We are grateful to Alina Li and Seiko Benno for their technical assistance. We would also like to thank Ms. Harumi Watanabe for providing administrative assistance and Dr. Peter Karagiannis for editing the paper. This work was supported by the Core Center for iPS Cell Research of Research Center Network for Realization of Regenerative Medicine from the Japan Agency for Medical Research and Development (AMED) [M.K.S.], the Program for Intractable Diseases Research utilizing. Disease-specific iPS cells of AMED (17935423) [M.K.S.] and Center for Innovation program of Japan Science and Technology Agency (JST) [R.O. and M.K.S.].

1. Reagents
<ol>
	<li>Cell lines (human PSCs): obtain either embryonic stem cells (ESCs) or iPSC lines 409B2 and 201B7(317-9)) and CBA11.</li>
	<li>Reagent preparation
	<ol>
		<li>PSC plating medium: mix PSC maintenance medium with 10 &#181;M Y-27632 and store at 4 &#176;C.</li>
		<li>PSC Spheroid tiling medium: mix PSC maintenance medium with 625&#8211;1,250 ng/mL human recombinant Laminin-511 E8 fragment (LM511-E8)(depending on cell line) just prior to use. 
		NOTE: LM511-E8 can be mixed in media15. Other matrix such as full-length laminin-511 needs to be pre-coated, and even did so, they do not sustain mesodermal organoids adhered during differentiation process.</li>
		<li>Day 0 differentiation medium: mix Mesodermal basal medium with 2 &#181;M CHIR99021, 80 ng/mL Bone morphogenetic protein 4 (BMP4) and 80 ng/mL Vascular endothelial growth factor (VEGF).</li>
		<li>Day 2 differentiation medium: mix Hemogenic basal medium with 1 &#181;M SB431542, 80 ng/mL VEGF and 100 ng/mL Stem cell factor (SCF).</li>
		<li>Prepare phosphate buffer saline (PBS) without calcium or magnesium (See Table of Materials).</li>
		<li>1 mM ethylenediaminetetraacetic acid (EDTA) buffer: add 1 mL of 0.5 M EDTA to 500 mL of PBS.</li>
		<li>EHT medium: mix Hematopoietic basal medium with 50 ng/mL SCF, 20 ng/mL thrombopoietin (TPO), 50 ng/mL Fms-related tyrosine kinase 3 ligand (Flt-3L), 20 ng/mL Interleukin-6/Interleukin-6 receptor alpha (IL-6/IL-6R&#945;), insulin-transferrin-selenium-ethanolamine, L-glutamine and Penicillin/Streptomycin/Amphotericin B.</li>
		<li>FACS buffer: mix PBS with 2% fetal calf serum and 1 mM EDTA.</li>
		<li>Prepare magnetic separation buffer (See Table of Materials).</li>
		<li>Prepare methylcellulose-based media (See Table of Materials).</li>
	</ol>
	</li>
</ol>
2. PSC Colony Formation
<ol>
	<li>Grow hPSCs to 70 &#8211; 80% confluency on a 0.5 &#181;g cm-2 LM511-E8-coated 6-well plate in mTeSR1 in a 37 &#176;C incubator with 5% CO2.</li>
	<li>PSC dissociation (Day -4)
	<ol>
		<li>Aspirate the medium and wash the cells with PBS twice. Rinse the cells with dissociation solution. Incubate at 37 &#176;C for 15 min (cells will not desiccate in this time).</li>
		<li>Suspend the cells in PSC maintenance medium, transfer the suspension to an appropriately sized tube and centrifuge the cells at 200 x g for 3 min. Aspirate the supernatant and suspend the cells in PSC plating medium.</li>
	</ol>
	</li>
	<li>Plate the PSC suspension at a density of 48,000 &#8211; 70,000 cells cm-2 (depending on the cell line) onto a micro-fabricated plastic vessel and incubate overnight in the 37 &#176;C incubator with 5% CO2. 
	NOTE: We recommend 100 &#956;L well-1 of cell suspension in a 96-well micro-fabricated plastic vessel. You will typically seed 20,000 cells well-1 in a 96-well plate to yield approximately 100 spheroids.</li>
	<li>Tiling PSC spheroids on LM511-E8 (Day -3)
	<ol>
		<li>Collect the PSC spheroids in a 15 mL conical tube by gently pipetting using P1000 micropipetter and leave the spheroids to stand at room temperature for 2 min to precipitate by gravity. Aspirate the supernatant.</li>
		<li>Suspend in spheroid plating medium, dispense the suspension into a non-coated 6-well culture plate at a density of 4-spheroids cm-2 and culture in the 37 &#176;C incubator with 5% CO2 for three days.</li>
	</ol>
	</li>
</ol>
3. Hemogenic Induction (Day 0)
<ol>
	<li>Aspirate the medium, add Day0 differentiation medium and culture in the 37 &#176;C incubator with 5% O2 and 5% CO2 (hypoxic incubator).</li>
	<li>After two days of culture in Day0 differentiation medium, aspirate the medium, then add Day2 differentiation medium and culture in the hypoxic incubator.</li>
</ol>
4. Isolation of Hemogenic Endothelium (Day 4)
<ol>
	<li>Two days later, aspirate the medium and wash the cells with PBS twice. Rinse the cells with dissociation solution. Incubate in the 37 &#176;C incubator with 5% CO2 for 30 min.</li>
	<li>Gently suspend the cells in 1 mM EDTA to dissociate to the single-cell level, transfer the suspension into a 50 mL conical tube and centrifuge the cells at 200 x g for 3 min. Aspirate the supernatant and suspend the cell pellet with 300 &#181;L of magnetic separation buffer.</li>
	<li>Add 100 &#181;L of CD34+ microbeads and gently pipet. Incubate for 30 min at room temperature. Up to 20 million cells per 100 &#956;L CD34+ beads can be used.</li>
	<li>Filter the suspension through a 40 &#181;m cell strainer.</li>
	<li>Separate the CD34+ cells using a magnetic separator.</li>
</ol>
5. Induction of Endothelial-to-hematopoietic Transition (EHT)
<ol>
	<li>Fibronectin coating
	<ol>
		<li>Prepare the required volume of 5 &#181;g/mL fibronectin-coating solution by diluting 1 mg/mL fibronectin in PBS.</li>
		<li>Dispense 0.5 mL of the coating solution into each well of a 24-well culture plate. Incubate the plate at room temperature for 30 min.</li>
	</ol>
	</li>
	<li>Centrifuge the sorted CD34+ cells at 200 x g for 3 min. Resuspend the cell pellet in 1 mL of EHT medium.</li>
	<li>Determine the viable cell density with the cell counter. Adjust the cell density to 200,000 cells mL-1 by adding EHT medium.</li>
	<li>Aspirate and discard the coating solution from the fibronectin-coated well. Dispense 0.5 mL of CD34+ cell suspension into each fibronectin-coated well.</li>
	<li>Incubate in the hypoxic incubator for one week.</li>
</ol>
6. Flow Cytometry Analysis of Hematopoietic Cells
<ol>
	<li>Floating cell collection: Transfer the culture medium to a 15 mL conical tube. Centrifuge the medium at 200 x g for 3 min. Aspirate the supernatant.</li>
	<li>Adherent cell collection
	<ol>
		<li>Wash the cells with 0.5 mL of PBS twice.</li>
		<li>Rinse the cells with dissociating solution and aspirate the surplus liquid.</li>
		<li>Incubate the plate in the 37 &#176;C incubator for 5 min.</li>
		<li>Resuspend the cells in 1 mL of FACS buffer.</li>
	</ol>
	</li>
	<li>Mix the floating cells and adherent cells.</li>
	<li>Centrifuge the cells at 200 x g for 3 min. Aspirate the supernatant. Resuspend in 50 &#181;L of FACS buffer. Incubate the cells with anti-CD34 and anti-CD45 antibodies for 1 h at room temperature in the dark.</li>
	<li>Wash the cells with PBS twice and centrifuge at 200 x g for 3 min. Aspirate the supernatant and resuspend in 0.5 mL of FACS buffer with 0.5 &#181;g/mL 4&#39;,6-diamidino-2-phenylindole.</li>
	<li>Measure CD34 and CD45 expression by flow cytometer.</li>
</ol>
7. Colony Forming Unit (CFU) Assay of Hematopoietic Cells
<ol>
	<li>Hematopoietic cell collection: Transfer the medium from the culture to a 15 mL conical tube. Gently rinse the well twice with PBS.</li>
	<li>Centrifuge the cells at 200 x g for 3 min. Aspirate the supernatant. Resuspend in 1 mL of EHT medium. Determine cell number with the cell counter.</li>
	<li>Add a suspension volume equivalent of 10,000 cells to 3 mL of methylcellulose-based media supplemented with antibiotics and mix well 5 times with a 16 G or 18 G syringe.</li>
	<li>Dispense the whole methylcellulose-based media suspension into each well of a 6-well plate.</li>
	<li>Incubate in the 37 &#176;C incubator with 5% CO2 for two weeks while keeping moist. Do not disturb the dish. Colonies are motion-sensitive.</li>
	<li>After two weeks, count the colonies under a microscope.</li>
</ol>

<ol><li>Pereira, C. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+a+hemogenic+program+in+mouse+fibroblasts%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Induction of a hemogenic program in mouse fibroblasts.</a> Cell Stem Cell. 13, 205-218 (2013).</li>
<li>Lancrin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+haemangioblast+generates+haematopoietic+cells+through+a+haemogenic+endothelium+stage%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The haemangioblast generates haematopoietic cells through a haemogenic endothelium stage.</a> Nature. 457, 892-895 (2009).</li>
<li>Loh, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+the+Pairwise+Choices+Leading+from+Pluripotency+to+Human+Bone%2C+Heart%2C+and+Other+Mesoderm+Cell+Types%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.</a> Cell. , 451-467 (2016).</li>
<li>Ng, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiation+of+human+embryonic+stem+cells+to+HOXA%2B+hemogenic+vasculature+that+resembles+the+aorta-gonad-mesonephros%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.</a> Nature Biotechnology. 34, 1168-1179 (2016).</li>
<li>Batta, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Direct+reprogramming+of+murine+fibroblasts+to+hematopoietic+progenitor+cells%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells.</a> Cell Reports. 9, 1871-1884 (2014).</li>
<li>Sturgeon, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defining+the+path+to+hematopoietic+stem+cells%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Defining the path to hematopoietic stem cells.</a> Nature Biotechnology. 31, 416-418 (2013).</li>
<li>Ivanovs, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+haematopoietic+stem+cell+development%3A+from+the+embryo+to+the+dish%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Human haematopoietic stem cell development: from the embryo to the dish.</a> Development. 144, 2323-2337 (2017).</li>
<li>Blaser, B. W., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Making+HSCs+in+vitro%3A+don%27t+forget+the+hemogenic+endothelium%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Making HSCs in vitro: don't forget the hemogenic endothelium.</a> Blood. , (2018).</li>
<li>Speck, N., Dzierzak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Of+lineage+and+legacy%3A+the+development+of+mammalian+hematopoietic+stem+cells%5BTitle%5D)+AND+%22Nature+imunology%22%5BJournal%5D)">Of lineage and legacy: the development of mammalian hematopoietic stem cells.</a> Nature imunology. , (2008).</li>
<li>Niwa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+serum-free+monolayer+culture+for+orderly+hematopoietic+differentiation+of+human+pluripotent+cells+via+mesodermal+progenitors%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">A novel serum-free monolayer culture for orderly hematopoietic differentiation of human pluripotent cells via mesodermal progenitors.</a> PLoS One. 6, e22261(2011).</li>
<li>Sturgeon, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wnt+signaling+controls+the+specification+of+definitive+and+primitive+hematopoiesis+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">Wnt signaling controls the specification of definitive and primitive hematopoiesis from human pluripotent stem cells.</a> Nature Biotechnology. 32, 554-561 (2014).</li>
<li>Ditadi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+definitive+haemogenic+endothelium+and+arterial+vascular+endothelium+represent+distinct+lineages%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages.</a> Nature Cell Biology. 17, 580-591 (2015).</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+Reports%22%5BJournal%5D)">T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures.</a> Cell Reports. 2, 1722-1735 (2012).</li>
<li>Sugimura, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Haematopoietic+stem+and+progenitor+cells+from+human+pluripotent+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Haematopoietic stem and progenitor cells from human pluripotent stem cells.</a> Nature. 545, 432-438 (2017).</li>
<li>Miyazaki, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+Adhesion+Culture+of+Human+Pluripotent+Stem+Cells+Using+Laminin+Fragments+in+an+Uncoated+Manner%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Efficient Adhesion Culture of Human Pluripotent Stem Cells Using Laminin Fragments in an Uncoated Manner.</a> Scientific Reports. 7, 41165(2017).</li>
</ol>

The authors declare no conflicts of interest.

Our feeder- and xeno-free defined induction method offers a precise and cost-effective platform to induce HE cells in a scalable manner. The faithful formation of hemogenic endothelium in conventional protocols10,11,12,13,14 have been hampered by lack of precise control of PSC colony density and size, which affects efficiency of morphogen/cytokine-based directed differentiation. We had experienced inconsistency of hemogenic endothelium induction in each independent batch from conventional protocols. The new protocol enables tight regulation of PSC colony density and size, thus overcomes the inconsistency of hemogenic endothelium induction.
We exploited the uniformed formation of spheroids and subsequently conveyed a feeder- and xeno-free defined condition to form HE in a total of 4 days. We confirmed using three independent cell lines that the spheroid formation assures the consistent formation of HE cells. As a representative result, the 409B2 cell lines yielded 12.7%-23.6% CD34+ cell efficiency based on three independent experiments. The critical factor to tile PSC spheroids is LM511-E8. We do not recommend substituting this with other matrix, such as Matrigel or full-length laminin products in our experiences. We experienced that mesodermal organoids were easily detached from Matrigel and lost during differentiation process. And neither Matrigel or full-length laminin do not anchor cells without pre-coating.
The potential limitation of this protocol is that we depend on PSC maintenance medium to maintain PSCs. We have tested other media such as StemFit, but we compromised hemogenic induction. Presumably cells were resistant to exit from pluripotent state in our short time (4 days) induction protocol. If one maintains PSCs in other media, we recommend adapting cells in PSC maintenance medium and conduct a couple of passages prior to differentiation. This platform will allow the systematic manipulation and analysis of cells, and the off-the-shelf production and large-scale induction of hematopoietic cells for potential clinical use. A long-term goal of this system is to model immunodeficiency diseases in humanized mouse models to investigate the responsible cellular and molecular mechanisms and conduct drug screenings.

The development of new therapeutics for hematological and immunological disorders has been hampered by the lack of robust platforms for disease modeling and high throughput drug screening with autologous hematopoietic stem cells (HSCs) derived from patient pluripotent stem cells (PSCs). The breakthrough of induced (i)PSC technology holds promise to model diseases from patients&#39; cells, but the establishment of functional HSCs from patient iPSCs has been elusive. In order to derive HSCs from human PSCs, the proper induction of embryonic patterning and transcriptional programs is key1,2,3,4,5,6,7,8. Recent progress in hematopoietic development has demonstrated the role of hemogenic endothelium (HE) in HSC development9. HE can be induced from PSCs and is amenable to downstream intervention such as gene transfer10,11,12,13. Using HE as a platform, the combination of 5 transcription factors (ERG, HOXA5, HOXA9, LCOR, RUNX1) successfully induced functional HSCs that engraft long-term and differentiate into multiple lineages14. However, variable and inefficient generation of HE from PSCs have hampered the systematic manipulation and analysis of cells along with the off-the-shelf production and large-scale induction of hematopoietic cells for clinical use.
Here, we describe a cost-effective and precise method to derive HE in 4 days with a feeder- and xeno-free defined condition. In this method, spheroid formation and spontaneous re-flattening on iMarix-511 ensures a consistent density of PSC colonies, which is crucial for the efficient induction of HE cells.

<table><tbody><tr><td>0.5 M EDTA</td><td>Thermo Fisher Scientific</td><td>15575</td><td>0.5 M EDTA</td></tr><tr><td>40 &amp;mu;m cell strainer</td><td>Corning</td><td>352340</td><td>40&amp;mu;M cell strainer</td></tr><tr><td>6 well plate</td><td>Corning</td><td>353046</td><td>6 well plate</td></tr><tr><td>Antibiotic-antimycotic</td><td>Thermo Fisher Scientific</td><td>15240-112</td><td>Penicillin/Streptomycin/Amphotericin B</td></tr><tr><td>anti-CD34 antibody</td><td>Beckman Coulter</td><td>A07776</td><td>anti-CD34 antibody</td></tr><tr><td>anti-CD45 antibody</td><td>Biolegend</td><td>304012</td><td>anti-CD45 antibody</td></tr><tr><td>BMP4</td><td>R&amp;amp;D Systems</td><td>314-BP-010</td><td>BMP4</td></tr><tr><td>CD34 microbeads</td><td>Miltenyi</td><td>130-046-703</td><td>CD34 microbeads</td></tr><tr><td>CHIR99021</td><td>Wako</td><td>038-23101</td><td>CHIR99021</td></tr><tr><td>Essential 6</td><td>Thermo Fisher Scientific</td><td>A15165-01</td><td>Hemogenic basal medium</td></tr><tr><td>Essential 8</td><td>Thermo Fisher Scientific</td><td>A15169-01</td><td>Mesodermal basal medium</td></tr><tr><td>Ezsphere SP Microplate 96well</td><td>AGC</td><td>4860-900SP</td><td>micro-fabricated plastic vessel&amp;nbsp;</td></tr><tr><td>FCS</td><td>Biosera</td><td>FB-1365/500</td><td>FCS</td></tr><tr><td>Fibronectin</td><td>Millipore</td><td>FC010-100MG</td><td>Fibronectin</td></tr><tr><td>Flt-3L</td><td>R&amp;amp;D Systems</td><td>308-FK-005</td><td>Flt-3L</td></tr><tr><td>Glutamax</td><td>Thermo Fisher Scientific</td><td>35050-061</td><td>L-Glutamine</td></tr><tr><td>IL-6/IL-6R&amp;alpha;</td><td>R&amp;amp;D Systems</td><td>8954-SR</td><td>IL-6/IL-6R&amp;alpha;</td></tr><tr><td>iMatrix-511</td><td>Matrixome</td><td>892 001</td><td>LM511-E8</td></tr><tr><td>ITS-X</td><td>Thermo Fisher Scientific</td><td>51500-056</td><td>Insulin-Transferrin-Selenium-Ethanolamine</td></tr><tr><td>MACS buffer</td><td>Miltenyi</td><td>130-091-221</td><td>magnetic separation buffer</td></tr><tr><td>Methocult 4435MethoCult&amp;trade; H4435 Enriched</td><td>STEMCELL TECHNOLOGIES</td><td>04435</td><td>Methylcellulose-based media</td></tr><tr><td>mTeSR1</td><td>STEMCELL TECHNOLOGIES</td><td>85850</td><td>PSC maintenance medium</td></tr><tr><td>PBS</td><td>nacalai tesque</td><td>14249-24</td><td>PBS</td></tr><tr><td>SB431542</td><td>Wako</td><td>031-24291</td><td>SB431542</td></tr><tr><td>SCF</td><td>R&amp;amp;D Systems</td><td>255-SC-010</td><td>SCF</td></tr><tr><td>Stemline II Hematopoietic Stem Cell Expansion Medium</td><td>Sigma Aldrich</td><td>S0192-500ML</td><td>Hematopoietic basal medium</td></tr><tr><td>TPO</td><td>R&amp;amp;D Systems</td><td>288-TPN</td><td>TPO</td></tr><tr><td>TrypLE Express</td><td>Thermo Fisher Scientific</td><td>12604-021</td><td>dissociation solution</td></tr><tr><td>VEGF</td><td>R&amp;amp;D Systems</td><td>293-VE-010</td><td>VEGF</td></tr><tr><td>Y-27632</td><td>Wako</td><td>253-00513</td><td>Y-27632</td></tr></tbody></table>

hemogenic endothelium differentiation from human pluripotent stem cells in a feeder- and xeno-free defined condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

A schematic of forming PSC colonies is depicted in Figure 1A. PSC spheroids are formed on a micro-fabricated plastic vessel for one day. As a representative result, 20,000 409B2 cells could be handled per 96-well of micro-fabricated plastic vessel, although other cell lines may require a higher density (30,000-40,000 cells per 96-well of micro-fabricated plastic vessel). Those spheres are spontaneously flattened to a nearly two-dimensional culture when plated onto a culture dish in the presence of LM511-E8.
A schematic of hemogenic induction is illustrated in Figure 1B. When PSC colonies grow to a diameter of 750 &#181;m, the medium is sequentially changed to induce mesodermal organoids. The PSC colonies will gradually become a sunny side up structure during differentiation to mesodermal organoids by sequential medium change until Day 4. On Day 4, hemogenic endothelia are specified from mesodermal organoids by magnetic sorting and subsequently plated onto fibronectin in EHT medium. 5-10 million CD34+CD45-&#160;cells are expected from spheroids containing 1 million PSCs (Figure 1C).
It is observed that the cells morphologically change from endothelial to hematopoietic cells (Supplementary Video 1). A representative phase-contrast image of hematopoietic progenitor cells upon stimulation with hematopoietic cocktail on day 7 is shown in Figure 2A. Hematopoietic cell colonies emerged in the culture.
A representative flow cytometry plot is shown in Figure 2B. The whole culture is stimulated with hematopoietic cocktail and on day 7 is analyzed for the expression of CD34 and CD45 by flow cytometry. Hematopoietic progenitor cells express CD34 and&#160;CD45.
A CFU assay showed the generation of granulocytes/macrophage colonies from the CD34+ CD45+ hematopoietic progenitor cells (Figure 2C). The estimated colony number is 38 CFU-G/M per 10,000 cells (Figure 2D)
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59823/59823fig1v2.jpg" /> 
Figure 1: Schematic of tiling PSC colonies and subsequent hematopoietic differentiation via hemogenic endothelia. (A) Schematic process of forming PSC colonies. PSCs are maintained on a LM511-E8-coated 6-well plate in PSC maintenance medium. On Day -4, cells are detached and dissociated to the single-cell level using dissociating solution, subsequently plated onto micro-fabricated plastic vessel in PSC maintenance medium with Y-27632 and cultured overnight to form spheroids. On Day -3, spheroids are plated in PSC maintenance medium with LM511-E8 and cultured for three days. By Day 0, spheroids are spontaneously flattened to a nearly two-dimensional culture. Scale bars = 200 &#181;m. (B) On Day 0, the medium is replaced with Day0 differentiation medium and cultured in the hypoxic incubator. On Day 2 of the differentiation, the medium is replaced with Day2 differentiation medium. On Day 4 of the hemogenic endothelium enrichment, CD34+ cells are magnetically sorted and cultured on a fibronectin-coated 12-well plate in EHT medium for 6 days. (C) Representative flowcytometry plot of CD45 and CD34 expression of day 4 cells sorted by CD34. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59823/59823fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59823/59823fig2.jpg" /> 
Figure 2: Analysis of hematopoietic property. (A) Representative phase-contrast image of hematopoietic progenitor cells after stimulation with hematopoietic cocktail on day 7. Scale bar = 200 &#181;m. (B) Representative flow cytometry plot of CD45 and CD34 expression of whole culture upon stimulation with hematopoietic cocktail on day 7. (C) Representative phase-contrast image of a granulocyte/macrophage colony generated from Day 11 hematopoietic progenitor cells. Scale bar = 500 &#181;m. (D) Number of CFUs per 10,000 cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59823/59823fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Video" src="/files/ftp_upload/59823/59823vid1.jpg" /> 
Supplementary video 1: EHT video. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59823/EHT_video.avi">Please click here to download this file.</a>

Watch this Scientific Journal Video about Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition at JoVE.com

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

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.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

hemogenic-endothelium-differentiation-from-human-pluripotent-stem

Ateneo de Manila University

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9.5K Views.  Center for iPS cell research and application (CiRA). 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.

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

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

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

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

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

Feeder-free Derivation of Melanocytes from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can direct the differentiation of hPSCs into the cell type of interest by manipulating the culture conditions to recapitulate signals seen during development. One such cell type is the melanocyte, a pigment-producing cell of neural crest (NC) origin responsible for protecting the skin against UV irradiation. This protocol presents an extension of a currently available in vitro Neural Crest differentiation protocol from hPSCs to further differentiate NC into fully pigmented melanocytes. Melanocyte precursors can be enriched from the Neural Crest protocol via a timed exposure to activators of WNT, BMP, and EDN3 signaling under dual-SMAD-inhibition conditions. The resultant melanocyte precursors are then purified and matured into fully pigmented melanocytes by culture in a selective medium. The resultant melanocytes are fully pigmented and stain appropriately for proteins characteristic of mature melanocytes.

This work describes an in vitro differentiation protocol to produce pigmented, mature melanocytes from human pluripotent stem cells via a neural crest and melanoblast intermediate stage using a feeder-free, 25 day protocol.

Human pluripotent stem cells (hPSCs) represent a platform to study human development in vitro under both normal and disease conditions. Researchers can ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

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

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

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

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

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

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.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

The production of specialized retinal cells from pluripotent stem cells is a turning point in the development of stem cell-based therapy for retinal diseases. The present paper describes a simple method for an efficient generation of retinal organoids and retinal pigmented epithelium for basic, translational, and clinical research.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

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

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

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

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Neuroscience

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.

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 ...

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/&#946;-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

Here, we describe a protocol, the extended endothelial cell culture method (EECM), that allows differentiation of pluripotent stem cells to brain microvascular endothelial cell (BMEC)-like cells. These cells show endothelial cell adhesion molecule expression and are thus a human blood-brain barrier model suitable to study immune cell interactions in vitro.

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous ...

Bioengineering

Derivation, Expansion, Cryopreservation and Characterization of Brain Microvascular Endothelial Cells from Human Induced Pluripotent Stem Cells

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models for studying blood-brain barrier (BBB) function. This modified protocol provides detailed steps to derive, expand, and cryopreserve BMECs from human iPSCs using a different donor and reagents than those reported in previous protocols. iPSCs are treated with essential 6 medium for 4 days, followed by 2 days of human endothelial serum-free culture medium supplemented with basic fibroblast growth factor, retinoic acid, and B27 supplement. At day 6, cells are sub-cultured onto a collagen/fibronectin matrix for 2 days. Immunocytochemistry is performed at day 8 for BMEC marker analysis using CLDN5, OCLN, TJP1, PECAM1, and SLC2A1. Western blotting is performed to confirm BMEC marker expression, and absence of SOX17, an endodermal marker. Angiogenic potential is demonstrated with a sprouting assay. Trans-endothelial electrical resistance (TEER) is measured using chopstick electrodes and voltohmmeter starting at day 7. Efflux transporter activity for ATP binding cassette subfamily B member 1 and ATP binding cassette subfamily C member 1 is measured using a multi-plate reader at day 8. Successful derivation of BMECs is confirmed by the presence of relevant cell markers, low levels of SOX17, angiogenic potential, transporter activity, and TEER values ~2000 &#937; x cm2. BMECs are expanded until day 10 before passaging onto freshly coated collagen/fibronectin plates or cryopreserved. This protocol demonstrates that iPSC-derived BMECs can be expanded and passaged at least once. However, lower TEER values and poorer localization of BMEC markers was observed after cryopreservation. BMECs can be utilized in co-culture experiments with other cell types (neurons, glia, pericytes), in three-dimensional brain models (organ-chip and hydrogel), for vascularization of brain organoids, and for studying BBB dysfunction in neuropsychiatric disorders.

This protocol details an adapted method to derive, expand, and cryopreserve brain microvascular endothelial cells obtained by differentiating human induced pluripotent stem cells, and to study blood brain barrier properties in an ex vivo model.

Brain microvascular endothelial cells (BMECs) can be differentiated from human induced pluripotent stem cells (iPSCs) to develop ex vivo cellular models ...