1. Bulger, M., and Groudine, M. (2011).
Functional and mechanistic diversity of distal
transcription enhancers. Cell 144, 327–339.
https://doi.org/10.1016/j.cell.2011.01.024.
2. Hnisz, D., Abraham, B.J., Lee, T.I., Lau, A.,
Saint-Andre´, V., Sigova, A.A., Hoke, H.A., and
Young, R.A. (2013). Super-Enhancers in the
Control of Cell Identity and Disease. Cell 155,
934–947. https://doi.org/10.1016/j.cell.2013.
09.053.
3. Heinz, S., Romanoski, C.E., Benner, C., and
Glass, C.K. (2015). The selection and function
of cell type-specific enhancers. Nat. Rev. Mol.
Cell Biol. 16, 144–154. https://doi.org/10.
1038/nrm3949.
4. Spitz, F., and Furlong, E.E.M. (2012).
Transcription factors: from enhancer binding
to developmental control. Nat. Rev. Genet.
13, 613–626. https://doi.org/10.1038/
nrg3207.
5. Davis, R.L., Weintraub, H., and Lassar, A.B.
(1987). Expression of a single transfected
cDNA converts fibroblasts to myoblasts. Cell
51, 987–1000. https://doi.org/10.1016/00928674(87)90585-X.
6. Kim, H.S., Tan, Y., Ma, W., Merkurjev, D.,
Destici, E., Ma, Q., Suter, T., Ohgi, K.,
Friedman, M., Skowronska-Krawczyk, D., and
Rosenfeld, M.G. (2018). Pluripotency factors
functionally premark cell-type-restricted
enhancers in ES cells. Nature 556, 510–514.
https://doi.org/10.1038/s41586-018-0048-8.
7. Medvinsky, A., and Dzierzak, E. (1996).
Definitive Hematopoiesis Is Autonomously
Initiated by the AGM Region. Cell 86,
897–906. https://doi.org/10.1016/S00928674(00)80165-8.
8. de Bruijn, M.F., Speck, N.A., Peeters, M.C.,
and Dzierzak, E. (2000). Definitive
hematopoietic stem cells first develop within
the major arterial regions of the mouse
embryo. EMBO J. 19, 2465–2474. https://doi.
org/10.1093/emboj/19.11.2465.
9. Clements, W.K., and Traver, D. (2013).
Signalling pathways that control vertebrate
haematopoietic stem cell specification. Nat.
Rev. Immunol. 13, 336–348. https://doi.org/
10.1038/nri3443.
10. Nakagawa, M., Ichikawa, M., Kumano, K.,
Goyama, S., Kawazu, M., Asai, T., Ogawa, S.,
Kurokawa, M., and Chiba, S. (2006). AML1/
Runx1 rescues Notch1-null mutation-induced
deficiency of para-aortic splanchnopleural
hematopoiesis. Blood 108, 3329–3334.
https://doi.org/10.1182/blood-2006-04019570.
11. Robert-Moreno, A., Guiu, J., Ruiz-Herguido,
C., Lo´pez, M.E., Ingle´s-Esteve, J., Riera, L.,
Tipping, A., Enver, T., Dzierzak, E., Gridley, T.,
et al. (2008). Impaired embryonic
haematopoiesis yet normal arterial
development in the absence of the Notch
ligand Jagged1. EMBO J. 27, 1886–1895.
https://doi.org/10.1038/emboj.2008.113.
12. Burns, C.E., Galloway, J.L., Smith, A.C.H.,
Keefe, M.D., Cashman, T.J., Paik, E.J.,
Mayhall, E.A., Amsterdam, A.H., and Zon, L.I.
(2009). A genetic screen in zebrafish defines a
hierarchical network of pathways required for
hematopoietic stem cell emergence. Blood
113, 5776–5782. https://doi.org/10.1182/
blood-2008-12-193607.
13. Monteiro, R., Pinheiro, P., Joseph, N.,
Peterkin, T., Koth, J., Repapi, E., Bonkhofer,
F., Kirmizitas, A., and Patient, R. (2016).
Transforming Growth Factor b Drives
Hemogenic Endothelium Programming and
the Transition to Hematopoietic Stem Cells.
Dev. Cell 38, 358–370. https://doi.org/10.
1016/j.devcel.2016.06.024.
14. Jung, H.S., Uenishi, G., Park, M.A., Liu, P.,
Suknuntha, K., Raymond, M., Choi, Y.J.,
Thomson, J.A., Ong, I.M., and Slukvin, I.I.
(2021). SOX17 integrates HOXA and arterial
programs in hemogenic endothelium to drive
definitive lympho-myeloid hematopoiesis.
Cell Rep. 34, 108758. https://doi.org/10.
1016/j.celrep.2021.108758.
15. Zeng, Y., He, J., Bai, Z., Li, Z., Gong, Y., Liu, C.,
Ni, Y., Du, J., Ma, C., Bian, L., et al. (2019).
Tracing the first hematopoietic stem cell
generation in human embryo by single-cell
RNA sequencing. Cell Res. 29, 881–894.
https://doi.org/10.1038/s41422-019-0228-6.
16. Niwa, A., Heike, T., Umeda, K., Oshima, K.,
Kato, I., Sakai, H., Suemori, H., Nakahata, T.,
and Saito, M.K. (2011). A novel serum-free
monolayer culture for orderly hematopoietic
differentiation of human pluripotent cells via
mesodermal progenitors. PLoS One 6,
e22261. https://doi.org/10.1371/journal.
pone.0022261.
17. Ohta, R., Sugimura, R., Niwa, A., and Saito,
M.K. (2019). Hemogenic Endothelium
Differentiation from Human Pluripotent Stem
Cells in A Feeder- and Xeno-free Defined
Condition. J. Vis. Exp. https://doi.org/10.
3791/59823.
18. Choi, K.-D., Vodyanik, M.A., Togarrati, P.P.,
Suknuntha, K., Kumar, A., Samarjeet, F.,
Probasco, M.D., Tian, S., Stewart, R.,
Thomson, J.A., and Slukvin, I.I. (2012).
Identification of the hemogenic endothelial
progenitor and its direct precursor in human
pluripotent stem cell differentiation cultures.
Cell Rep. 2, 553–567. https://doi.org/10.1016/
j.celrep.2012.08.002.
19. Ditadi, A., Sturgeon, C.M., Tober, J., Awong,
G., Kennedy, M., Yzaguirre, A.D., Azzola, L.,
Ng, E.S., Stanley, E.G., French, D.L., et al.
(2015). HUMAN DEFINITIVE HAEMOGENIC
ENDOTHELIUM AND ARTERIAL VASCULAR
ENDOTHELIUM REPRESENT DISTINCT
LINEAGES. Nat. Cell Biol. 17, 580–591.
https://doi.org/10.1038/ncb3161.
20. Uenishi, G.I., Jung, H.S., Kumar, A., Park,
M.A., Hadland, B.K., McLeod, E., Raymond,
M., Moskvin, O., Zimmerman, C.E., Theisen,
D.J., et al. (2018). NOTCH signaling specifies
21.
22.
23.
24.
25.
26.
27.
28.
29.
arterial-type definitive hemogenic
endothelium from human pluripotent stem
cells. Nat. Commun. 9, 1828. https://doi.org/
10.1038/s41467-018-04134-7.
Nottingham, W.T., Jarratt, A., Burgess, M.,
Speck, C.L., Cheng, J.-F., Prabhakar, S.,
Rubin, E.M., Li, P.-S., Sloane-Stanley, J.,
Kong-a-San, J., and de Bruijn, M.F.T.R. (2007).
Runx1-mediated hematopoietic stem-cell
emergence is controlled by a Gata/Ets/SCLregulated enhancer. Blood 110, 4188–4197.
https://doi.org/10.1182/blood-2007-07100883.
Xiang, P., Wei, W., Lo, C., Rosten, P., Hou, J.,
Hoodless, P.A., Bilenky, M., Bonifer, C.,
Cockerill, P.N., Kirkpatrick, A., et al. (2014).
Delineating MEIS1 cis-regulatory elements
active in hematopoietic cells. Leukemia 28,
433–436. https://doi.org/10.1038/leu.
2013.287.
Shlyueva, D., Stampfel, G., and Stark, A.
(2014). Transcriptional enhancers: from
properties to genome-wide predictions. Nat.
Rev. Genet. 15, 272–286. https://doi.org/10.
1038/nrg3682.
Verschueren, K., Remacle, J.E., Collart, C.,
Kraft, H., Baker, B.S., Tylzanowski, P., Nelles,
L., Wuytens, G., Su, M.-T., Bodmer, R., et al.
(1999). SIP1, a Novel Zinc Finger/
Homeodomain Repressor, Interacts with
Smad Proteins and Binds to 50 -CACCT
Sequences in Candidate Target Genes.
J. Biol. Chem. 274, 20489–20498. https://doi.
org/10.1074/jbc.274.29.20489.
Challen, G.A., and Goodell, M.A. (2010).
Runx1 isoforms show differential expression
patterns during hematopoietic development
but have similar functional effects in adult
hematopoietic stem cells. Exp. Hematol. 38,
403–416. https://doi.org/10.1016/j.exphem.
2010.02.011.
Wang, H., Liu, C., Liu, X., Wang, M., Wu, D.,
Gao, J., Su, P., Nakahata, T., Zhou, W., Xu, Y.,
et al. (2018). MEIS1 Regulates Hemogenic
Endothelial Generation, Megakaryopoiesis,
and Thrombopoiesis in Human Pluripotent
Stem Cells by Targeting TAL1 and FLI1. Stem
Cell Rep. 10, 447–460. https://doi.org/10.
1016/j.stemcr.2017.12.017.
Arner, E., Daub, C.O., Vitting-Seerup, K.,
Andersson, R., Lilje, B., Drabløs, F.,
Lennartsson, A., Ro¨nnerblad, M.,
Hrydziuszko, O., Vitezic, M., et al. (2015).
Transcribed enhancers lead waves of
coordinated transcription in transitioning
mammalian cells. Science 347, 1010–1014.
https://doi.org/10.1126/science.1259418.
Kim, Y.W., Lee, S., Yun, J., and Kim, A. (2015).
Chromatin looping and eRNA transcription
precede the transcriptional activation of gene
in the b-globin locus. Biosci. Rep. 35, e00179.
https://doi.org/10.1042/BSR20140126.
Menendez, P., Navarro-Montero, O., RamosMejia, V., Ayllo´n, V., Stanley, E., Elefanty, A.,
and Real, P. (2016). RUNX1C regulates
iScience 26, 107893, October 20, 2023
15
iScience
ll
Article
OPEN ACCESS
30.
31.
32.
33.
34.
35.
36.
16
hematopoietic specification of human
embryonic stem cells. Exp. Hematol. 44, S89.
https://doi.org/10.1016/j.exphem.2016.
06.186.
Navarro-Montero, O., Ayllon, V., Lamolda,
M., Lo´pez-Onieva, L., Montes, R., Bueno, C.,
Ng, E., Guerrero-Carreno, X., Romero, T.,
Romero-Moya, D., et al. (2017). RUNX1c
Regulates Hematopoietic Differentiation of
Human Pluripotent Stem Cells Possibly in
Cooperation with Proinflammatory Signaling.
Stem Cell. 35, 2253–2266. https://doi.org/10.
1002/stem.2700.
Lichtinger, M., Ingram, R., Hannah, R., Mu¨ller,
D., Clarke, D., Assi, S.A., Lie-A-Ling, M.,
Noailles, L., Vijayabaskar, M.S., Wu, M., et al.
(2012). RUNX1 reshapes the epigenetic
landscape at the onset of haematopoiesis.
EMBO J. 31, 4318–4333. https://doi.org/10.
1038/emboj.2012.275.
Park, M.A., Kumar, A., Jung, H.S., Uenishi, G.,
Moskvin, O.V., Thomson, J.A., and Slukvin, I.I.
(2018). Activation of Arterial Program Drives
Development of Definitive Hemogenic
Endothelium with Lymphoid Potential. Cell
Rep. 23, 2467–2481. https://doi.org/10.1016/
j.celrep.2018.04.092.
Goossens, S., Janzen, V., Bartunkova, S.,
Yokomizo, T., Drogat, B., Crisan, M., Haigh,
K., Seuntjens, E., Umans, L., Riedt, T., et al.
(2011). The EMT regulator Zeb2/Sip1 is
essential for murine embryonic
hematopoietic stem/progenitor cell
differentiation and mobilization. Blood 117,
5620–5630. https://doi.org/10.1182/blood2010-08-300236.
Wang, L., Menendez, P., Shojaei, F., Li, L.,
Mazurier, F., Dick, J.E., Cerdan, C., Levac, K.,
and Bhatia, M. (2005). Generation of
hematopoietic repopulating cells from
human embryonic stem cells independent of
ectopic HOXB4 expression. J. Exp. Med. 201,
1603–1614. https://doi.org/10.1084/jem.
20041888.
Azcoitia, V., Aracil, M., Martı´nez-A, C., and
Torres, M. (2005). The homeodomain protein
Meis1 is essential for definitive
hematopoiesis and vascular patterning in the
mouse embryo. Dev. Biol. 280, 307–320.
https://doi.org/10.1016/j.ydbio.2005.01.004.
Cvejic, A., Serbanovic-Canic, J., Stemple,
D.L., and Ouwehand, W.H. (2011). The role of
meis1 in primitive and definitive
iScience 26, 107893, October 20, 2023
37.
38.
39.
40.
41.
42.
43.
44.
hematopoiesis during zebrafish
development. Haematologica 96, 190–198.
https://doi.org/10.3324/haematol.2010.
027698.
Doss, M.X., and Sachinidis, A. (2019). Current
Challenges of iPSC-Based Disease Modeling
and Therapeutic Implications. Cells 8, 403.
https://doi.org/10.3390/cells8050403.
Chen, B., Teng, J., Liu, H., Pan, X., Zhou, Y.,
Huang, S., Lai, M., Bian, G., Mao, B., Sun, W.,
et al. (2017). Inducible overexpression of
RUNX1b/c in human embryonic stem cells
blocks early hematopoiesis from mesoderm.
J. Mol. Cell Biol. 9, 262–273. https://doi.org/
10.1093/jmcb/mjx032.
Riddell, J., Gazit, R., Garrison, B.S., Guo, G.,
Saadatpour, A., Mandal, P.K., Ebina, W.,
Volchkov, P., Yuan, G.-C., Orkin, S.H., and
Rossi, D.J. (2014). Reprogramming
committed murine blood cells to induced
hematopoietic stem cells with defined
factors. Cell 157, 549–564. https://doi.org/10.
1016/j.cell.2014.04.006.
Yanagimachi, M.D., Niwa, A., Tanaka, T.,
Honda-Ozaki, F., Nishimoto, S., Murata, Y.,
Yasumi, T., Ito, J., Tomida, S., Oshima, K.,
et al. (2013). Robust and highly-efficient
differentiation of functional monocytic cells
from human pluripotent stem cells under
serum- and feeder cell-free conditions. PLoS
One 8, e59243. https://doi.org/10.1371/
journal.pone.0059243.
Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto,
R., Habib, N., Hsu, P.D., Wu, X., Jiang, W.,
Marraffini, L.A., and Zhang, F. (2013).
Multiplex Genome Engineering Using
CRISPR/Cas Systems. Science 339, 819–823.
https://doi.org/10.1126/science.1231143.
Martin, M. (2011). Cutadapt removes adapter
sequences from high-throughput sequencing
reads. EMBnet. J. 17, 10–12. https://doi.org/
10.14806/ej.17.1.200.
Kim, D., Langmead, B., and Salzberg, S.L.
(2015). HISAT: a fast spliced aligner with low
memory requirements. Nat. Methods 12,
357–360. https://doi.org/10.1038/
nmeth.3317.
Liao, Y., Smyth, G.K., and Shi, W. (2014).
featureCounts: an efficient general purpose
program for assigning sequence reads to
genomic features. Bioinformatics 30,
923–930. https://doi.org/10.1093/
bioinformatics/btt656.
45. Vera Alvarez, R., Pongor, L.S., Marin˜oRamı´rez, L., and Landsman, D. (2019).
TPMCalculator: one-step software to quantify
mRNA abundance of genomic features.
Bioinformatics 35, 1960–1962. https://doi.
org/10.1093/bioinformatics/bty896.
46. Gu, Z., Eils, R., and Schlesner, M. (2016).
Complex heatmaps reveal patterns and
correlations in multidimensional genomic
data. Bioinformatics 32, 2847–2849. https://
doi.org/10.1093/bioinformatics/btw313.
47. Langmead, B., and Salzberg, S.L. (2012). Fast
gapped-read alignment with Bowtie 2. Nat.
Methods 9, 357–359. https://doi.org/10.
1038/nmeth.1923.
48. Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J.,
Johnson, D.S., Bernstein, B.E., Nusbaum, C.,
Myers, R.M., Brown, M., Li, W., and Liu, X.S.
(2008). Model-based Analysis of ChIP-Seq
(MACS). Genome Biol. 9, R137. https://doi.
org/10.1186/gb-2008-9-9-r137.
49. Heinz, S., Benner, C., Spann, N., Bertolino, E.,
Lin, Y.C., Laslo, P., Cheng, J.X., Murre, C.,
Singh, H., and Glass, C.K. (2010). Simple
combinations of lineage-determining
transcription factors prime cis-regulatory
elements required for macrophage and B cell
identities. Mol. Cell 38, 576–589. https://doi.
org/10.1016/j.molcel.2010.05.004.
50. Yu, G., Wang, L.-G., Han, Y., and He, Q.-Y.
(2012). clusterProfiler: an R Package for
Comparing Biological Themes Among Gene
Clusters. OMICS A J. Integr. Biol. 16,
284–287. https://doi.org/10.1089/omi.
2011.0118.
51. Guo, J., Ma, D., Huang, R., Ming, J., Ye, M.,
Kee, K., Xie, Z., and Na, J. (2017). An inducible
CRISPR-ON system for controllable gene
activation in human pluripotent stem cells.
Protein Cell 8, 379–393. https://doi.org/10.
1007/s13238-016-0360-8.
52. Butler, A., Hoffman, P., Smibert, P., Papalexi,
E., and Satija, R. (2018). Integrating single-cell
transcriptomic data across different
conditions, technologies, and species. Nat.
Biotechnol. 36, 411–420. https://doi.org/10.
1038/nbt.4096.
53. Binns, D., Dimmer, E., Huntley, R., Barrell, D.,
O’Donovan, C., and Apweiler, R. (2009).
QuickGO: a web-based tool for Gene
Ontology searching. Bioinformatics 25, 3045–
3046. https://doi.org/10.1093/
bioinformatics/btp536.
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
SOURCE
IDENTIFIER
Antibodies
Anti-H3K27ac
GeneTex
GTX60815; RRID: AB_2888004
Anti-H3K4me1
ActiveMotif
39297; RRID: AB_2615075
Anti-H3K27me3
Millipore
07-449; RRID: AB_310624
Anti-SIP1 (ZEB2)
Bethyl Laboratories
A302474A; RRID: AB_1944271
Anti-MEIS1
Abcam
ab19867; RRID: AB_776272
Anti-NOTCH1
Cell Signaling Technology
3608S; RRID: AB_2153354
BV421 anti-CD34
BD Biosicneces
744904; RRID: AB_2742572
APC anti-CD309
BIOLEGEND
359916; RRID: AB_2565928
PECy7 anti-CD45
BIOLEGEND
304016; RRID: AB_314404
FITC anti-CD73
BIOLEGEND
344015; RRID: AB_2561808
PE anti-DLL4
Miltenyi Biotec
130-096-567; RRID: AB_10831209
APC anti-CD184 (CXCR4)
BD Biosicneces
555976; RRID: AB_398616
Anti-b-Actin
Cell Signaling Technology
5125S; RRID: AB_1903890
Anti-rabbit-HRP
Cell Signaling Technology
7074S; RRID: AB_2099233
BMP-8
R&D
314-BP-010
VEGF
R&D
293-VE-010
Chemicals, peptides, and recombinant proteins
CHIR99021
Wako
038-23101
SCF
R&D
255-SC/CF
bFGF
Wako
064-05381
SB431542
Wako
031-24291
TPO
R&D
288-TPN
FLT3L
R&D
308-GMP
FP6
R&D
8954-SR
iMatrix-511
Matrixome
892 012
iMatrix-511 silk
Matrixome
892 021
RetroNectin
TAKARA
T100B
mTeSR1
STEMCELL Technologies
ST-85850
Essential 8
Thermo Fisher Scientific
A1517001
Essential 6
Thermo Fisher Scientific
A1516401
StemLine II
Sigma-Aldrich
S0192
TrypLE Select
Gibco
A1217702
Puromycin
InvivoGen
ant-pr-1
G418
Nacalai Tesque
09380-44
Critical commercial assays
SMART-seq v4 Ultra Low Input RNA Kit for Sequencing
Takara
Z4891N
SMARTer ThruPLEX DNA-seq 48S Kit
Takara
RB4427
NextSeq 500/550 High Output Kit v2.5 (75 Cycles)
Illumina
20024906
CD34 MicroBead Kit, human
Miltenyi Biotec
130-046-702
ChIP DNA Clean & Concentrator
Zymo Research
D5205
(Continued on next page)
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OPEN ACCESS
Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
This study
PRJNA783033
This study
N/A
pX330-U6-Chimeric_BB-CBh-hSpCas9
Cong et al.41
Addgene #42230
AAVS1-idCas9-vpr
Guo et al.51
Addgene #89985
Cutadapt (v1.15)
EMBnet.Journal42
https://cutadapt.readthedocs.io/en/stable/index.html
Bowtie2 (v2.2.5)
Langmead and Salzberg47
http://bowtie-bio.sourceforge.net/bowtie2/index.shtml
Hisat2 (v2.1.0)
Kim et al.43
http://daehwankimlab.github.io/hisat2/
BEDTools (v.2.27.1)
Bedtools
https://github.com/arq5x/bedtools2
MACS2 (v2.1.1.20160309)
Zhang et al.48
https://github.com/taoliu/MACS
samtools (v1.7)
Samtools
http://www.htslib.org/
featureCounts (v1.6.0)
Liao et al.44
http://subread.sourceforge.net/
R (v3.6.1)
The R Project
https://www.r-project.org/
R: DESeq2 (v1.24.0)
Bioconductor
http://bioconductor.org/packages/release/bioc/html/
R: ClusterProfiler (v3.12.0)
Bioconductor
R: ComplexHeatmap (v2.0.0)
Bioconductor
Deposited data
RNA-seq and ChIP-seq data
Oligonucleotides
gRNA for CRISPR, see method details for
oligonucleotide sequences
Recombinant DNA
Software and algorithms
DESeq2.html
https://bioconductor.org/packages/release/bioc/html/
clusterProfiler.html
https://bioconductor.riken.jp/packages/3.2/bioc/html/
ComplexHeatmap.html
R: ggplot2 (v3.2.1)
CRAN
https://cran.r-project.org/web/packages/ggplot2/index.html
GenomeJack (v3.1)
Mitsubishi Space Software
http://genomejack.net/japanese/index.html
Homer
Heinz et al.49
http://homer.ucsd.edu/homer/
TPMCalculator
Vera Alvarez et al.45
https://github.com/ncbi/TPMCalculator
GraphPad Prism 7
GraphPad Software
https://www.graphpad.com/scientific-software/prism/
FlowJo
BD Biosicneces
https://www.flowjo.com/
CountBright Absolute Counting Beads
Thermo Fisher Scientific
C36950
Dynabeads M-280 Sheep anti-Rabbit IgG
Thermo Fisher Scientific
11203D
Other
RESOURCE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Megumu K. Saito
(msaito@cira.kyoto-u.ac.jp).
Materials availability
All requests for resources and reagents should be directed to the lead contact author. All reagents will be made available on request after
completion of a Materials Transfer Agreement.
Data and code availability
RNA-seq and ChIP-seq data have been deposited at NCBI SRA and are publicly available as of the date of publication. Accession
numbers are listed in the key resources table.
18
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All original code is available in this paper’s supplemental information (Methods S1).
Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell lines
The human ESC line KhES-1 and iPSC line 201B2 were kindly provided by Norio Nakatsuji (Kyoto University, Kyoto, Japan) and Shinya Yamanaka (Kyoto University, Kyoto, Japan), respectively. Both lines were generated from female donors. All cells were regularly karyotyped and
tested for the presence of mycoplasma. The use of human embryonic stem cells (ESCs) in Kyoto University was approved by the Ministry
of Education Culture, Sports, Science and Technology of Japan (MEXT). The study plan for recombinant DNA research was approved by
the recombinant DNA experiments safety committee of Kyoto University.
METHOD DETAILS
Monolayer hematopoietic cell differentiation via HE
PSCs spheroids were formed on an EZSPHERE SP microplate (IWAKI) to tightly control colony numbers and speed up the time required to
obtain colonies of sufficient size. They were transferred to iMatrix-511-coated plates the next day and maintained in mTeSR1 (STEMCELL
Technologies) for three more days. When undifferentiated colonies reached 750–1000 mm in diameter (day 0), hematopoietic cell differentiation was initiated based on previously described protocols.16,17,40 Briefly, the culture media was replaced with Essential-8 medium
(#A1517001, Thermo Fisher Scientific) containing 80 ng/mL BMP4 (#314-BP-010, R&D), 80 ng/mL VEGF 165 (#293-VE-010, R&D), and 4 mM
GSK-3 inhibitor CHIR99021 (#038-23101, Wako). On day 2, the media was changed to Essential-6 medium (#A1516401, Thermo Fisher Scientific) with 80 ng/mL VEGF, 25 ng/mL bFGF (#064-05381, Wako), 2 mM ALK5 inhibitor SB431542 (#031-24291, Wako), and 50 ng/mL SCF (#255SC/CF, R&D). On day 4, CD34+ cells were isolated using a human CD34 Microbead Kit (Miltenyi Biotec) and re-plated on Retronectin-coated
plates at a density of 2.5 x 104 cells/cm2 in hematopoietic cell differentiation medium, which contains Stemline-II medium (#S0192, SigmaAldrich), 50 ng/mL SCF, 50 ng/mL Flt-3 Ligand (#308-GMP, R&D), 5 ng/mL TPO (#288-TPN, R&D) and 20 ng/mL FP6 (#8954-SR, R&D). The
media was thereafter changed every 3 days. 10 mM DAPT or 2 mM SB431542 were added on day 4 and/or 7 for the inhibition of Notch
and TGF-b signaling, respectively, in Figures 3 and S3.
Flow cytometric analysis and cell sorting
For the isolation of cells from day 0 and 2, adherent cells were treated with 50% TrypLE Select (Gibco) in PBS supplemented with 0.5 mM EDTA
(Gibco) and harvested by pipetting. Viability of cells was ensured to be > 90% for the RNA-seq and ChIP-seq experiments. On days 4 and 7, the
supernatant was removed, and adherent cells were collected with 50% TrypLE Select. After staining with DAPI and antibodies, CD34+KDR+
cells were sorted or analyzed using BD FACSAriaII (BD Biosciences). To analyze both CD34+CD45– and CD34+CD45+ cells on day 10, the supernatant and adherent cells were collected. Dead cells were removed by DAPI staining, and the gating was set so that the purity of the sorted
cells was over 90%. The following antibodies were used: BV421 anti-CD34 (744904, BD Biosciences), APC anti-CD309 (3559916, Biolegend),
PECy7 anti-CD45 (304016, Biolegend), FITC anti-CD73 (344015, Biolegend), PE anti-DLL4 (130-096-567, Miltenyi Biotec), and APC anti-CD184
(555976, BD Biosciences). Flow cytometric data were analyzed using FlowJo (version 10.6.1, BD Biosciences).
Generation of ZEB2-deficient and MEIS1-deficient PSCs
A pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid41 (Addgene #42230) was modified to replace Cas9 with Cas9-T2A-puromycin. This plasmid
was digested by BbsI, and the following oligonucleotides were annealed and inserted.
ZEB2: (forward) CACCGATCCAGACCGCAATTAACAA
(reverse) AAACTTGTTAATTGCGGTCTGGATC
MEIS1: (forward) CACCG TACTTGTACCCCCCGCGAGC
(reverse) AAACGCTCGCGGGGGGTACAAGTAC
The plasmids were transfected into human ESCs or human iPSCs using a NEPA21 electroporator (NEPAGENE) and cuvettes of 2-mm gap.
The transfected cells were transiently selected by adding 1 mg/mL puromycin from 24 to 48 hours after the transfection. Single colonies were
harvested for genotyping by Sanger sequencing. At least two knockout colonies were expanded for further analysis. To generate ZEB2/MEIS1
double-deficient ESCs, ZEB2-deficient ESCs were transfected with the pX330-U6-Chimeric_BB-CBh-hSpCas9 plasmid containing MEIS1-targeting gRNA.
Loss of the target protein was confirmed by western blotting. Briefly, CD34+ cells on day 4 were isolated using the human CD34 Microbead
Kit (Miltenyi Biotec) and lysed with RIPA buffer (Wako) for 30 minutes on ice. After removing cell debris by centrifugation, the lysate was mixed
with 23 Laemmli Sample Buffer (Bio-Rad Laboratories) containing 5% total volume 2-mercaptoethanol (Nacalai tesque) and boiled for 5 minutes at 95 C. Polyacrylamide gel electrophoresis was performed on SDS-polyacrylamide gels, and proteins were transferred to a nitrocellulose membrane (Merck Millipore). The membrane was then incubated with 5% BSA in Tris-buffered saline with tween 20 (Santa Cruz Biotechnology, lnc.) for blocking. The primary antibody reaction was performed at 4 C overnight. The secondary antibody incubation was performed
for 90 minutes at room temperature. The target protein was detected using ECL chemiluminescence reagents (Thermo Fisher Scientific). An
iScience 26, 107893, October 20, 2023
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antibody against b-Actin was reacted for 60 minutes at room temperature. The following primary and secondary antibodies were used: antiZEB2 (1:1000, 61095, Active Motif), anti-MEIS1 (1:1000, abcam ab19867), anti-b-Actin (1:5000, #5125S, Cell Signaling Technology), and antirabbit-HRP (1:2500, #7074S, Cell Signaling Technology).
RNA-seq
FACS-sorted cells were lysed in RLT buffer (Qiagen), and RNA was extracted using RNAClean XP beads (Beckman Coulter). Reverse transcription was performed using a SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara). cDNA was then fragmented using a Covaris
Focused-ultrasonicator M220 (M&S Instruments Inc.). The library was constructed using a SMARTer ThruPLEX DNA-seq 48S Kit (Takara)
and sequenced on a NextSeq 500 System (Illumina) with 75-bp single-end reads. No technical replicates were generated.
Reads were trimmed by Cutadapt (version 1.15)42 and mapped to the human genome hg19 by Hisat2 (version 2.1.0).43 Tags were counted
by featureCounts (version 1.6.0)44 and normalized using the DESeq2 package (version 1.24.0) in R. Differentially expressed genes were defined
as those with FDR < 0.01 and log2 fold-change > 2. The expression of RUNX1 splice variants was determined by TPMCalculator (version
0.0.3).45 Heatmaps showing gene expression patterns were generated using the ComplexHeatmap package (version 2.0.0)46 in R. Data
from individual samples were treated independently in all PCA plots and heatmaps.
ChIP-seq
The following number of cells were used for the ChIP-seq: 1 x 105 for H3K27ac, 2 x 105 for H3K4me1 and H3K27me3, and 1-5 x 106 for transcription factor ChIP-seq. The antibodies used were anti-H3K27ac (GeneTex, GTX60815), anti-H3K4me1 (Active Motif, 39297), anti-H3K27me3
(Millipore, 07-449), anti-ZEB2 (Bethyl Laboratories, A302474A), anti-MEIS1 (Abcam, ab19867), anti-Notch1 (Cell Signaling Technology, 3608S),
anti-SOX6 (Abcam, ab30455), anti-SOX17 (R&D Systems, AF1924-SP), and anti-SPI1 (Cell Signaling Technology, 2266). Cells were cross-linked
in 1% (w/v) formaldehyde solution for 5 min (histone ChIP-seq) or 30 min (transcription factor ChIP-seq) and lysed. Cross-linked DNA was fragmented by sonication using a Digital Sonifier (Branson) and incubated overnight at 4 C with 50 mL DynaBeads IgG magnetic beads (Thermo
Fisher) conjugated with 2.5 mg antibodies in the presence of 1% FBS and 10-30 mg salmon sperm DNA. Samples were then washed, eluted,
reverse cross-linked at 65 C overnight, and purified using ChIP DNA Clean & Concentrator (Zymo Research). For transcription factor ChIPseq, purified ChIP DNA was fragmented using a Covaris Focused-ultrasonicator M220 (M&S Instruments Inc.). The library was prepared using
a SMARTer ThruPLEX DNA-seq 48S Kit (Takara) and sequenced on a NextSeq 500 System (Illumina) with 75-bp single-end reads.
ChIP-seq reads were trimmed by Cutadapt and mapped by bowtie247 to hg19 after removing the reads mapped to salmon. For visualization, ChIP peaks were called and normalized by the number of mapped reads (-SPMR option) using MACS248 with input reads as a control
and are presented in GenomeJACK Browser (version 3.1, Mitsubishi Space Software). The y-axis of a ChIP-seq track indicates the count per
million (CPM). To identify ChIP peaks, the findPeaks program in the Homer package49 was used. For H3K27ac ChIP-seq, the program was
used with -region option and 20-fold enrichment over input as a cutoff. For transcription factor ChIP-seq, both the peak size and minimum
distance of peaks were set to 500 bp, the local fold change cutoff was disabled, and the default settings were applied for all other parameters.
ChIP-seq tag counts at the target regions were determined by featureCounts (version 1.6.0) and normalized by the number of uniquely mapped reads (tag counts per region O uniquely mapped reads 3 1,000,000). Heatmaps showing relative signal intensities were generated using
the ComplexHeatmap package (version 2.0.0) in R.
Definition of hematopoietic enhancers
First, regions with H3K27ac marks in day 10 CD34+CD45+ cells were identified by the findPeaks program in the Homer package. Regions overlapping promoter regions (defined as up to 2.5 kb upstream of the transcription start site indicated in NCBI RefSeq) were removed. Regions
with H3K27ac marks were similarly identified in day 0 and day 2 cells and H3K27ac-marked regions in day 10 CD34+CD45+ cells which overlap
with H3K27ac-marked regions in day 0 and day 2 cells were also removed to define hematopoietic enhancers.
Gene ontology analysis
A gene ontology analysis of defined gene sets was conducted using ClusterProfiler (version 3.12.0)50 in R. All gene sets in the Gene Ontology
database were used, except for the data in Figure 1B, where the gene sets whose name contain ‘development’ or ‘differentiation’ were
selected.
Motif analysis
A motif analysis was performed using the findMotifsGenome program in the Homer package with default settings. For the background
sequence, proximal enhancers [ENCFF036NSJ] and distal enhancers [ENCFF535MKS] from candidate cis-Regulatory Elements predicted
in the ENCODE project (https://www.encodeproject.org/) were combined, and the genome coordinates were converted from hg38 to
hg19 using UCSC LiftOver. Results with q-value < 0.0001, log P-value < -30 and the ratio of % target sequences with motifs to % background
sequences with motifs > 1.5 in at least one cluster of hematopoietic enhancers were selected for the data presentation.
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