Abstract

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Gene Expression Omnibus

References

  1. 1.

    et al. CellNet: network biology applied to stem cell engineering. Cell 158, 903–915 (2014)

  2. 2.

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

  3. 3.

    et al. Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature 511, 312–318 (2014)

  4. 4.

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

  5. 5.

    et al. Direct conversion of human fibroblasts to sub-lethal blood progenitors. Nature 468, 521–526 (2010)

  6. 6.

    , , & Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells. Cell Rep. 9, 1871–1884 (2014)

  7. 7.

    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)

  8. 8.

    On the origin of haemopoietic stem cells in the avian embryo: an experimental approach. J. Embryol. Exp. Morphol. 33, 607–619 (1975)

  9. 9.

    et al. Highly potent human hematopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region. J. Exp. Med. 208, 2417–2427 (2011)

  10. 10.

    et al. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464, 108–111 (2010)

  11. 11.

    et al. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature 464, 116–120 (2010)

  12. 12.

    & Of lineage and legacy: the development of mammalian hematopoietic stem cells. Nat. Immunol. 9, 129–136 (2008)

  13. 13.

    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)

  14. 14.

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

  15. 15.

    et al. Direct induction of haematoendothelial programs in human pluripotent stem cells by transcriptional regulators. Nat. Commun. 5, 4372 (2014)

  16. 16.

    et al. A stem cell molecular signature. Science 298, 601–604 (2002)

  17. 17.

    et al. Revised map of the human progenitor hierarchy shows the origin of macrophages and dendritic cells in early lymphoid development. Nat. Immunol. 11, 585–593 (2010)

  18. 18.

    et al. Densely interconnected transcriptional circuits control cell states in human hematopoiesis. Cell 144, 296–309 (2011)

  19. 19.

    et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J. Immunol. 174, 6477–6489 (2005)

  20. 20.

    , , , & The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells. Stem Cells 33, 1130–1141 (2015)

  21. 21.

    & Directed differentiation of definitive hemogenic endothelium and hematopoietic progenitors from human pluripotent stem cells. Methods 101, 65–72 (2016)

  22. 22.

    et al. Nonirradiated NOD,B6.SCID Il2rγ−/− KitW41/W41 (NBSGW) mice support multilineage engraftment of human hematopoietic cells. Stem Cell Rep. 4, 171–180 (2015)

  23. 23.

    et al. Long non-coding RNA profiling of human lymphoid progenitor cells reveals transcriptional divergence of B cell and T cell lineages. Nat. Immunol. 16, 1282–1291 (2015)

  24. 24.

    . et al. Distinct routes of lineage development reshape the human blood hierarchy across ontogeny. Science 351, aab2116 (2016)

  25. 25.

    et al. Focal adhesion kinase is required for CXCL12-induced chemotactic and pro-adhesive responses in hematopoietic precursor cells. Leukemia 21, 1723–1732 (2007)

  26. 26.

    et al. α4 integrin levels on mobilized peripheral blood stem cells predict rapidity of engraftment in patients receiving autologous stem cell transplantation. Blood 118, 2362–2365 (2011)

  27. 27.

    et al. Robo4 cooperates with CXCR4 to specify hematopoietic stem cell localization to bone marrow niches. Cell Stem Cell 8, 72–83 (2011)

  28. 28.

    et al. Medial HOXA genes demarcate haematopoietic stem cell fate during human development. Nat. Cell Biol. 18, 595–606 (2016)

  29. 29.

    et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187–1201 (2015)

  30. 30.

    Controlling the fetal globin switch in man. Nature 301, 108–109 (1983)

  31. 31.

    & The thymus as an inductive site for T lymphopoiesis. Annu. Rev. Cell Dev. Biol. 23, 463–493 (2007)

  32. 32.

    et al. VDJ recombination. Immunol. Today 13, 306–314 (1992)

  33. 33.

    et al. The host genomic environment of the provirus determines the abundance of HTLV-1-infected T-cell clones. Blood 117, 3113–3122 (2011)

  34. 34.

    et al. Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo. Immunity 16, 661–672 (2002)

  35. 35.

    et al. Runx1 is essential for hematopoietic commitment at the hemangioblast stage of development in vitro. Blood 100, 458–466 (2002)

  36. 36.

    et al. Ligand-dependent nuclear receptor corepressor LCoR functions by histone deacetylase-dependent and -independent mechanisms. Mol. Cell 11, 139–150 (2003)

  37. 37.

    et al. An RCOR1 loss-associated gene expression signature identifies a prognostically significant DLBCL subgroup. Blood 125, 959–966 (2015)

  38. 38.

    & PU.1 induces myeloid lineage commitment in multipotent hematopoietic progenitors. Genes Dev. 12, 2403–2412 (1998)

  39. 39.

    , & In vitro and in vivo expansion of hematopoietic stem cells. Oncogene 23, 7223–7232 (2004)

  40. 40.

    et al. Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros. Nat. Biotechnol. 34, 1168–1179 (2016)

  41. 41.

    , , & Additive and global functions of HoxA cluster genes in mesoderm derivatives. Dev. Biol. 341, 488–498 (2010)

  42. 42.

    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)

  43. 43.

    et al. Trisomy of Erg is required for myeloproliferation in a mouse model of Down syndrome. Blood 115, 3966–3969 (2010)

  44. 44.

    et al. The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells. Nat. Immunol. 9, 810–819 (2008)

  45. 45.

    et al. Identification and characterization of Hoxa9 binding sites in hematopoietic cells. Blood 119, 388–398 (2012)

  46. 46.

    et al. Identification of Notch target genes in uncommitted T-cell progenitors: no direct induction of a T-cell specific gene program. Leukemia 20, 1967–1977 (2006)

  47. 47.

    et al. RUNX1 regulates corepressor interactions of PU.1. Blood 117, 6498–6508 (2011)

  48. 48.

    et al. Hematopoietic stem/progenitor cell conversion from human pluripotent stem cells (2017)

  49. 49.

    et al. Reprogramming of T cells from human peripheral blood. Cell Stem Cell 7, 15–19 (2010)

  50. 50.

    et al. Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451, 141–146 (2008)

  51. 51.

    et al. The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc. Natl Acad. Sci. USA 108, 3665–3670 (2011)

  52. 52.

    et al. Developmental and species-divergent globin switching are driven by BCL11A. Nature 460, 1093–1097 (2009)

  53. 53.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

  54. 54.

    , & HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015)

  55. 55.

    , & edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010)

  56. 56.

    et al. The transcriptional architecture of early human hematopoiesis identifies multilevel control of lymphoid commitment. Nature Immunol. 14, 756–763 (2013)

  57. 57.

    , & Amplification, next-generation sequencing, and genomic DNA mapping of retroviral integration sites. J. Vis. Exp. (109): (2016)

Download references

Acknowledgements

We thank T. Schlaeger and the Boston Children’s Hospital Human ESC Core Facility, R. Mathieu from the flow cytometry core, R. Renee, A. Ratner, and S. Boswell from Harvard Medical School for RNA-seq, the orchestra team at Harvard Medical School for providing high-performance computing, D. Kaufman for providing the RUNX1c+24 hPSC line, N. Gerry for microarray and SNP array analysis, and T. North, C. Brendel and J. Powers for reading the manuscript. This work was supported by grants from the US National Institute of Diabetes and Digestive and Kidney Diseases (R24DK092760), the National Institute of Allergy and Infectious Diseases (R37AI039394), and the National Heart, Lung, Blood Institute Progenitor Cell Biology Consortium (UO1-HL100001) and the Progenitor Cell Translation Consortium (UO1-HL134812); Alex’s Lemonade Stand Foundation; and the Doris Duke Medical Foundation. G.Q.D. is an associate member of the Broad Institute and an investigator of the Howard Hughes Medical Institute and the Manton Center for Orphan Disease Research. R.S. is supported by an American Society of Hematology Scholar Fellowship. C.S.V. is an EMBO (ALTF 1240-2015) fellow. S.D. is supported by the K99/R00HL123484. J.A.G. is supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (DK106311) and the Crohn’s and Colitis Foundation of America CDA 352644 (J.A.G.), S.B.S. is supported by NIDDK (DK034854), the Helmsley Charitable Trust, and the Wolpow Family Chair in IBD Treatment and Research.

Author information

Author notes

    • Sergei Doulatov

    Present address: Department of Medicine, Division of Hematology, University of Washington, Seattle, Washington 98195, USA.

    • Deepak Kumar Jha
    •  & Areum Han

    These authors contributed equally to this work.

Affiliations

  1. Stem Cell Transplantation Program, Division of Pediatric Hematology and Oncology, Dana-Farber Cancer Institute, Boston Children’s Hospital and Dana-Farber Cancer Institute, Boston, Massachusetts 02115, USA

    • Ryohichi Sugimura
    • , Deepak Kumar Jha
    • , Areum Han
    • , Clara Soria-Valles
    • , Edroaldo Lummertz da Rocha
    • , Yi-Fen Lu
    • , R. Grant Rowe
    • , Patricia Sousa
    • , Sergei Doulatov
    •  & George Q. Daley
  2. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Ryohichi Sugimura
    • , Deepak Kumar Jha
    • , Areum Han
    • , Clara Soria-Valles
    • , Edroaldo Lummertz da Rocha
    • , Yi-Fen Lu
    • , Patricia Sousa
    • , Sergei Doulatov
    •  & George Q. Daley
  3. Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA

    • Ryohichi Sugimura
    • , Deepak Kumar Jha
    • , Clara Soria-Valles
    • , Edroaldo Lummertz da Rocha
    • , Yi-Fen Lu
    • , Patricia Sousa
    • , Sergei Doulatov
    •  & George Q. Daley
  4. Manton Center for Orphan Disease Research, Boston, Massachusetts 02115, USA

    • Ryohichi Sugimura
    • , Deepak Kumar Jha
    • , Clara Soria-Valles
    • , Edroaldo Lummertz da Rocha
    • , Yi-Fen Lu
    • , Patricia Sousa
    • , Sergei Doulatov
    •  & George Q. Daley
  5. Department of Pediatrics, Division of Gastroenterology, Hepatology and Nutrition, Boston Children’s Hospital, Boston, Massachusetts, USA

    • Jeremy A. Goettel
    •  & Scott B. Snapper
  6. Department of Medicine, Harvard Medical School, Boston, Massachusetts, USA

    • Jeremy A. Goettel
    •  & Scott B. Snapper
  7. Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts, 02215, USA

    • Erik Serrao
    •  & Alan N. Engelman
  8. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    • Mohan Malleshaiah
  9. Department of Biology, Brandeis University, Waltham, Massachusetts 02453, USA

    • Irene Wong
  10. Program in Computer Science, Harvard University, Cambridge, Massachusetts, USA

    • Ted N. Zhu
  11. McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario M5G 1L7, Canada

    • Andrea Ditadi
    •  & Gordon Keller
  12. Division of Gastroenterology, Brigham and Women’s Hospital, Boston, Massachusetts, USA

    • Scott B. Snapper
  13. Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA

    • George Q. Daley

Authors

  1. Search for Ryohichi Sugimura in:

  2. Search for Deepak Kumar Jha in:

  3. Search for Areum Han in:

  4. Search for Clara Soria-Valles in:

  5. Search for Edroaldo Lummertz da Rocha in:

  6. Search for Yi-Fen Lu in:

  7. Search for Jeremy A. Goettel in:

  8. Search for Erik Serrao in:

  9. Search for R. Grant Rowe in:

  10. Search for Mohan Malleshaiah in:

  11. Search for Irene Wong in:

  12. Search for Patricia Sousa in:

  13. Search for Ted N. Zhu in:

  14. Search for Andrea Ditadi in:

  15. Search for Gordon Keller in:

  16. Search for Alan N. Engelman in:

  17. Search for Scott B. Snapper in:

  18. Search for Sergei Doulatov in:

  19. Search for George Q. Daley in:

Contributions

R.S. designed, performed, interpreted experiments, and wrote the paper. D.J., A.H., E.L.R., and T.N.Z. performed computational analysis of RNA-seq data. C.S.V., I.W., and P.S. assisted in iPSC culture, differentiation, and analyses of mice. C.S.V. replicated the entire process, independently. Y.L. performed globin expression analysis. R.R. performed cytospin of cells. M.M. performed flow cytometric analysis. D.J. contributed to writing and C.S.V. contributed to editing. E.S. and A.N.E. designed and interpreted integration sequencing experiments. J.A.G. and S.B.S. designed and interpreted TCRB rearrangement experiments. A.D. and G.K. instructed haemogenic endothelium induction. S.D. assisted in design and interpretation of experiments. G.Q.D. designed, interpreted experiments, and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to George Q. Daley.

Reviewer Information Nature thanks B. Gottgens and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Figure 1, the uncropped gels and Supplementary Tables 1-2.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature22370

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.