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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a+hemogenic+program+in+mouse+fibroblasts.">Induction of a hemogenic program in mouse fibroblasts.</a> Cell Stem Cell. 13, 205-218 (2013).</li><li>Lancrin, C., 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=The+haemangioblast+generates+haematopoietic+cells+through+a+haemogenic+endothelium+stage.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+Pairwise+Choices+Leading+from+Pluripotency+to+Human+Bone,+Heart,+and+Other+Mesoderm+Cell+Types.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+human+embryonic+stem+cells+to+HOXA++hemogenic+vasculature+that+resembles+the+aorta-gonad-mesonephros.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+reprogramming+of+murine+fibroblasts+to+hematopoietic+progenitor+cells.">Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells.</a> Cell Reports. 9, 1871-1884 (2014).</li><li>Sturgeon, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+path+to+hematopoietic+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+haematopoietic+stem+cell+development:+from+the+embryo+to+the+dish.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+HSCs+in+vitro:+don't+forget+the+hemogenic+endothelium.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Of+lineage+and+legacy:+the+development+of+mammalian+hematopoietic+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+serum-free+monolayer+culture+for+orderly+hematopoietic+differentiation+of+human+pluripotent+cells+via+mesodermal+progenitors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+controls+the+specification+of+definitive+and+primitive+hematopoiesis+from+human+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+definitive+haemogenic+endothelium+and+arterial+vascular+endothelium+represent+distinct+lineages.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+lymphocyte+potential+marks+the+emergence+of+definitive+hematopoietic+progenitors+in+human+pluripotent+stem+cell+differentiation+cultures.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+and+progenitor+cells+from+human+pluripotent+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Adhesion+Culture+of+Human+Pluripotent+Stem+Cells+Using+Laminin+Fragments+in+an+Uncoated+Manner.">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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15575</td>
			<td>0.5M EDTA</td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Corning</td>
			<td>352340</td>
			<td>40&#956;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&#38;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&#160;</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&#38;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&#945;</td>
			<td>R&#38;D Systems</td>
			<td>8954-SR</td>
			<td>IL-6/IL-6R&#945;</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&#8482; 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&#38;D Systems</td>
			<td>255-SC-010</td>
			<td>SCF</td>
		</tr>
		<tr>
			<td>Stemline &#8545; 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&#38;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&#38;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/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/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/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

Differenziazione dell'endotelio endogenico dalle cellule staminali pluripotenti umane in una condizione definita senza alimentatore e Xeno

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

Research

JoVE Journal

Developmental Biology

9.4K Views.  Center for iPS cell research and application (CiRA). Offriamo la produzione di cellule del sangue da cellule staminali pluripotenti umane. Il principale vantaggio di questa tecnica è che otteniamo rese coerenti e precise di cellule endoteliali emogeniche. Prevediamo che la nostra piattaforma consentirà ampie applicazioni nel monitoraggio e nello screening delle malattie per i composti terapeutici.Questa tecnica è particolarmente utile per la ricerca ematologia e immunologica. Il metodo può essere applicato all'intervento genetico o a...

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.

Induzione diretta di Hemogenic endotelio e Sangue da sovraespressione di fattori di trascrizione in cellule staminali pluripotenti umane

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

Ricerca

Biologia dello sviluppo

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

Generazione di pluripotenti indotte cellule staminali da umani periferici T Cellule con Sendai virus in condizioni di libera-Feeder

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

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.

Derivazione senza alimentatore di melanociti da cellule staminali umane pluripotenti

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

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Produzione su larga scala dei cardiomiociti da cellule staminali umane pluripotenti usando un altamente riproducibile piccole molecole-Based Differenziazione protocollo

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

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.

Regia di differenziazione dei progenitori ematopoietici primitivi e definitivo da cellule staminali pluripotenti umane

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

La differenziazione in vitro di pluripotenti umane Cellule Staminali in cellule trofoblastiche

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Definito e Generazione scalabile di cellule epatociti-come da cellule staminali umane pluripotenti

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

Rapida, regia di differenziazione delle cellule epiteliali del pigmento retinico da cellule staminali pluripotenti umane embrionali o indotto

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

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.

Condizioni di coltura privo di alimentatore per i modelli di cellula retinica iPSC-derivato generazione umana e definito privo di Xeno

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

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Efficiente e scalabile di differenziazione diretto di clinicamente compatibile corneale Limbal cellule staminali epiteliali da cellule staminali pluripotenti umane

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...