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Medial HOXA genes demarcate haematopoietic stem cell fate during human development

Nature Cell Biology volume 18, pages 595–606 (2016)Cite this article

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Abstract

Pluripotent stem cells (PSCs) may provide a potential source of haematopoietic stem/progenitor cells (HSPCs) for transplantation; however, unknown molecular barriers prevent the self-renewal of PSC-HSPCs. Using two-step differentiation, human embryonic stem cells (hESCs) differentiated in vitro into multipotent haematopoietic cells that had the CD34+CD38−/loCD90+CD45+GPI-80+ fetal liver (FL) HSPC immunophenotype, but exhibited poor expansion potential and engraftment ability. Transcriptome analysis of immunophenotypic hESC-HSPCs revealed that, despite their molecular resemblance to FL-HSPCs, medial HOXA genes remained suppressed. Knockdown of HOXA7 disrupted FL-HSPC function and caused transcriptome dysregulation that resembled hESC-derived progenitors. Overexpression of medial HOXA genes prolonged FL-HSPC maintenance but was insufficient to confer self-renewal to hESC-HSPCs. Stimulation of retinoic acid signalling during endothelial-to-haematopoietic transition induced the HOXA cluster and other HSC/definitive haemogenic endothelium genes, and prolonged HSPC maintenance in culture. Thus, medial HOXA gene expression induced by retinoic acid signalling marks the establishment of the definitive HSPC fate and controls HSPC identity and function.

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Figure 1: Two-step culture of hESCs generates immunophenotypic HSPCs that engraft poorly.
Figure 2: hESC-derived haematopoietic cells have limited proliferative potential in vitro.
Figure 3: Identification of differentially expressed programs in hESC- and FL-HSPCs.
Figure 4: Medial HOXA genes govern the function and identity of human fetal HSPCs.
Figure 5: Overexpression of medial HOXA genes enhances proliferative potential in FL-HSPCs but does not confer HSC properties to hESC-HSPCs.
Figure 6: Retinoic acid signalling activates medial HOXA genes during human haematopoietic development.
Figure 7: Retinoic acid signalling pulse in EB-derived haemato-vascular cells induces transcriptional programs associated with definitive haemogenic endothelium and HSC fate.

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References

  1. Bordignon, C. Stem-cell therapies for blood diseases. Nature 441, 1100–1102 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Doulatov, S. et al. Induction of multipotential hematopoietic progenitors from human pluripotent stem cells via respecification of lineage-restricted precursors. Cell Stem Cell 13, 459–470 (2013).

    Article  CAS  PubMed  Google Scholar 

  3. Pereira, C. F. et al. Induction of a hemogenic program in mouse fibroblasts. Cell Stem Cell 13, 205–218 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Riddell, J. et al. Reprogramming committed murine blood cells to induced hematopoietic stem cells with defined factors. Cell 157, 549–564 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Olsen, A. L., Stachura, D. L. & Weiss, M. J. Designer blood: creating hematopoietic lineages from embryonic stem cells. Blood 107, 1265–1275 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Slukvin, I. I. Hematopoietic specification from human pluripotent stem cells: current advances and challenges toward de novo generation of hematopoietic stem cells. Blood 122, 4035–4046 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. McGrath, K. E. & Palis, J. Hematopoiesis in the yolk sac: more than meets the eye. Exp. Hematol. 33, 1021–1028 (2005).

    Article  PubMed  Google Scholar 

  8. Kyba, M. & Daley, G. Q. Hematopoiesis from embryonic stem cells: lessons from and for ontogeny. Exp. Hematol. 31, 994–1006 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Mikkola, H. K. & Orkin, S. H. The journey of developing hematopoietic stem cells. Development 133, 3733–3744 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Swiers, G., Rode, C., Azzoni, E. & de Bruijn, M. F. A short history of hemogenic endothelium. Blood Cells Mol. Dis. 51, 206–212 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Vodyanik, M. A., Bork, J. A., Thomson, J. A. & Slukvin, I. I. Human embryonic stem cell-derived CD34 + cells: efficient production in the coculture with OP9 stromal cells and analysis of lymphohematopoietic potential. Blood 105, 617–626 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Vodyanik, M. A., Thomson, J. A. & Slukvin, I. I. Leukosialin (CD43) defines hematopoietic progenitors in human embryonic stem cell differentiation cultures. Blood 108, 2095–2105 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Van Handel, B. et al. The first trimester human placenta is a site for terminal maturation of primitive erythroid cells. Blood 116, 3321–3330 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Prashad, S. L. et al. GPI-80 defines self-renewal ability in hematopoietic stem cells during human development. Cell Stem Cell 16, 80–87 (2015).

    Article  CAS  PubMed  Google Scholar 

  15. Keller, G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 19, 1129–1155 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Pick, M., Azzola, L., Mossman, A., Stanley, E. G. & Elefanty, A. G. Differentiation of human embryonic stem cells in serum-free medium reveals distinct roles for bone morphogenetic protein 4, vascular endothelial growth factor, stem cell factor, and fibroblast growth factor 2 in hematopoiesis. Stem Cells 25, 2206–2214 (2007).

    Article  CAS  PubMed  Google Scholar 

  17. Ditadi, A. et al. Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages. Nat. Cell Biol. 17, 580–591 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zambidis, E. T., Peault, B., Park, T. S., Bunz, F. & Civin, C. I. Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development. Blood 106, 860–870 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wang, L. et al. Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. Immunity 21, 31–41 (2004).

    Article  CAS  PubMed  Google Scholar 

  20. Dravid, G., Zhu, Y., Scholes, J., Evseenko, D. & Crooks, G. M. Dysregulated gene expression during hematopoietic differentiation from human embryonic stem cells. Mol. Ther. 19, 768–781 (2011).

    Article  CAS  PubMed  Google Scholar 

  21. Shojaei, F. & Menendez, P. Molecular profiling of candidate human hematopoietic stem cells derived from human embryonic stem cells. Exp. Hematol. 36, 1436–1448 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Martin, C. H., Woll, P. S., Ni, Z., Zuniga-Pflucker, J. C. & Kaufman, D. S. Differences in lymphocyte developmental potential between human embryonic stem cell and umbilical cord blood-derived hematopoietic progenitor cells. Blood 112, 2730–2737 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Qiu, C. et al. Differentiation of human embryonic stem cells into hematopoietic cells by coculture with human fetal liver cells recapitulates the globin switch that occurs early in development. Exp. Hematol. 33, 1450–1458 (2005).

    Article  CAS  PubMed  Google Scholar 

  24. Tian, X. & Kaufman, D. S. Differentiation of embryonic stem cells towards hematopoietic cells: progress and pitfalls. Curr. Opin. Hematol. 15, 312–318 (2008).

    Article  PubMed  Google Scholar 

  25. Wang, L., Cerdan, C., Menendez, P. & Bhatia, M. Derivation and characterization of hematopoietic cells from human embryonic stem cells. Methods Mol. Biol. 331, 179–200 (2006).

    PubMed  Google Scholar 

  26. Magnusson, M. et al. Expansion on stromal cells preserves the undifferentiated state of human hematopoietic stem cells despite compromised reconstitution ability. PLoS ONE 8, e53912 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kennedy, M. et al. T lymphocyte potential marks the emergence of definitive hematopoietic progenitors in human pluripotent stem cell differentiation cultures. Cell Rep. 2, 1722–1735 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Shaut, C. A., Keene, D. R., Sorensen, L. K., Li, D. Y. & Stadler, H. S. HOXA13 is essential for placental vascular patterning and labyrinth endothelial specification. PLoS Genet. 4, e1000073 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Aguilo, F. et al. Prdm16 is a physiologic regulator of hematopoietic stem cells. Blood 117, 5057–5066 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Klimmeck, D. et al. Transcriptome-wide profiling and posttranscriptional analysis of hematopoietic stem/progenitor cell differentiation toward myeloid commitment. Stem Cell Rep. 3, 858–875 (2014).

    Article  CAS  Google Scholar 

  31. Thorsteinsdottir, U. et al. Overexpression of the myeloid leukemia-associated Hoxa9 gene in bone marrow cells induces stem cell expansion. Blood 99, 121–129 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Lawrence, H. J. et al. Loss of expression of the Hoxa-9 homeobox gene impairs the proliferation and repopulating ability of hematopoietic stem cells. Blood 106, 3988–3994 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang, Y., Schulte, B. A., LaRue, A. C., Ogawa, M. & Zhou, D. Total body irradiation selectively induces murine hematopoietic stem cell senescence. Blood 107, 358–366 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hilpert, M. et al. p19 INK4d controls hematopoietic stem cells in a cell-autonomous manner during genotoxic stress and through the microenvironment during aging. Stem Cell Rep. 3, 1085–1102 (2014).

    Article  CAS  Google Scholar 

  35. Pluta, K., Luce, M. J., Bao, L., Agha-Mohammadi, S. & Reiser, J. Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters. J. Gene Med. 7, 803–817 (2005).

    Article  CAS  PubMed  Google Scholar 

  36. Lois, C., Hong, E. J., Pease, S., Brown, E. J. & Baltimore, D. Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Marshall, H., Morrison, A., Studer, M., Pöpperl, H. & Krumlauf, R. Retinoids and Hox genes. FASEB J. 10, 969–978 (1996).

    Article  CAS  PubMed  Google Scholar 

  38. Gavalas, A. & Krumlauf, R. Retinoid signalling and hindbrain patterning. Curr. Opin. Genet. Dev. 10, 380–386 (2000).

    Article  CAS  PubMed  Google Scholar 

  39. Chanda, B., Ditadi, A., Iscove, N. N. & Keller, G. Retinoic acid signaling is essential for embryonic hematopoietic stem cell development. Cell 155, 215–227 (2013).

    Article  CAS  PubMed  Google Scholar 

  40. Delescluse, C. et al. Selective high affinity retinoic acid receptor alpha or beta-gamma ligands. Mol. Pharmacol. 40, 556–562 (1991).

    CAS  PubMed  Google Scholar 

  41. Kishimoto, H. et al. Molecular mechanism of human CD38 gene expression by retinoic acid. Identification of retinoic acid response element in the first intron. J. Biol. Chem. 273, 15429–15434 (1998).

    Article  CAS  PubMed  Google Scholar 

  42. Balmer, J. E. & Blomhoff, R. Gene expression regulation by retinoic acid. J. Lipid Res. 43, 1773–1808 (2002).

    Article  CAS  PubMed  Google Scholar 

  43. Salvagiotto, G. et al. Molecular profiling reveals similarities and differences between primitive subsets of hematopoietic cells generated in vitro from human embryonic stem cells and in vivo during embryogenesis. Exp. Hematol. 36, 1377–1389 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ramos-Mejía, V. et al. HOXA9 promotes hematopoietic commitment of human embryonic stem cells. Blood 124, 3065–3075 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Wang, L. et al. Generation of hematopoietic repopulating cells from human embryonic stem cells independent of ectopic HOXB4 expression. J. Exp. Med. 201, 1603–1614 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Beachy, S. H. et al. Isolated Hoxa9 overexpression predisposes to the development of lymphoid but not myeloid leukemia. Exp. Hematol. 41, 518–529 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Alharbi, R. A., Pettengell, R., Pandha, H. S. & Morgan, R. The role of HOX genes in normal hematopoiesis and acute leukemia. Leukemia 27, 1000–1008 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. McKinney-Freeman, S. et al. The transcriptional landscape of hematopoietic stem cell ontogeny. Cell Stem Cell 11, 701–714 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Chen, F., Greer, J. & Capecchi, M. R. Analysis of Hoxa7/Hoxb7 mutants suggests periodicity in the generation of the different sets of vertebrae. Mech. Dev. 77, 49–57 (1998).

    Article  CAS  PubMed  Google Scholar 

  50. Boucherat, O. et al. Partial functional redundancy between Hoxa5 and Hoxb5 paralog genes during lung morphogenesis. Am. J. Physiol. Lung Cell. Mol. Physiol. 304, L817–L830 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Lebert-Ghali, C. E. et al. HoxA cluster is haploinsufficient for activity of hematopoietic stem and progenitor cells. Exp. Hematol. 38, 1074–1086 (2010).

    Article  CAS  PubMed  Google Scholar 

  52. Lebert-Ghali, C. E. et al. Hoxa cluster genes determine the proliferative activity of adult mouse hematopoietic stem and progenitor cells. Blood 127, 87–90 (2016).

    Article  CAS  PubMed  Google Scholar 

  53. Muramoto, G. G. et al. Inhibition of aldehyde dehydrogenase expands hematopoietic stem cells with radioprotective capacity. Stem Cells 28, 523–534 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Szatmari, I., Iacovino, M. & Kyba, M. The retinoid signaling pathway inhibits hematopoiesis and uncouples from the Hox genes during hematopoietic development. Stem Cells 28, 1518–1529 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Rönn, R. E. et al. Retinoic acid regulates hematopoietic development from human pluripotent stem cells. Stem Cell Rep. 4, 269–281 (2015).

    Article  CAS  Google Scholar 

  56. Cano, E., Ariza, L., Muñoz-Chápuli, R. & Carmona, R. Signaling by retinoic acid in embryonic and adult hematopoiesis. J. Dev. Biol. 2, 18–33 (2014).

    Article  CAS  Google Scholar 

  57. Dou, D. R., Calvanese, V., Saarikoski, P., Galic, Z. & Mikkola, H. K. A. Induction of HOXA genes in hESC-derived HSPC by two-step differentiation and RA signalling pulse. Protocol Exchange http://dx.doi.org/10.1038/protex.2016.035 (2016).

  58. Thoma, S. J., Lamping, C. P. & Ziegler, B. L. Phenotype analysis of hematopoietic CD34 + cell populations derived from human umbilical cord blood using flow cytometry and cDNA-polymerase chain reaction. Blood 83, 2103–2114 (1994).

    CAS  PubMed  Google Scholar 

  59. Bauchwitz, R. & Costantini, F. Developmentally distinct effects on human epsilon-, gamma- and delta-globin levels caused by the absence or altered position of the human beta-globin gene in YAC transgenic mice. Hum. Mol. Genet. 9, 561–574 (2000).

    Article  CAS  PubMed  Google Scholar 

  60. Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Gentleman, R. C. et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 5, R80 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  62. de Hoon, M. J., Imoto, S., Nolan, J. & Miyano, S. Open source clustering software. Bioinformatics 20, 1453–1454 (2004).

    Article  CAS  PubMed  Google Scholar 

  63. Saldanha, A. J. Java Treeview–extensible visualization of microarray data. Bioinformatics 20, 3246–3248 (2004).

    Article  CAS  PubMed  Google Scholar 

  64. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Quinlan, A. R. & Hall, I. M. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26, 841–842 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Ramírez, F., Dündar, F., Diehl, S., Grüning, B. A. & Manke, T. deepTools: a flexible platform for exploring deep-sequencing data. Nucleic Acids Res. 42, W187–W191 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Zhang, Y. et al. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank BSCRC FACS Core at UCLA, the UCLA Clinical Pathology Microarray Core, BSCRC Sequencing Core, UCLA Tissue and Pathology Core and CFAR Gene and Cell Therapy Core (NIH grant AI028697-21) and Novogenix LLC. We thank H. Coller for discussions, T. Bolan for assistance with experiments, Y. Xing and Y.-T. Tseng for consultation on RNA sequencing analysis, and T. Stoyanova, D. Johnson and O. Witte for help with NSG mice. This work was supported by CIRM RN1-00557 and RT3-07763, NIH RO1 DK100959, LLS Scholar award and Rose Hills Foundation Scholar Award to H.K.A.M; Broad Stem Cell Research Center at UCLA and JCC Foundation; NIH P01 GM081621 to J.A.Z. and Z.G.; and NIH PO1 HL073104 and CIRM RB3-05217 to G.M.C. D.R.D. was supported by the NSF GRFP and Ruth L. Kirschstein National Research Service Award GM007185, V.C. by an LLS Special Fellow Award and a BSCRC post-doctoral fellow award, M.I.S. by Ruth L. Kirschstein National Research Service Award HL086345, A.T.N. by a Beckman Scholarship, and P.S. by the Eugene V. Cota-Robles fellowship.

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  1. Diana R. Dou and Vincenzo Calvanese: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, Los Angeles, California 90095, USA

    Diana R. Dou, Vincenzo Calvanese, Maria I. Sierra, Andrew T. Nguyen, Arazin Minasian, Pamela Saarikoski, Rajkumar Sasidharan, Gay M. Crooks & Hanna K. A. Mikkola

  2. Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA

    Diana R. Dou, Vincenzo Calvanese, Maria I. Sierra, Arazin Minasian, Pamela Saarikoski, Rajkumar Sasidharan, Jerome A. Zack, Gay M. Crooks, Zoran Galic & Hanna K. A. Mikkola

  3. Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA

    Diana R. Dou & Hanna K. A. Mikkola

  4. Department of Biostatistics, Fielding School of Public Health, University of California, Los Angeles, Los Angeles, California 90095, USA

    Christina M. Ramirez

  5. Department of Medicine, Division of Hematology-Oncology, University of California, Los Angeles, Los Angeles, California 90095, USA

    Jerome A. Zack & Zoran Galic

  6. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA

    Jerome A. Zack

  7. Department of Pathology and Laboratory Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA

    Gay M. Crooks

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Contributions

D.R.D., V.C., M.I.S., P.S., Z.G., J.A.Z., G.M.C. and H.K.A.M. designed experiments and interpreted data. D.R.D., V.C., M.I.S., A.T.N., A.M. and P.S. performed experiments. R.S. performed bioinformatics analysis of the microarray data and C.M.R. assisted with statistical analysis, D.R.D., V.C. and H.K.A.M. wrote the manuscript, which all authors edited and approved.

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Correspondence to Hanna K. A. Mikkola.

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Integrated supplementary information

Supplementary Figure 1 FL derived, but not hESC derived haematopoietic cells can reconstitute human HSPC compartment in recipient BM.

(A) Schematic of transplantation of CD34+ cells into irradiated NSG mice. (B) NSG mice were transplanted with CD34+ cells from hESCs (EB and EB-OP9) and fetal liver (FL or FL-OP9) and human engraftment in the BM assessed at 12 weeks for CD45+CD34+CD38−CD90+ immunophenotypic HSPCs. Results shown are from representative animals for each group of transplanted mice (5 mice transplanted with EB cells, 4 with EB-OP9 cells, 4 with FL cells, and 3 with FL-OP9 cells).

Supplementary Figure 2 hESC-derived haematopoietic cells can upregulate adult haemoglobin-beta (HBB) and differentiate into T-lymphoid cells.

(A) Representative FACS plots and quantification of BrdU incorporation and 7-AAD to determine cell cycle distribution in EB and FL CD90+ immunophenotypic HSPCs and CD90− cells is shown (mean ± s.e.m. from n = 3 independent experiments). (B) Comparison between CD34+ haematopoietic cells and immunophenotypic (CD34+CD38−CD90+CD45+) HSPCs, all seeded at an initial density of 10,000 cells per sample, from FL and hESC-derived cells (mean ± s.e.m. of n = 5 independent experiments). (C) CFU-C expansions from 10,000 hESC-derived or FL-derived CD34+ cells in methylcellulose following 0, 1, 2 and 3 additional weeks on OP9-M2 co-culture (mean ± s.e.m. of n = 3 independent experiments). (D) Haemoglobin levels (expression measured from colonies derived from CD34+ cells) of embryonic epsilon (HBE), fetal gamma (HBG), and adult beta (HBB) measured through qRT-PCR and normalized to Glycophorin A levels (mean ± s.e.m. shown from n = 5 independent experiments). (E) FACS staining of hESC- and FL-derived CD34+ haematopoietic cells grown on OP9-DL1 stroma for 4 weeks is shown. Cells were stained for CD45, the myeloid exclusion marker CD14, and T-cell markers CD4 and CD8 (mean ± s.e.m. shown from n = 3 independent experiments. Statistics source data for graphs shown in A, B, C, and E can be found in Supplementary Table 7. Statistical significance was assessed using the Wilcoxon Rank Sum test for A, B, C and E.

Supplementary Figure 3 Knockdown of HOXA5 or HOXA7 does not lead to changes in BRDU incorporation in FL immunophenotypic HSPCs.

(A) Representative FACS plots and quantification of cell cycle analyses based on BrdU incorporation (mean from one experiment with 2 independent donors, statistics source data can be found in Supplementary Table 7) of control vector and HOXA5 and HOXA7 shRNA vector transduced FL-HSPCs. (B,C) Examples of cell cycle activators (B) and inhibitors (C) from RNA-seq analyses of FL immunophenotypic HSPCs with HOXA7 knockdown compared to empty vector controls (showing mean from 4 independent experiments, values used to generate graphs can be found in Supplementary Table 4 and GEO database GSE76685).

Supplementary Figure 4 Lentiviral overexpression of HOXA5, HOXA7 and HOXA9 in EB-derived CD34+ cells is not sufficient for rescuing HSC function.

(A) Schematic showing the strategy for tet-inducible overexpression of HOXA5 or HOXA7 in FL-HSPCs using a PNL vector. (B) q-RT-PCR showing induction of HOXA5 or HOXA7 expression in FL-HSPCs overexpressing HOXA5 or HOXA7, compared to empty vector control 1 week post-transduction (plotting one representative experiment). (C,D) Representative FACS plots (C) and quantification (D) of FL-HSPCs overexpressing HOXA5 or HOXA7 (mean from 3 independent experiments, except for 2 independent experiments for 7–8 weeks timepoint). (E) Representative FACS plots assessing concurrent overexpression of HOXA5, HOXA7 and HOXA9 using PNL vector. EB and FL CD34 + cells transduced with empty-vector were used as controls (mean from 2 independent experiments, except for EB-control and HOXA5/7/9 at day 14, 1 independent experiment). Statistics source data for values used to generate graphs shown in b, d, and e can be found in Supplementary Table 7.

Supplementary Figure 5 AM580 treatment prolongs CFU-C potential in hESC-derived cells.

(A) Quantification of CFU-Cs generated from 10,000 EB- or FL-derived haematopoietic cells at day 24 ± 1 of OP9-M2 culture (mean ± s.e.m. from n = 4 independent experiments, statistics source data can be found in Supplementary Fig. 5B). (B) Table showing CFU counts for the indicated samples. Counts were rounded to the closest integer value (DM = DMSO, AM = AM580).

Supplementary Figure 6 Analysis of gene expression changes in hESC-HSPCs on AM580 treatment shows partial conversion to definitive HSC transcriptome.

(A) RNA-seq genome browser screenshot of the HOXA cluster of day 12 EB and FL derived immunophenotypic HSPCS that were treated with AM580 for 6 days (6 days of treatment and 6 additional days in culture). (B) Representative genes upregulated by AM580 treatment at day 6, shown at day 12 as compared to FL-HSPCs. (C) Representative genes upregulated by HOXA7 shRNA knockdown (see Fig. 4l) shown in day 12 EB derived cells treated with AM580 (6 days of treatment and 6 additional days in culture) as compared to FL-HSPCs (showing mean from 2 independent experiments, values used to generate graphs in B and C can be found in Supplementary Table 5 and GEO database GSE76685).

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Dou, D., Calvanese, V., Sierra, M. et al. Medial HOXA genes demarcate haematopoietic stem cell fate during human development. Nat Cell Biol 18, 595–606 (2016). https://doi.org/10.1038/ncb3354

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