 (2016).</li><li>Ikuno, 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=Efficient+and+robust+differentiation+of+endothelial+cells+from+human+induced+pluripotent+stem+cells+via+lineage+control+with+VEGF+and+cyclic+AMP.">Efficient and robust differentiation of endothelial cells from human induced pluripotent stem cells via lineage control with VEGF and cyclic AMP.</a> PLOS One. 12 (3), 0173271 (2017).</li><li>Aoki, H., 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=Efficient+differentiation+and+purification+of+human+induced+pluripotent+stem+cell-derived+endothelial+progenitor+cells+and+expansion+with+the+use+of+inhibitors+of+ROCK,+TGF-B,+and+GSK3B.">Efficient differentiation and purification of human induced pluripotent stem cell-derived endothelial progenitor cells and expansion with the use of inhibitors of ROCK, TGF-B, and GSK3B.</a> Heliyon. 6 (3), 03493 (2020).</li><li>Lian, X., 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=Efficient+differentiation+of+human+pluripotent+stem+cells+to+endothelial+progenitors+via+small-molecule+activation+of+Wnt+signaling.">Efficient differentiation of human pluripotent stem cells to endothelial progenitors via small-molecule activation of Wnt signaling.</a> Stem Cell Reports. 3 (5), 804-816 (2014).</li><li>Orlova, V. V., 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=Generation,+expansion+and+functional+analysis+of+endothelial+cells+and+pericytes+derived+from+human+pluripotent+stem+cells.">Generation, expansion and functional analysis of endothelial cells and pericytes derived from human pluripotent stem cells.</a> Nature Protocols. 9 (6), 1514-1531 (2014).</li><li>Kusuma, S., Gerecht, S., Turksen, K. <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+endothelial+cells+and+pericytes+from+human+pluripotent+stem+cells.">Derivation of endothelial cells and pericytes from human pluripotent stem cells.</a> Human Embryonic Stem Cell Protocols. , 213-222 (2014).</li><li>Bao, X., Lian, X., Palecek, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directed+endothelial+progenitor+differentiation+from+human+pluripotent+stem+cells+via+wnt+activation+under+defined+conditions.">Directed endothelial progenitor differentiation from human pluripotent stem cells via wnt activation under defined conditions.</a> Methods in Molecular Biology. 1481, 183-196 (2016).</li><li>Xu, M., He, J., Zhang, C., Xu, J., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+derivation+of+endothelial+lineages+from+human+stem+cells.">Strategies for derivation of endothelial lineages from human stem cells.</a> Stem Cell Research &amp; Therapy. 10 (1), 200 (2019).</li><li>Arora, S., Yim, E. K. F., Toh, Y. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+specification+of+pluripotent+stem+cell+derived+endothelial+cells+toward+arterial+and+venous+subtypes.">Environmental specification of pluripotent stem cell derived endothelial cells toward arterial and venous subtypes.</a> Frontiers in Bioengineering and Biotechnology. 7, 143 (2019).</li><li>Fang, J. 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=Shear-induced+Notch-Cx37-p27+axis+arrests+endothelial+cell+cycle+to+enable+arterial+specification.">Shear-induced Notch-Cx37-p27 axis arrests endothelial cell cycle to enable arterial specification.</a> Nature Communications. 8 (1), 2149 (2017).</li><li>Dalton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+the+cell+cycle+to+cell+fate+decisions.">Linking the cell cycle to cell fate decisions.</a> Trends in Cell Biology. 25 (10), 592-600 (2015).</li><li>Wolf, K., Hu, H., Isaji, T., Dardik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identity+of+arteries,+veins,+and+lymphatics.">Molecular identity of arteries, veins, and lymphatics.</a> Journal of Vascular Surgery. 69 (1), 253-262 (2019).</li><li>Rocha, S. F., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+differentiation+and+specialization+of+vascular+beds.">Molecular differentiation and specialization of vascular beds.</a> Angiogenesis. 12 (2), 139-147 (2009).</li><li>Goldie, L. C., Lucitti, J. L., Dickinson, M. E., Hirschi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+signaling+directing+the+formation+and+function+of+hemogenic+endothelium+during+murine+embryogenesis.">Cell signaling directing the formation and function of hemogenic endothelium during murine embryogenesis.</a> Blood. 112 (8), 3194-3204 (2008).</li><li>Chanda, B., Ditadi, A., Iscove, N. N., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+signaling+is+essential+for+embryonic+hematopoietic+stem+cell+development.">Retinoic acid signaling is essential for embryonic hematopoietic stem cell development.</a> Cell. 155 (1), 215-227 (2013).</li><li>Marcelo, K. L., 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=Hemogenic+endothelial+cell+specification+requires+c-kit,+notch+signaling,+and+p27-mediated+cell-cycle+control.">Hemogenic endothelial cell specification requires c-kit, notch signaling, and p27-mediated cell-cycle control.</a> Developmental Cell. 27 (5), 504-515 (2013).</li><li>Dejana, E., Hirschi, K. K., Simons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+basis+of+endothelial+cell+plasticity.">The molecular basis of endothelial cell plasticity.</a> Nature Communications. 8 (1), 14361 (2017).</li><li>Ottersbach, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-to-haematopoietic+transition:+an+update+on+the+process+of+making+blood.">Endothelial-to-haematopoietic transition: an update on the process of making blood.</a> Biochemical Society Transactions. 47 (2), 591-601 (2019).</li><li>Pauklin, S., Vallier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell-cycle+state+of+stem+cells+determines+cell+fate+propensity.">The cell-cycle state of stem cells determines cell fate propensity.</a> Cell. 155 (1), 135-147 (2013).</li><li>Qiu, J., Nordling, S., Vasavada, H. H., Butcher, E. C., Hirschi, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+promotes+endothelial+cell+cycle+early+g1+state+to+enable+human+hemogenic+endothelial+cell+specification.">Retinoic acid promotes endothelial cell cycle early g1 state to enable human hemogenic endothelial cell specification.</a> Cell Reports. 33 (9), 108465 (2020).</li><li>Sriram, G., Tan, J. Y., Islam, I., Rufaihah, A. J., Cao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+differentiation+of+human+embryonic+stem+cells+to+arterial+and+venous+endothelial+cells+under+feeder-+and+serum-free+conditions.">Efficient differentiation of human embryonic stem cells to arterial and venous endothelial cells under feeder- and serum-free conditions.</a> Stem Cell Research &amp; Therapy. 6 (1), 261 (2015).</li><li>Ohta, R., Sugimura, R., Niwa, A., Saito, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemogenic+endothelium+differentiation+from+human+pluripotent+stem+cells+in+a+feeder-+and+xeno-free+defined+condition.">Hemogenic endothelium differentiation from human pluripotent stem cells in a feeder- and xeno-free defined condition.</a> Journal of Visualized Experiments: JoVE. 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Herein, the steps for producing hemogenic endothelial cells from human embryonic stem cells in approximately 1 week using a murine feeder- and serum-free 2D culture system (Figure 1) are outlined. This protocol expands on a method described by Sriram et al. (2015) to obtain primordial ECs38. The primordial nature and specification potential of the CD31+ CD45- ECs is demonstrated by culturing these cells on DLL4-coated plates and observing gene ex...

Endothelial cells (ECs) are a heterogeneous population of cells that perform multiple functions throughout the human body and in engineered tissues. In addition to supporting and regulating other cell types (i.e., cardiomyocytes1, osteoblastic cells2), these functions include forming a selective barrier between blood and tissues and assisting in tissue formation3. Differentiation of mature ECs during normal development requires diverse signaling pathways. Primordial ECs are derived from mesoderm progenitors, and are then specified toward mature arterial, venous, capillary and lymphatic phenotypes4. Additionally, a small subset of ECs in the extraembryonic yolk sac and embryonic Aorta-Gonad-Mesonephros (AGM) region are also specified to become hemogenic ECs, which give rise to hematopoietic stem and progenitor cells (HSPCs) that migrate to the fetal liver and fetal bone marrow, where they remain postnatally and generate all blood cell types throughout life4. The diverse range of EC phenotypes is essential for all tissue development and maintenance.
Thus, ECs and their derivatives are critical components of studies aimed at modeling, and elucidating mechanisms of, human development and/or disease, as well as regenerative medicine and tissue engineering applications5,6,7,8. However, the main limitation for these types of studies is the lack of availability of primary human ECs in the quantity required. It has been estimated that a minimum of 3 x 108 ECs would be required for the majority of therapeutic applications6. To solve this problem, the use of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) has been proposed due to their diverse lineage potential and their ability to generate large numbers of progeny6,9.
Indeed, the usefulness of cells derived from hESCs or hiPSCs has been demonstrated in multiple studies focused on disease modeling and drug screening10,11,12. Organ-on-a-Chip (OOC) technology has been used to more faithfully recapitulate the physiology of the human body by integrating cells and tissues into three-dimensional scaffolds. Furthermore, connection of multiple individual OOCs (a so-called body- or human-on-a-chip, BOC/HOC) can be accomplished via microfluidics to allow for crosstalk between the organs of interest13,14,15. Supporting tissues, such as the vasculature, are critical components of OOCs and BOC/HOCs; incorporating vasculature allows for the transport of nutrients, oxygen, and paracrine factors throughout the tissues, thereby promoting the requisite tissue-specific microenvironment3,12. Thus, methods for deriving mature human ECs, such as arterial, venous, lymphatic, and hemogenic ECs, are crucial to advancing these tissue engineering approaches.
Multiple protocols have been published detailing steps for the derivation of human primordial or progenitor ECs from hESCs or hiPSCs5,16,17,18,19,20,21,22,23,24,25,26. Many of these protocols rely on embryoid body (EB) formation or co-culture of ESCs/iPSCs with a murine feeder layer of stromal cells. These strategies tend to be difficult and time consuming, with low EC yields and/or contamination of human ECs with murine cells. Protocols that rely strictly on 2D culture without the use of stromal cells often require long inductions, utilize complex combinations of growth factors and/or inhibitors for induction, have extended expansion periods following cell separation, or a combination of these factors. Advancing knowledge of signaling pathways and factors involved in the derivation of mature EC types in vivo provides the groundwork for a straightforward and robust in vitro differentiation protocol.
Previously, key roles for Notch and Retinoic Acid (RA) signaling pathways in the specification of murine arterial and hemogenic ECs, respectively, during development were identified. The Notch signaling pathway plays multiple roles in the specification and maintenance of the arterial EC phenotype. Work using the murine retinal vascularization model identified a pathway in which fluid shear stress induces a Notch-Cx37-p27 signaling axis, promoting G1 cell cycle arrest, which enables arterial EC specification27. Cell cycle states have been hypothesized to play a role in cell fate decisions by providing distinct windows of opportunity in which cells are receptive to certain signals that can induce gene expression and phenotypic changes28. This Notch-mediated G1 arrest enabled the expression of genes enriched in arterial ECs, including ephrinB2, Cx40, DLL4, Notch1, and Notch 4 (reviewed in29,30). It has also been shown that hemogenic EC specification is promoted in vivo via RA signaling31,32. Additional studies identified that, downstream of RA signaling, expression of c-Kit and Notch upregulate p27, which enables hemogenic specification in the murine yolk sac and AGM33. Murine hemogenic ECs can be minimally identified by expression of both endothelial (i.e., CD31, KDR) and hematopoietic (i.e., c-Kit, CD34) markers4. Finally, hemogenic ECs undergo an endothelial-to-hematopoietic transition (EHT) to form HSPCs, which can give rise to all blood cell types4,34,35.
Recent work tested whether this same signaling hierarchy can promote human hemogenic EC specification. To do so, a serum- and feeder-free 2D culture protocol to derive hemogenic ECs from hESCs was developed, and these hemogenic ECs were characterized on a single cell level as CD31+ KDR+ c-Kit+ CD34+ VE-Cadherin- CD45-. This study also took advantage of the Fluorescent Ubiquitination Cell Cycle Indicator (FUCCI) reporter, which identifies different cell cycle states, using H9-hESCs that express the FUCCI reporter construct (H9-FUCCI-hESC)36. In studies with these cells, it was demonstrated that RA promotes early G1 cell cycle arrest in ECs, and early G1 state enables hemogenic specification in vitro37. Herein, a detailed protocol for the differentiation of these human hemogenic endothelial cells and assays confirming their identity are provided. This straightforward method provides a useful means of generating this specialized subset of ECs for future studies of mechanisms of human blood cell development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 cm dishes</td>
			<td>Corning</td>
			<td>430599</td>
			<td>tissue culture treated</td>
		</tr>
		<tr>
			<td>35 mm dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td>tissue culture treated</td>
		</tr>
		<tr>
			<td>6-well plates</td>
			<td>Corning</td>
			<td>3516</td>
			<td>tissue culture treated</td>
		</tr>
		<tr>
			<td>Antimicrobial reagent 
			Brand Name: Normocin</td>
			<td>Invitrogen</td>
			<td>ant-nr-1</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>R&#38;D systems</td>
			<td>233-FB-025</td>
			<td>use at 50 ng/mL</td>
		</tr>
		<tr>
			<td>BMP4</td>
			<td>BioLegend</td>
			<td>595202</td>
			<td>use at 25 ng/mL</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Detatchment Solution 
			Brand Name: vAccutase</td>
			<td>Stemcell Technologies</td>
			<td>7920</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2650-100mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Stemcell Technologies</td>
			<td>7913</td>
			<td></td>
		</tr>
		<tr>
			<td>DLL4</td>
			<td>R&#38;D systems</td>
			<td>1506-D4/CF</td>
			<td>recombinant human; use at 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>DMEM:F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell growth medium 
			Brand Name: EGM-2 Endothelial Cell Growth Medium-2 BulletKit (EGM-2)</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Corning</td>
			<td>352235</td>
			<td>polystyrene round bottom with filter cap</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gemini Bio</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>ThermoFisher Scientific</td>
			<td>33016015</td>
			<td>use at 4 mg/cm2</td>
		</tr>
		<tr>
			<td>GSK3i/CHIR99021</td>
			<td>Stemgent</td>
			<td>04-0004-02</td>
			<td>10 mM stock; use at 5 &#956;M</td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14175-095</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid (HCl)</td>
			<td>Fisher Scientific</td>
			<td>A144S-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix protein&#160; 
			Brand Name: Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Growth factor reduced. Refer to the Certificate of Analysis for the lot to determine the protein (Matrigel) concentration. This concentration is required to calculate the volume of Matrigel that contains 1 mg of protein.</td>
		</tr>
		<tr>
			<td>Methylcellulose-based medium 
			Brand Name: MethoCult H4435 Enriched</td>
			<td>Stemcell Technologies</td>
			<td>4435</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluripotent stem cell differentiation medium 
			Brand Name: STEMdiff APEL 2</td>
			<td>Stemcell Technologies</td>
			<td>5270</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluripotent stem cells: H1, H9, H9-FUCCI</td>
			<td>WiCell</td>
			<td>WA09 (H9), WA01 (H1)</td>
			<td>human; H9-FUCCI were obtained from Dr. Ludovic Vallier&#39;s lab at Cambridge Stem Cell Institute</td>
		</tr>
		<tr>
			<td>Protein-Free Hybridoma Medium (PFMH)</td>
			<td>Gibco</td>
			<td>12040077</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>R2625-50mg</td>
			<td>use at 0.5 &#956;M</td>
		</tr>
		<tr>
			<td>Reverse transcription master mix 
			Brand Name: iScript Reverse Transcription Supermix</td>
			<td>BioRad</td>
			<td>1708840</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA extraction kit 
			Brand Name: RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide (NaOH)</td>
			<td>Fisher Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem cell growth medium 
			Brand Name: mTeSR1</td>
			<td>Stemcell Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Green master mix 
			Brand Name: iTaq Universal SYBR Green Master Mix</td>
			<td>BioRad</td>
			<td>1725121</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25299956</td>
			<td>0.25%</td>
		</tr>
		<tr>
			<td>VEGF165 (VEGF-A)</td>
			<td>PeproTech</td>
			<td>100-20</td>
			<td>use at 50 ng/mL</td>
		</tr>
		<tr>
			<td>&#945;-CD31-FITC</td>
			<td>BioLegend</td>
			<td>303104</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-CD34-Pacific Blue</td>
			<td>BioLegend</td>
			<td>343512</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-CD45-APC/Cy7</td>
			<td>BioLegend</td>
			<td>304014</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-c-Kit-APC</td>
			<td>BioLegend</td>
			<td>313206</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-Flk-1-PE/Cy7</td>
			<td>BioLegend</td>
			<td>359911</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td>&#945;-VE-Cadherin-PE</td>
			<td>BioLegend</td>
			<td>348506</td>
			<td>2 &#956;g/mL*</td>
		</tr>
		<tr>
			<td colspan="4">* Antibody fluorescent conjugates should be optimized based on the cell sorter used. Presented here are the final concentrations utilized in this study.</td>
		</tr>
	</tbody>
</table>

directed differentiation of hemogenic endothelial cells from human pluripotent stem cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

A schematic outlining the specification of primordial ECs and hemogenic ECs from hESCs, and a representative image of cells 24 h after plating are shown in Figure 1. Following specification, primordial ECs and hemogenic ECs are FACS purified on days 5 and 8, respectively. Primordial ECs are defined as CD31+ CD45- and hemogenic ECs are defined as CD31+ KDR+ c-Kit+ CD34+ VE-Cadherin- CD45-. A representative...

Watch this Scientific Journal Video about Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells at JoVE.com

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

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

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

This protocol details a method to generate a well-characterized population of human hemogenic endothelial cells. These cells can be utilized to study the molecular regulation and endothelial-to-hematopoietic transition. Begin by culturing human embryonic stem cells for four days to differentiate them into primordial endothelial cells.On day five, aspirate the medium above the cells. Then gently wash the cells with one milliliter of DMEM/F-12 per well. Add one milliliter of freshly prepared hemogenic endothelial cell differentiation medium per well and incubate the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide.On day six and day seven, replace the medium above the cells with one milliliter of freshly prepared homogenic endothelial cell differentiation medium per well, and incubate the cells for another 24 hours. On day eight. after detaching and washing the cells, divide them evenly into microcentrifuge tubes on ice, each containing a minimum of 600 microliters of cells at a density of 1 times 10 to the 5th cells per milliliter.Add antibodies to the tubes containing cells as appropriate and incubate on ice protected from light for 30 minutes. Pellet the cells by centrifugation at 1000 times G and 4 degrees Celsius for five minutes. Remove the supernatant and resuspend the pellets in 600 microliters of ice-cold sorting buffer.Strain the samples through the mesh filter cap of five milliliter FACS tubes and store the cells on ice protected from light for immediate cell sorting. To isolate hemogenic endothelial cells by FACS, first gate the CD45-negative cell population. Then within the CD45-negative population, gate the CD31-positive cells.Next within the CD31-positive population, gate for the VE-cadherin-negative cell population. And then within the VE-cadherin-negative population, gate for the c-Kit-positive cells. From the c-Kit-positive population, gate the CD34-positive cell population.And finally from the CD34-positive cell population, identify and collect the KDR-positive cells. After seeding hemogenic endothelial cells in a Methylcellulose-based medium formulated for growth of Hematopoietic Progenitor Cells, Colony Forming Unit-Erythroid colonies, and Blast Forming Unit-Erythroid colonies are counted on day eight. Colony-Forming Unit-Granulocyte-Macrophage and Colony-Forming Unit-Granulocyte, Erythroid, Macrophage, and Megacaryocyte.Multipotent hematopoietic progenitor colonies are counted on day 14. Per 1000 hemogenic endothelial cells plated, approximately 20 colony forming units are generated. Cells with endothelial cell morphology are also visible in the cultures.These are the hemogenic endothelial cells that give rise to multilineage hematopoietic progenitors on a single-cell level. This protocol is a 2D serum and murine feeder-free culture system that can generate human hemogenic endothelial cells in roughly one week.

directed-differentiation-hemogenic-endothelial-cells-from-human

Research

JoVE Journal

Developmental Biology

2.0K visualizações.  University of Virginia. Este protocolo detalha um método para gerar uma população bem caracterizada de células endoteliais hemogênicas humanas. Essas células podem ser utilizadas para estudar a regulação molecular e a transição endotelial-hematopoiética. Comece cultivando células-tronco embrionárias humanas por quatro dias para diferenciá-las em células endoteliais primordiais.No quinto dia, aspirar o meio acima das células. Em seguida, lave suavemente as células com um mililitro de DMEM / F-...