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Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros

Abstract

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34+ cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34+ cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34+ hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17+ vessels from which RUNX1C+ blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34+ hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

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Figure 1: Characterization of RUNX1CGFP/w hESCs.
Figure 2: Differential HOXA gene expression between hESC-derived hematopoietic cells and cord blood modulated by patterning with SB/CHIR.
Figure 3: Transcriptional profiling reveals differential expression of HOXA and endothelial identity genes in SB/CHIR cultures.
Figure 4: Gene expression and hemogenic function of SB/CHIR endothelial cells.
Figure 5: Generation of fetal stage erythroid cells from HOXA+ cultures and transition from SOX17+ endothelial cells to RUNX1C+ blood cells.
Figure 6: Transcriptional similarity between d18 SOX/RUNX-expressing cells and human AGM.

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Acknowledgements

The assistance and cooperation of the Mercy Hospital for Women in the collection and provision of umbilical cord blood samples, and the expertise of M. Burton and P. Lau in the MCRI Flowcore facility, as well as FlowCore at Monash University, are gratefully acknowledged. The assistance of D. Cardozo with animal work is also gratefully acknowledged. This work was supported by grants from the Australian Stem Cell Centre, Stem Cells Australia, the Juvenile Diabetes Research Foundation, the National Health and Medical Research Council (NHMRC) of Australia, the California Institute for Regenerative Medicine (RT3-07763), the Stafford Fox Medical Research Foundation and the Victorian Government's Operational Infrastructure Support Program and Australian Government National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme (NHMRC IRIISS). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. A.G.E. and E.G.S. are supported as Senior Research Fellows of the NHMRC. Work in the laboratory of K.S.-L. was supported by grants from the Ministry of Science, Research and the Arts of Baden-Wuerttemberg (Az: 33-729.55-3/214-3, 33-729.55-3/214-9). Work in the laboratory of H.K.A.M. was also supported by the National Institutes of Health (1R01DK100959-01A1).

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Authors

Contributions

E.S.N. contributed to designing and performing experiments, analyzing data, and writing and editing the manuscript; L.A. contributed to designing and performing experiments concerning generation and differentiation of reporter hESCs, analyzing data, and editing the manuscript; F.F.B. contributed to designing and performing experiments concerning differentiation of hESCs, analyzing data, and editing the manuscript; V.C. contributed to designing and performing experiments concerning profiling of differentiated hESCs and analyzing data; B.P. contributed to designing experiments concerning profiling of differentiated hESCs, analyzing data, and editing the manuscript; K.V. contributed to designing and performing experiments concerning generation and differentiation of hESCs and analyzing data; C.H. contributed to designing experiments concerning profiling of differentiated hESCs and analyzing data; V.J.J. contributed to designing and performing experiments concerning generation and differentiation of hESCs and analyzing data; Q.C.Y. contributed to designing experiments concerning profiling of differentiated hESCs and analyzing data; J.M. contributed to designing experiments concerning profiling of differentiated hESCs, analyzing data, and editing the manuscript; S.L. contributed to designing and performing experiments concerning human tissues; V.J. contributed to designing experiments concerning profiling of differentiated hESCs and analyzing data; Z.Z. contributed to performing experiments concerning bone marrow homing of differentiated hESCs; B.W. contributed to performing experiments concerning bone marrow homing of differentiated hESCs; A.C. contributed to designing and performing experiments concerning generation of reporter hESCs; J.D. contributed to designing and performing experiments concerning differentiation of reporter hESCs; S.J. contributed to designing and performing experiments concerning generation of reporter hESCs; M.C. contributed to analyzing experiments concerning differentiation of reporter hESCs; D.E. contributed to designing and performing experiments, analyzing data, and editing the manuscript; D.N.H. contributed to experiments concerning comparison of umbilical cord blood with differentiated hESCs, analyzing data and editing the manuscript; S.K.N. contributed to designing and performing experiments concerning bone marrow homing, analyzing data and editing the manuscript; R.S. contributed to designing experiments concerning profiling of differentiated hESCs and analyzing data; K.S.-L. contributed to designing and performing experiments concerning human tissues, and editing the manuscript; A.O. contributed to designing experiments concerning profiling of differentiated hESCs, analyzing data, and editing the manuscript; H.K.A.M. contributed to designing and performing experiments, analyzing data, and editing the manuscript; E.G.S. contributed to designing experiments, analyzing data, and writing and editing the manuscript; A.G.E. contributed to designing and performing experiments, analyzing data, and writing and editing the manuscript.

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Integrated supplementary information

Supplementary Figure 1 Generation of RUNX1C and SOX17 reporter lines

(a) Schematic representation of the RUNX1 locus, the targeting vector and the targeted allele with GFP sequences inserted into exon 1(e1). The distal (D) and proximal (P) promoters are shown. Exons (e) are numbered with non-coding exons boxed in white, coding exons in black and exons encoding the runt homology domain in red. e7A and e7B are alternately spliced exons that give rise to RUNX1A and RUNX1B transcripts. RES indicates a cassette encoding either G418 (Neo) or hygromycin resistance flanked by loxP sites (black triangles). The positions of primers used to identify homologously integrated alleles are shown (p1-p7), with the sizes of PCR products indicated. p3 identifies the hygromycin resistance gene whilst p7 is specific for the G418 resistance gene (b) A representative example of the PCR screen to detect homologous integration of the left and right homology arms with the hygromycin cassette containing vector. '+' refers to a clone where the targeting construct was homologously integrated into the RUNX1 locus, and '–' indicates a randomly integrated clone. The combinations of p1/p2 and p3/p4 identify correctly integrated left and right homology arms of the vector. p1/p5 identifies sequences of a wild type allele and p3/p6 identifies vector sequences present even in random integrations. Sequences for each primer are indicated in Supplementary Table 9. (c) Homologous integration into the RUNX1 locus was further verified by cloning and sequencing the integration sites. An example using primers p1/p2 and p3/p4 is shown. Junction sequences from the genomic locus are shown in lower case. (d) RUNX1cGFP/w EBs harvested on day 13, 14 or 17 were flow-sorted on the basis of GFP and CD34 expression. cDNA synthesized from the sorted samples was analysed by RT-PCR using primers that detected both RUNX1B and RUNX1C (RUNX1B/C) or RUNX1C alone. Note that RUNX1B/C primers revealed expression in GFP-CD34+ cells, consistent with expression of the RUNX1B isoform in a subset of CD34+ endothelial cells. Gene expression is shown relative to GAPDH. UN, unfractionated cells. (e) Schematic outlining the strategy for generation of the RUNX1C GFP/GFP cell line by re-targeting the wild type RUNX1C allele with the hygromycin targeting vector. (f) Schematic outlining the PCR based screening strategy to identify doubly targeted cell lines, prior to excision of the targeting cassette. (g) PCR analysis using primers shown in (e) to identify GFPHygro, GFP and wild type (w) alleles. Clone 180 depicts an example where the GFPHygro targeting vector has replaced the previous GFP allele, leaving one wild type allele, clone 22 represents depicts one previously targeted GFP allele and one wild type allele and clone 253 represents a clone in which GFPHygro has correctly integrated into the wild type allele resulting in both RUNX1C alleles being targeted. (h) PCR analysis of sorted fractions of RUNX1C GFP/w and RUNX1C GFP/GFP differentiated cells confirming the absence of RUNX1C transcripts from the RUNX1C GFP/GFP line without disruption of the RUNX1B transcripts. Cells were sorted at day 14 based on GFP (G) and CD34 expression. UN, unfractionated cells. (i) Schematic representation of the SOX17 locus, the targeting vector and the targeted allele with mCHERRY sequences inserted into exon 1(e1). The loxP flanked Neo selectable marker was excised with CRE recombinase. See also Supplementary experimental methods in Loh et al. for further details64. (j) Intracellular flow cytometry profiles of SOX17mCHERRY/w hESC differentiated for 5d to endoderm as described in Loh et al64, showing specific staining of mCHERRY+ cells with FITC-conjugated SOX17 antibody.

Supplementary Figure 2 Analysis of cell surface markers expressed on differentiating RUNX1CGFP/w and RUNX1CGFP/GFP cells.

(ad) Expression of CD34, CD43 and CD41 precedes GFP expression in RUNX1CGFP/w EBs. Representative flow cytometry analyses of differentiating RUNX1CGFP/w EBs indicating that expression of CD34 at day 5 (a) followed by CD41 and CD43 at day 7 (b) antedated GFP expression. Correlation of GFP expression with hematopoietic cell surface markers at day 11 (c) and day 16 (d) revealed that all GFP+ cells initially co-expressed CD34, CD31, and most expressed CD41. At day 16, GFP expression remained confined to hematopoietic cells expressing the indicated cell surface markers. Red boxed areas represent CD34+CD31+CD41-CD43-CD33-CD45- endothelial cells and the blue boxes mark GYPA+ erythroid cells, both of which were GFP-. A high proportion of d16 GFP+ cells co-expressed VECAD and CD45, a combination of surface markers found in mouse hematopoietic stem/progenitor cells. (eh) Flow cytometry analysis of differentiation timecourse comparing RUNX1CGFP/w heterozygous cells (clone 67.4) with RUNX1CGFP/GFP nullizygous clone A5 at (e) day 7, (f) day11, (g) day 14 and (h) day 18. The percentage of GFP+ cells and CD34 and CD45 expression patterns were similar for all clones. (i) Strategy for flow cytometry sorting of d14 differentiating RUNXICGFP/w and RUNXICGFP/GFP cultures based on GFP and CD34 expression.

Supplementary Figure 3 Characterization of RUNX1CGFP/w and RUNX1CGFP/GFP hematopoietic cells.

(a) Brightfield (BF) and overlaid fluorescence images of d22 RUNXICGFP/w and RUNXICGFP/GFP cultures. Scale bar, 100μM. (b, c) Distribution of myeloid and erythroid colony forming cells in RUNXICGFP/w and RUNXICGFP/GFP cultures. Mean±SEM of 3 experiments. * p <0.05, repeated measures ANOVA comparing all groups to GFP+CD34lo population, Tukey's Multiple Comparison Test. (d) Distribution of colony types between RUNX1C heterozygous and nullizygous lines. Mean±SEM of 3 experiments. (e) Photomicrographs of erythroid (ERY), granulocytic (GRAN), granulocyte-macrophage (GM) and macrophage (MAC) or mast cell (MAST) colony types. Scale bar, 200μM. (f) Cytocentrifuge preparations of hematopoietic colonies stained with May-Grunwald-Giemsa. Nucleated erythroid (ERY), granulocyte (GRAN), mast cell (MAST), mixed macrophage and granulocyte (GM) and macrophage (MAC) colonies are shown. Scale bar, 20μM. (g) Bone marrow homing of d14 RUNX1CGFP/w and RUNX1CGFP/GFP EBs sorted on GFP (G) and CD34 (34) expression. Mean±SEM for 12 mice from 4 experiments. **, p<0.01, one way ANOVA comparing all groups to GFP-CD34- from RUNX1CGFP/w, Holm-Sidak's multiple comparison test.

Supplementary Figure 4 Comparison of gene expression between RUNX1CGFP/w and RUNX1CGFP/GFP EBs.

(a) Differentially expressed probe sets between the indicated fractions and GFP-CD34- cells for samples including both d14 RUNX1CGFP/w and RUNX1CGFP/GFP EBs. (b) Venn diagram showing overlap between probe sets up-regulated in each cell fraction in (a). (c) Hierarchical cluster analysis of the fractions sorted on the basis of CD34 and GFP expression, as well as undifferentiated hESCs isolated from the heterozygote RUNX1CGFP/w 67.4 line and the homozygous null RUNX1CGFP/GFP A5 line, based on the top 25% of probes with the greatest variance. (d) Scatter plot representations of the logarithmic signal intensities of the pairwise comparisons between the CD34 and GFP fractions as well as undifferentiated hESCs from the heterozygous and homozygous null lines. Numbers in the top left and bottom right corners indicate the number of probes that are outside of the 2-fold cut-off (indicated by red lines). The correlation co-efficient for each differentiated population is similar to the correlation co-efficient for the undifferentiated cells, indicating the similarity of the differentiated progeny. (e) Table indicating the Pearson correlation co-efficient for each of the crosswise comparisons. Note the high correlation co-efficients (r2≥0.96) between the equivalent fractions from heterogygous and homozygous RUNX1C cell lines and between the RUNX1C expressing populations (G+34- and G+34lo; r2≥0.94). (f) Heatmap showing HOX gene expression in sorted d14 RUNX1CGFP/w EBs and three samples of CB 34+ cells. (g) Heatmap depicting expression of HOX cluster genes during differentiation of RUNX1CGFP/w cells for 8 days. Note that the probes for HOXA7 and HOXB9 function poorly in this array, based on comparisons by RT-PCR and RNA-seq, Supplementary Fig. 8e. (h) Heatmap depicting expression of key pluripotency (POU5F1, NANOG, SOX2), mesendoderm (T, GSC, MIXL, SOX17, PDGFRA, FOXA2, FOXA1) and hematopoietic genes (GATA2, CD34, CDH5), the homeobox regulatory CDX genes, and transcription factors BCL11A and HLF, expressed in definitive hematopoietic lineages. Heatmap legend represents log2 transformed values of signal intensity applied to (g-i). (i) Heatmap showing HOX gene expression in d4 MIXL1GFP/w hESC differentiated in BMP4 and sorted into undifferentiated (ECADHERIN+MIXL1- [E+MIXL-]), primitive streak-like (E+MIXL+) and nascent mesoderm (E-MIXL+). Also shown is the expression of selected mesendoderm genes (T, MIXL1), CDX genes, BCL11A and HLF. As a comparison, gene expression in EBs differentiated in the absence of growth factors is shown (No GF). In this case, EBs adopt an early neural fate and HOX genes are not expressed.

Supplementary Figure 5 HOXA expression in differentiating cultures and generation of RNA-Seq transcriptional profiling data.

(a) Relative expression by RT-PCR of HOXA and CDX genes at d7 of differentiation in cultures supplemented with SB431542 (SB), CHIR99021 (CHIR) and the combination of these agents (SB/CHIR). Mean±SEM for 3-4 experiments. *, p<0.05; **, p<0.01; ***, p<0.001 compared to control using 2-way ANOVA with Holm-Sidak's multiple comparisons test. Data for the d4 timepoint is shown in Fig. 2d. (b) Time course correlating CDX and HOXA gene induction in control and SB/CHIR cultures. An independent CDX time course experiment is shown in Fig. 2e. (c) Gating strategy and representative FACS plots showing cell fractions sorted from Control and SB/CHIR treated cultures at day 10. Cells were sorted on the basis of CD43, CD34 and SOX17 (mCHERRY) to identify hematopoietic (CD43+), and SOX17+ and SOX17- endothelial (abbreviated to SOX17+34+ and SOX17-34+) and adherent cells (abbreviated to SOX17+34- and SOX17-34-). (d) Venn diagrams showing overlap in genes whose expression was selectively increased in CD34+ endothelium or CD34- adherent cells between Control and SB/CHIR cultures. Gene lists are provided in Supplementary Table 3. (e) Gene ontology (GO) terms indicating that similar biological processes were enriched in control and SB/CHIR cultures in the CD34+ and CD34- fractions from panel (d). (f, g) Venn diagrams showing overlap in (f) SB/CHIR and (g) Control culture differentially up-regulated genes between CD34+ and CD34- fractions. Genes up-regulated in CD43+ hematopoietic cells clustered more closely to the CD34+ endothelial cells. Gene lists are provided in Supplementary Table 4.

Supplementary Figure 6 Differential HOX gene expression and expression of vascular genes in sorted fractions from control and SB/CHIR cultures.

(a-e) Bar plots showing fold change (log2) in HOX gene expression in SB/CHIR versus control cultures for each sorted fraction. Red fill indicates the difference was statistically significant (p<0.05). Bars are arranged in order of increasing p value from top to bottom. (f, g) Bar plot showing the expression by RNA-Seq of HOXA genes in (f) CD43+ blood cells and (g) SOX17+ and SOX17- CD34- adherent cells in control and SB/CHIR cultures. Mean±SEM, 3 experiments. Expression in SB/CHIR cultures was higher for asterisked (*) genes (p<0.05). (h) Expression of genes by RNA-Seq analysis discriminating arterial and venous gene signatures in umbilical artery and vein endothelium as reported by Aranguren et al35. Genes shown in red type were selectively expressed in CD34+ sorted fractions as shown in Supplementary Fig. 5d and listed in Supplementary Table 3. The sorted cell fraction is indicated at the top of each pair of columns and the Control (C) or SB/CHIR (S/C) culture condition at the bottom.

Supplementary Figure 7 Increased accessibility of HOXA chromatin in SB/CHIR endothelial cells.

RNA-Seq, DNA methylation (ME) array, and ATAC-Seq, comparing the HOXA cluster in control and SB/CHIR CD34+ endothelial fractions. In the DNA ME array, the blue and red lines represent the fraction of methylated probe in control and SB/CHIR cultures, respectively. Shading represents 95% confidence intervals for the DNA ME value, scored for mean probe methylation from 0 (unmethylated) to 1 (methylated). Scale represents counts per million for RNA-Seq and ATAC-Seq.

Supplementary Figure 8 DNA methylation, RNA-Seq and ATAC-Seq analyses of HOX genes in d10 SOX17mCHERRY/wRUNX1CGFP/w cells.

(a) Multidimensional scaling plots of DNA methylation array data from d10 control (C) and SB/CHIR (S/C) cultures sorted into SOX17+ and SOX17- endothelium, and SOX17-34- adherent cell fractions demonstrating the clustering of sorted fractions by phenotype and by culture condition. The closer clustering of the control and SB/CHIR samples for the SOX17+CD34+ endothelium suggests more similar levels of methylation in these samples. (b) Heatmap of the 1002 methylation probes that encompassed the HOXA-D loci showing global hypomethylation in all samples. Hierarchical clustering indicates additional grouping of samples by treatment type. (c) Multidimensional scaling plots of ATAC-Seq data from d10 control (C) and SB/CHIR (S/C) cultures sorted into SOX17+ and SOX17- endothelium, demonstrating the clustering of sorted fractions by phenotype and by culture condition. (df) RNA-Seq and DNA methylation (ME) array, and chromatin accessibility assessed by ATAC-Seq, comparing the (d) HOXB, (e) HOXC, and (f) HOXD clusters in control and SB/CHIR CD34+ endothelial fractions. In the DNA ME array, the blue and red lines represent the fraction of methylated probe at each locus in control and SB/CHIR cultures cells respectively.

Supplementary Figure 9 Analysis of T cell generation in SOX17mCHERRY/wRUNX1CGFP/w cells.

(a, b) Strategy used to sort RUNX1CGFP/w cells differentiated for 7 days in spinner cultures under Control (a) and SB/CHIR (b) conditions into RUNX1C+34+, SOX17+34+, RUNX1C-43+ and SOX17-34+ fractions prior to co culture with OP9 DL4 stromal cells. (c) Bar plot comparing the frequency of cells in each fraction indicating that RUNX1C-43+ (R-43+) cells were less frequent in SB/CHIR cultures, consistent with suppression of primitive hematopoieisis, and that SOX17+34+ (S+34+) cells increased in response to SB/CHIR. Mean±SEM from 4 experiments. *, p<0.05 Student's t test. (d) Day 7 SOX17+CD34+ and RUNX1C+CD34lo populations from control and SB/CHIR cultures after 5d on OP9 DL4 showing emerging hematopoietic cells. Scale bar, 50μM (e) Image of SOX17-CD34+ sorted cells from SB/CHIR cultures after 9 days on OP9 DL4 showing the generation of SOX17+ cells and emergence of hematopoietic cells. Scale bar, 50μM. (f, g) Representative flow cytometry profiles of control (f) and SB/CHIR (g) d7 sorted fractions after 21 days co-culture on OP9 DL4. Live cells were gated for CD45, then for the NK cell marker CD56 (left panels). T cell progenitors (CD45+CD56-CD7+CD5±) are shown in red and NK cells (CD45+CD56hiCD7±) are shown in blue. Whilst T cells and NK cells were generated from all sorted fractions, the yield appeared greater from the SB/CHIR treated cultures. (h, i) Bar plot showing the percentages of cells falling into the CD7 and CD5 quadrants shown in (e, f) for the CD56- T cell progenitors (h) and the CD56hi NK cells (i). (j) Generation of CD4+CD8+ T cells following 27 days co-culture on OP9 DL4 of SOX17-CD34+ cells from SB/CHIR treated cultures.

Supplementary Figure 10 RUNX1C+ blood cells are contained within SOX17+ endothelial walls.

(a, b). Serial optical sections through hemogenic regions of SOX17+ vessels at (a) d18 and (b) d21 of differentiation were used to create a z-stack. Examination of the x-z and y-z planes demonstrates that the RUNX1C+ blood cells are contained within SOX17+ endothelial walls (En).

Supplementary Figure 11 Increased generation of CD34+ hematopoietic cells in late SB/CHIR cultures.

(a) viability of floating hematopoietic cells in d23-d29 control and SB/CHIR cultures, as assessed by propidium iodide exclusion.Viable cells expressed the hematopoietic marker, CD43. Mean±SEM of 6 experiments. *, p<0.05, Student's t test. (b) Flow cytometry plots showing frequency of hematopoietic cells in control and SB/CHIR cultures expressing the indicated stem cell markers in d23 cultures. (c) Percentage of CD43+ and CD45+ hematopoietic cells expressing high levels of CD34 (34br), as shown in the gated regions in panel (b). Mean±SEM of 4 experiments. *, p<0.05, Student's t test. (d) Percentage of hematopoietic cells co-expressing CD34 with GPI80 (VNN2), FCER1A and ACE in d23-29 cultures. Mean±SEM of 4 experiments. *, p<0.05, Student's t test.

Supplementary Figure 12 RNA-Seq analysis of human FL, AGM and sorted SOX/RUNX d18 and d22 samples.

Sorting strategy for generating (a) SOX17mCHERRY/wRUNX1CGFP/w d18 endothelial (S+R-34+90+)(d18 S+R- En), stem/progenitor (S+R+34+90+)(d18 S+R+S/P) and progenitor (S-R+34+90lo)(d18 S-R+ Pr1) populations, (b) SOX17mCHERRY/wRUNX1CGFP/w d22 progenitor (S+R+34+K+)(d22 S+R+ Pr2) and (S-R+34+K+) (d22 S-R+Pr3) populations, (c) 5 week AGM endothelium (34+90+43-)(En), cells transiting from hemogenic endothelium to hematopoietic stem/progenitors (34+90+43+)(S/P) and committed progenitor cell (34+90-43+) (Pr1) populations and (d) 17 week fetal liver (FL) sample (CD43+CD45+) enriched for hematopoietic stem/progenitor cells (34+38lo/-90+)(S/P). For panels (a-d), the percentage of live cells sorted for each fraction is shown in red type. The RUNX1C+ fractions in panels (a, b) also expressed CD43 or CD45. (e) Heatmap showing expression of HOX genes by RNA-Seq in FL, AGM, and d18 and d22 SOX/RUNX fractions sorted as shown in panels (a-d). (f) Comparison of log2 RPKM values for HOXA expression in FL, AGM and d18 S+R+ S/P populations. This bar plot shows the anterior HOXA expression bias in FL and AGM samples (higher expression of HOXA2-HOXA4) compared to the d18 hESC-derived S+R+ S/P cells, which express higher levels of the posterior genes (HOXA11 and HOXA13). (g, h) Heatmaps showing similar patterns of expression for BMP/TGFb□signaling genes and globin genes in AGM, and d18 SOX/RUNX fractions. Red shaded gene groups are expressed in FL S/P cells, and blue shaded genes are selectively expressed in the AGM S/P fraction. Scale is log2 RPKM.

Supplementary Figure 13 Human AGM and hESC-derived d18 SOX/RUNX sorted hematopoietic cells express a similar pattern of genes that are enriched in CB CD34+ cells.

Expression of the list of 32 genes differentially expressed between cord blood and d14 RUNX1C+34+ cells (see Fig. 2c) by RNA-Seq in FL, AGM, and d18 and d22 SOX/RUNX fractions sorted as shown in Supplementary Fig. 12. HBB was not detected in any sample. These data highlight the similarity between fetal liver and cord blood expression of these genes and indicate that the human AGM and hESC-derived d18 samples express a similar pattern of expression that differs from FL. The number of genes expressed at ≥ 0 RPKM in each sort fraction is indicated. Scale is in log2 RPKM.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–13, Supplementary Results and Supplementary Tables 6 and 9 (PDF 5222 kb)

Supplementary Table 1

Differentially expressed genes between sorted cell populations from d14 RUNX1CGFP/w and RUNX1CGFP/GFP EBs (XLSX 858 kb)

Supplementary Table 2

Differentially expressed genes between CD34+ cord blood and GFP+CD34lo cells sorted from RUNX1CGFP/w and RUNX1CGFP/GFP EBs (XLSX 187 kb)

Supplementary Table 3

Differentially expressed genes between CD34+ endothelial and CD34-adherent cells from d10 SOX17mCHERRY/wRUNX1CGFP/w cells (XLSX 1263 kb)

Supplementary Table 4

Differentially expressed genes between endothelial, hematopoieticand CD34-negative adherent cell fractions from control and SB/CHIR treated d10 SOX17mCHERRY/wRUNX1CGFP/w cells (XLSX 1372 kb)

Supplementary Table 5

Differentially expressed genes between SOX17+ and SOX17-endothelial fractions from SB/CHIR treated d10 SOX17mCHERRY/wRUNX1CGFP/w cells (XLSX 1003 kb)

Supplementary Table 7

Gene loci showing increased chromatin accessibility by ATAC-Seq (XLSX 225 kb)

Supplementary Table 8

RNA-Seq analysis of human fetal liver, AGM and SOX/RUNX sorted d18 and d22 populations (XLSX 3905 kb)

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Ng, E., Azzola, L., Bruveris, F. et al. Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros. Nat Biotechnol 34, 1168–1179 (2016). https://doi.org/10.1038/nbt.3702

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