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Conversion of adult endothelium to immunocompetent haematopoietic stem cells

Abstract

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0–8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8–20), RUNX1+ FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20–28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

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Figure 1: Conditional expression of FGRS in adult mECs generates haematopoietic cells.
Figure 2: Conditional FGRS expression supports long-term primary and secondary HSPC engraftment.
Figure 3: Clonal and limiting-dilution quantification of rEC-HSPCs and rEC-HSCs.
Figure 4: Multi-dimensional clustering of control and reprogrammed haematopoietic cell populations.
Figure 5: Reconstitution of T cell immunity in Rag1−/− mice engrafted with rEC-HSPCs.
Figure 6: Deconvolution of vascular niche angiocrine signals in rEC-HSPC generation.

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References

  1. Wahlster, L. & Daley, G. Q. Progress towards generation of human haematopoietic stem cells. Nat. Cell Biol. 18, 1111–1117 (2016)

    CAS  PubMed  Article  Google Scholar 

  2. Sturgeon, C. M., Ditadi, A., Clarke, R. L. & Keller, G. Defining the path to hematopoietic stem cells. Nat. Biotechnol. 31, 416–418 (2013)

    CAS  PubMed  Article  Google Scholar 

  3. Tashiro, K. et al. Promotion of hematopoietic differentiation from mouse induced pluripotent stem cells by transient HoxB4 transduction. Stem Cell Res. 8, 300–311 (2012)

    CAS  PubMed  Article  Google Scholar 

  4. Rafii, S. et al. Human ESC-derived hemogenic endothelial cells undergo distinct waves of endothelial to hematopoietic transition. Blood 121, 770–780 (2013)

    CAS  PubMed  Article  Google Scholar 

  5. Batta, K., Florkowska, M., Kouskoff, V. & Lacaud, G. Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells. Cell Reports 9, 1871–1884 (2014)

    CAS  PubMed  Article  Google Scholar 

  6. 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)

    CAS  Article  PubMed  Google Scholar 

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

    ADS  CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. Pulecio, J. et al. Conversion of human fibroblasts into monocyte-like progenitor cells. Stem Cells 32, 2923–2938 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. Butler, J. M. et al. Development of a vascular niche platform for expansion of repopulating human cord blood stem and progenitor cells. Blood 120, 1344–1347 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. Ding, B. S. et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468, 310–315 (2010)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. Butler, J. M. & Rafii, S. Generation of a vascular niche for studying stem cell homeostasis. Methods Mol. Biol. 904, 221–233 (2012)

    CAS  PubMed  Google Scholar 

  14. Kobayashi, H. et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat. Cell Biol. 12, 1046–1056 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  15. Mendelson, A. & Frenette, P. S. Hematopoietic stem cell niche maintenance during homeostasis and regeneration. Nat. Med. 20, 833–846 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. Morrison, S. J. & Scadden, D. T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. Rafii, S., Butler, J. M. & Ding, B. S. Angiocrine functions of organ-specific endothelial cells. Nature 529, 316–325 (2016)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  18. Sasine, J. P., Yeo, K. T. & Chute, J. P. Concise review: paracrine functions of vascular niche cells in regulating hematopoietic stem cell fate. Stem Cells Transl. Med. 6, 482–489 (2017)

    CAS  PubMed  Article  Google Scholar 

  19. Butler, J. M. et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell 6, 251–264 (2010)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    ADS  CAS  PubMed  Article  Google Scholar 

  22. Zovein, A. C. et al. Fate tracing reveals the endothelial origin of hematopoietic stem cells. Cell Stem Cell 3, 625–636 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. Nguyen, P. D. et al. Haematopoietic stem cell induction by somite-derived endothelial cells controlled by meox1. Nature 512, 314–318 (2014)

    ADS  CAS  PubMed  Article  Google Scholar 

  24. Li, Y. et al. Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production. Genes Dev. 28, 2597–2612 (2014)

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  25. Espín-Palazón, R. et al. Proinflammatory signaling regulates hematopoietic stem cell emergence. Cell 159, 1070–1085 (2014)

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  26. Gritz, E. & Hirschi, K. K. Specification and function of hemogenic endothelium during embryogenesis. Cell. Mol. Life Sci. 73, 1547–1567 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  27. Slukvin, I. I. Generating human hematopoietic stem cells in vitro exploring endothelial to hematopoietic transition as a portal for stemness acquisition. FEBS Lett. 590, 4126–4143 (2016)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. Seandel, M. et al. Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene. Proc. Natl Acad. Sci. USA 105, 19288–19293 (2008)

    ADS  CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. Rafii, S. et al. Human bone marrow microvascular endothelial cells support long-term proliferation and differentiation of myeloid and megakaryocytic progenitors. Blood 86, 3353–3363 (1995)

    CAS  PubMed  Article  Google Scholar 

  30. Lorsbach, R. B. et al. Role of RUNX1 in adult hematopoiesis: analysis of RUNX1-IRES-GFP knock-in mice reveals differential lineage expression. Blood 103, 2522–2529 (2004)

    CAS  PubMed  Article  Google Scholar 

  31. Zhou, F. et al. Tracing haematopoietic stem cell formation at single-cell resolution. Nature 533, 487–492 (2016)

    ADS  CAS  PubMed  Article  Google Scholar 

  32. Kinkel, S. A. et al. Jarid2 regulates hematopoietic stem cell function by acting with polycomb repressive complex 2. Blood 125, 1890–1900 (2015)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Redmond, D., Poran, A. & Elemento, O. Single-cell TCRseq: paired recovery of entire T-cell alpha and beta chain transcripts in T-cell receptors from single-cell RNAseq. Genome Med. 8, 80 (2016)

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  34. Orkin, S. H. & Zon, L. I. Hematopoiesis and stem cells: plasticity versus developmental heterogeneity. Nat. Immunol. 3, 323–328 (2002)

    CAS  PubMed  Article  Google Scholar 

  35. Jaffredo, T. et al. From hemangioblast to hematopoietic stem cell: an endothelial connection? Exp. Hematol. 33, 1029–1040 (2005)

    PubMed  Article  Google Scholar 

  36. Hirschi, K. K. Hemogenic endothelium during development and beyond. Blood 119, 4823–4827 (2012)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  38. Souilhol, C. et al. Inductive interactions mediated by interplay of asymmetric signalling underlie development of adult haematopoietic stem cells. Nat. Commun. 7, 10784 (2016)

    ADS  CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Yzaguirre, A. D., de Bruijn, M. F. & Speck, N. A. The role of Runx1 in embryonic blood cell formation. Adv. Exp. Med. Biol. 962, 47–64 (2017)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  40. Rafii, S. et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood 84, 10–19 (1994)

    CAS  PubMed  Article  Google Scholar 

  41. Roden, M. M., Lee, K. H., Panelli, M. C. & Marincola, F. M. A novel cytolysis assay using fluorescent labeling and quantitative fluorescent scanning technology. J. Immunol. Methods 226, 29–41 (1999)

    CAS  PubMed  Article  Google Scholar 

  42. Israely, E. et al. Akt suppression of TGFβ signaling contributes to the maintenance of vascular identity in embryonic stem cell-derived endothelial cells. Stem Cells 32, 177–190 (2014)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  43. Raphael Lis, Karrasch C. C. & Rafii, S. In vitro conversion of endothelial cells into haematopoietic stem cells. Protoc. Exch. http://dx.doi.org/10.1038/protex.2017.033 (2017)

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Acknowledgements

We thank J. Downing at St Jude Hospital for providing the Runx1-IRES-GFP reporter mice. Floxed Cxcr4 mice were provided by Y.-R. Zou (the Feinstein Institute for Medical Research). We are grateful to V. Sandler for constructive discussions. R.L., W.S., K.S., and S.R. are supported by Ansary Stem Cell Institute (ASCI), New York State Department of Health grants (NYSDOH) (C026878, C028117, C029156, C030160), NIH-R01 (DK095039, HL119872, HL128158, HL115128, HL099997) and U54 CA163167, the Starr foundation TRI-Institution stem cell core project, Tri-Institutional Stem Cell Initiative grants (TRI-SCI#2013-032, #2014-023, #2016-013, and fellowships), R.L., A.R.T., and S.R. by the Qatar National Priorities Research Program (NPRP 8-1898-3-392, NPRP 6-131-3-268), B.K. by NIH-T32 HD060600. J.M.B. is supported by the ASCI, TRI-SCI #2013-022 and #2014-004, Leukemia & Lymphoma Society (LLS) grant 0859-15, and NIH-R01 (CA204308, HL133021); J.M.S. by the ASCI, Taub Foundation Grants Program, TRI-SCI#2014-023 and #2016-024, LLS grant 2299-14, (NYSDOH) C029156, C030160, ECRIP, and NIH R01 (HL119872, HL128158) and by Cancer Research & Treatment Fund (CR&T), J.M.S. and S.R. by the ECRIP and NYSDOH, and N.A.S. by NIH-RO1 HL091724.

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Authors and Affiliations

Authors

Contributions

R.L., J.M.S. and S.R. designed the study. R.L. and C.C.K. performed endothelial cell isolation, conversion, transplantation, and transplant analysis. M.G.P. analysed haematopoietic recovery after irradiation, C.R.B. irradiated recipients, W.S. and J.G.B.D. prepared lentiviral particles. M.G. isolated endothelial cells. B.K., D.R., J.X., and A.R.T. performed RNA-seq and statistical analysis, supervised by O.E. Z.R., K.S., N. A.S., J.M.B., and J.M.S. interpreted results and edited the manuscript. R.L., C.C.K., J.M.S. and S.R. wrote the manuscript.

Corresponding author

Correspondence to Shahin Rafii.

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Competing interests

S.R. is the founder of and a non-paid consultant for Angiocrine Bioscience. J.M.B. receives research funding from Angiocrine Bioscience, and M.G. is an employee of Angiocrine Bioscience.

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Extended data figures and tables

Extended Data Figure 1 Phenotypic and quality control characterization of the rEC-HSPCs.

a, 8.0 × 105 freshly isolated adult lung mouse CD45.2 endothelial cells (mECs), depleted of lymphatic endothelial cells or contaminating haematopoietic cells were purified and injected in lethally irradiated CD45.1+ recipients, graph indicates donor contribution to peripheral blood at indicated time points after transplant. Data represent independent transplantations, n = 5. b, Wild-type normal LT-HSCs (CD45.2+ LKS-SLAM cells) were sorted, transduced with FGRS transgenes and expanded in endothelial cell growth medium containing FGF-2, serum and TGFβ inhibitor, without haematopoietic growth factors. Expanded FGRS-transduced LT-HSCs were then plated in co-culture with VN-ECs and FGRS were turned on (dox-on) for 28 days and then transplanted into lethally irradiated CD45.1+ recipients. Graphs represent peripheral blood contribution 16 weeks after transplantation. Data represent independent transplantations, n = 3. c, qRT–PCR showing expression of FGRS in mECs upon doxycycline addition. All four factors were absent when adult mECs were not exposed to doxycycline, or exposed to doxycycline without rtTA. Data represent mean ± s.d., n = 3. d, Time-course of CD45+ cell formation during the stage-specific conversion process. e, Time-course analysis of linc-Kit+Sca-1+ (LKS) converted cells. f, Diff-Quik stain of cells from day 28 in vitro cultures (original magnification, ×60; scale bar, 50 μm). g, Absolute quantification of LKS cells present whether in the fraction adherent to VN-ECs or in supernatant. Representative picture of rEC-HSPC adherent fraction with prototypical ‘cobblestone’ structures is shown on the left-hand side, n = 3 independent reprogramming experiments. Data represent mean ± s.e.m., n = 3, individual data points are represented. h, FGRS-transduced mECs were grown on VN-ECs, OP9-DLL11, or in feeder-free conditions. Graph indicates absolute quantification (cell number) of CD45+ rEC-HSPC. Data represent mean ± s.e.m. (n = 5 for conversion experiments run in technical triplicates for each conditions). i, FGRS-transduced OP9 were grown onto VN-ECs, OP9-DLL11, or in feeder-free conditions. Graph indicates absolute quantification (cell number) of CD45+ rEC-HSPCs (n = 5). Data represent mean ± s.e.m. (n = 5 conversion experiments run in technical triplicates for each conditions).

Source data

Extended Data Figure 2 rEC-HSPCs are composed of rEC-HSCs that have the potential for primary and secondary engraftment and regenerative haematopoiesis self-renewal.

a, Kaplan–Meier curve showing percentage survival over 16 weeks of lethally irradiated mice transplanted with either 8.0 × 105 cells (purified CD45+ rEC-HSPCs, green line (n = 20 mice); non-converted lung endothelial cells, blue line (n = 10 mice)) or PBS (black line, n = 15 mice). b, Representative plots of rEC-HSPC lineage contribution. c, Donor reconstitution of mice transplanted with CD45+ rEC-HSPCs at indicated time points after primary transplantation. Data represent individual data points (n = 20). d, Representative plots of donor contribution to LKS-SLAM cells. e, Donor reconstitution of mice transplanted with WBM from chimaeric WBM control mice or WBM from chimaeric rEC-HSPC primary transplanted mouse at indicated time points after transplantation. Data represent individual data points, n = 15, four independent reprogramming experiments. f, Schematic representation of haematopoietic recovery following sub-lethal irradiation assay. g, Analysis of white blood cell recovery of rEC-HSPC-engrafted versus control mice following sub-lethal irradiation (500 cGy) (n = 5 for duration of analysis). Data represent mean ± s.e.m., no significant differences were found using two-tailed unpaired t-test. h, Multi-lineage analyses during bone marrow recovery. Myeloid and lymphoid regeneration, including CD3+CD4+ T cells, and CD3+CD8+ T cells at baseline and 28 days post sub-lethal irradiation (500 cGy). Data represent individual data points; black bar represents mean (n = 5).

Source data

Extended Data Figure 3 Peripheral and splenic rEC-HSPC-derived T cell phenotyping.

a, Gating strategy to phenotype naive, effector and memory T cells from peripheral blood of transplanted mice. b, Boxplot showing the averaged frequency of naive, effector and memory T cells for both CD3+CD4+ and CD3+CD8+ subpopulations, following long-term primary or secondary transplant. WBM control samples are denoted in blue, rEC-HSPC in green. Data represent mean ± s.e.m. (n = 5). P values, two-tailed unpaired t-test. c, Gating strategy to phenotype γδ T cells from peripheral blood of transplanted mice. d, Boxplot showing the averaged frequency of γδ T cells following long-term primary or secondary transplant. Data represent mean ± s.e.m. (n = 5); two-tailed unpaired t-test. e, Boxplot showing the absolute number of B220+, CD3+CD4+, and CD3+CD8+ cells following long-term primary or secondary transplant. WBM control samples are denoted in blue, rEC-HSPC in green. Boxplot and whiskers represent median, 25th and 75th percentile, mean is represented by ‘+’ sign. (n = 5); two-tailed unpaired t-test. f, Phenotype of regulatory T cells (Treg) by flow cytometry.

Source data

Extended Data Figure 4 Endothelial to haematopoietic conversion capture by live microscopy.

(See also Supplementary Video 1). a, Schema detailing the experimental setting for live confocal image capture. Adult lung mECs were isolated from Runx1-IRES-GFP. Then, Runx1-IRES-GFP mECs were transduced with FGRS and co-cultured with VN-ECs (HUVEC-E4ORF1). VN-ECs were differentiated from FGRS-transduced Runx1-IRES-GFP adult lung mECs by anti-human CD31 live staining (hCD31) (red). Live confocal images were acquired every 45 min for the duration of the experiment (see also Supplementary Video 1). b, Representative flow cytometry plots of day 10 (d10) showing that a subset of VEcad+CD45 and VEcad+CD45+ cells also co-express Runx1-GFP. c, Single time points from live confocal image capture. Upon doxycycline-dependent conditional expression of FGRS, flat spindle-shaped adult mECs rapidly transition from RUNX1 to round haematopoietic-like RUNX1+ cells (day 0–8, white arrow). This induction phase is characterized by the transitioning endothelial cells towards haematopoietic fate. From day 8 to 20 of specification phase, RUNX1+ cells further moved towards a haematopoietic identity by assuming a prototypical fully formed round shape (white arrow). Following the emergence of this definite haematopoietic program, a phase of robust expansion in the vascular niche layers (VN-ECs) of these RUNX1+ committed converted cells is observed from day 20 to 28 (expansion phase).

Extended Data Figure 5 Quantification of rEC-HSCs arising from single-cell reprogramming.

a, Haematopoietic cluster arising from single-cell reprograming at specified time points. Wells were considered negative if no haematopoietic clusters were visible 36 days after single RUNX1+ endothelial cell inoculation (mECs were isolated from Runx1-IRES-GFP mice). Representative wells considered as positive or negative are shown (the magnification used was 10×). Cloning of RUNX1+ mECs resulted in 22 ± 11 clusters per 1,000 RUNX1+ mECs sorted. b, Long-term peripheral blood contribution of each reprogrammed day 8 RUNX1+ endothelial cells. Data represent individual data points for each clones (n = 7) c, Limiting-dilution transplantation (LDT) assay showing the frequency of LT-HSCs in expanded clonal conversion experiments. CRU per LKS cells were determined using Poisson statistics by ELDA software. d, Viral integration mapping. PCR against LTR-B1 repeated sequence was run in each indicated populations. PCR assays were analysed on 4% TBE gel. CLP, common lymphoid progenitor; GMP, granulocyte–macrophage progenitor; MEP, megakaryocytic–erythroid progenitors. Progenitors were isolated as described in ref 20.

Source data

Extended Data Figure 6 Gene expression profiling of rEC-HSPCs and rEC-HSCs.

a, Supervised principal component analysis of global gene expression data of rEC-HSPCs and rEC-HSCs and the indicated control cell types. Data for individual cells of given cell types are indicated. Each shape represents an independent replicate for the indicated cell types (embryonic day 11 (E11.0) aorta–gonad–mesonephros endothelium, E11.0 CD201 pre-HSC type 1, E11.0 CD201+ pre-HSC type 1, E11.0 CD201+ pre-HSC type 2, E12.5 fetal liver HSCs (linSca-1+Mac1loCD201+), E14.5 fetal liver HSCs (linCD45+CD150+CD48CD201), adult bone marrow HSC (LKS-SLAM), ‘sc-’ refers to the single-cell RNA-seq dataset adapted from ref. 31; adult lung endothelial cells, in vitro rEC-LKS, in vivo rEC-LKS-SLAM, control adult in vivo LKS-SLAM cells). b, Supervised clustering of canonical endothelial genes expression profiles of freshly isolated lung endothelial cells (n = 3), embryonic stem cell (ES)-derived endothelial cells (n = 2), embryonic stem cells (n = 1), rEC-LKS-SLAM cells isolated from transplanted mice (n = 3), LKS-SLAM cells isolated from transplant control mice (n = 3)32. day 28 in vitro rEC-LKS (n = 3), data points obtained from clonal reprogramming are denoted by an asterisk. c, Supervised clustering of prototypical haematopoietic genes expression demonstrates that haematopoietic genes are induced during FGRS-mediated reprograming of endothelial cells into rEC-HSPCs and rEC-HSCs. d, Supervised clustering of prototypical pluripotency genes expression demonstrates that pluripotency genes are not induced during FGRS-mediated reprograming of endothelial cells into rEC-HSPCs and rEC-HSCs (be, rEC* refers to clonal rEC-HSPCs). e, Dendrogram showing unsupervised hierarchical clustering of global gene expression data of representative control cells (ctl), and all rEC-HSCs, rEC-HSPCs, and their progenies. Dendrogram branches are colour-coded per cell types indicated in the legend.

Extended Data Figure 7 Immune function assessment and molecular profiling of TCR diversity of rEC-HSC-derived T cells.

a, CFSE dilution and intracellular interferon-γ (IFNγ) production upon CD45.2+ CD3/CD28 polyclonal activation and/or Treg addition. Representative flow cytometry plots. b, Quantification of intracellular IFNγ production upon CD3/CD28 polyclonal activation and/or Treg addition. Data represent mean ± s.e.m. (n = 5 independent experiments, 3 technical replicates); two-tailed unpaired t-test. c, Normalized counts for CD3+CD4+ cells. WBM control samples are denoted in blue, rEC-HSPC in green. d, Normalized counts for CD3+CD8+ cells. WBM control samples are denoted in blue, rEC-HSPC in green. e, Normalized counts for Jurkat cell samples. f, Analysis of TCR repertoire in Rag1−/− rEC-HSPC reconstituted mice upon chicken ovalbumin vaccination.

Source data

Extended Data Figure 8 Molecular deconvolution of vascular-niche-derived angiocrine factors during stepwise differentiation of endothelial cells into rEC-HSPCs and rEC-HSCs.

a, Adult lung mECs (VEcad+CD31+CD45) were isolated from Runx1-IRES-GFP mice. Human-derived VN-ECs were discriminated from FGRS-transduced Runx1-IRES-GFP adult lung mECs by anti-human CD31 (hCD31). FGRS-transduced Runx1-IRES-GFP adult lung mECs and their progeny were gated as hCD31. Flow cytometry plots showing the expression of VEcad and CD45 in mouse hCD31 FGRS-transduced endothelial cells and derivatives over the course of endothelial to haematopoietic cell reprogramming. b, Quantification of mouse hCD31 FGRS-transduced mECs and their derivatives over the course of reprogramming. Data represent mean ± s.e.m. (n = 5). c, Colony number arising in methylcellulose from FGRS-transduced mECs and derivatives (gated on human hCD31 to exclude VN-EC feeders). n = 4 independent experiments are shown and each condition performed in triplicate. Data represent mean ± s.e.m. (n = 5 technical replicates); two-tailed unpaired t-test. d, Adult mECs were treated with different small molecules at their known IC50 (CXCR4 antagonist, AMD3100, 44 μmol l−1; CXCR7 agonist, TC14012, 350 nmol l−1; BMP antagonist, Noggin, 0.5 μg ml−1; TGFβ antagonist, SB431542, 10 μmol l−1). Representative flow cytometry plots of apoptosis assays are presented. e, Quantification of apoptotic cells following treatment with each small molecule tested. Data represent mean ± s.e.m. (n = 3); two-tailed unpaired t-test. f, Quantification of hCD31 FGRS-transduced endothelial cells and their derivatives over the course of reprogramming in presence of CXCR4 inhibitors (AMD3100, 44 μmol l−1), CXCR7 agonists (TC14012, 350 nmol l−1), BMP inhibitor (Noggin, 0.5 μg ml−1), TGFβ/ALK5 inhibitor (SB431542, 10 μmol l−1). Data represent mean ± s.e.m. (n = 5); two-tailed unpaired t-test. g, Quantification of hCD31 FGRS-transduced mECs and their derivatives over the course of reprogramming in the presence of VN-ECs overexpressing mouse CXCL12 (n = 5 independent experiments, 3 technical triplicates each). Data represent mean ± s.e.m.; two-tailed unpaired t-test.

Source data

Extended Data Figure 9 Organ-specific adult mECs are amenable to hierarchical FGRS-mediated reprogramming to rEC-HSPCs.

a, Left, FGRS-transduced liver mECs were directly co-cultured with VN-ECs. Graph indicates absolute quantification (cell number) of CD45+ rEC-HSPC (n = 3 independent biological replicates, 3 technical replicates each). Right, quantification of phenotypically marked CD45+ LKS cells at day 28 of reprogramming absolute cell number is reported. Data represent mean ± s.e.m. b, Left, FGRS-transduced lung mECs were directly co-cultured with VN-ECs. Graph indicates absolute quantification (cell number) of CD45+ rEC-HSPC Right, quantification of phenotypically marked CD45+ LKS cells at day 28 of reprogramming absolute cell number is reported. Data represent mean ± s.e.m. (n = 3 independent biological replicates, 3 technical replicates each). c, Left, FGRS-transduced brain mECs were directly co-cultured with VN-ECs. Graph indicates absolute quantification (cell number) of CD45+ rEC-HSPCs (n = 3 independent biological replicates, 3 technical replicates each). Right, quantification of phenotypically marked CD45+ LKS cells at day 28 of reprogramming absolute cell number is reported. Data represent mean ± s.e.m. d, Left, FGRS-transduced kidney mECs were directly co-cultured with monolayers of confluent VN-ECs. Graph indicates absolute quantification (cell number) of CD45+ rEC-HSPCs. Right, quantification of phenotypically marked CD45+ LKS cells at day 28 of reprogramming absolute cell number is reported. Data represent mean ± s.e.m. (n = 3 independent biological replicates, 3 technical replicates each). e, Terminally differentiated rEC-HSPC-derived cells were purified and co-cultured on VN-ECs in presence of doxycycline for 28 days. Cells were transplanted into lethally CD45.1+ irradiated recipients in absence of doxycycline. Data represent individual data points for donor contribution to peripheral blood at indicated time point after transplant (n = 3 biological replicates). f, Experimental model for hierarchical differentiation of endothelial cells into rEC-HSPCs. D8 VEcad+RUNX1+CD45 cells were purified by flow cytometry and replated on inductive vascular niche in presence or absence of doxycycline. Subsequently, at day 15 VEcad+RUNX1+CD45+ haemogenic-like cells were purified by flow cytometry and replated on the inductive vascular niche in presence (dox-on) or absence (dox-off) of dox. g, h, Flow cytometry quantification of cell subsets during stepwise conversion in f. Data represent individual data points (n = 3 independent biological replicates, 3 technical replicates each).

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Extended Data Figure 10 Analyses of the rEC-HSPC- and rEC-HSC-engrafted organs for malignant transformation.

Although none of the recipient mice engrafted with rEC-HSPCs manifested any anatomical or symptomatic evidence of leukaemia, lymphoma or myeloproliferative neoplasm (MPN) (that is, lymphadenopathy, organomegaly, illness or haemorrhage), we analysed recipient organ architecture and histological profile after 20 weeks of primary transplantation, or after serial (an additional 20 weeks) secondary transplantation for any evidence of malignant alterations. For each organ, including bone marrow, lung, kidney, spleen, liver, intestine and brain, Wright–Giemsa (left), Masson (middle) and PicroSirius Red (right) staining is shown at 2 different magnifications (10×, top and 40× bottom; scale bars, 10 μm and 40 μm, respectively). We did not observe any evidence of aberrant infiltration of haematopoietic cells, abnormal inflammatory response, chloromas, or alteration of the geometry of any organs of the primary or secondary transplanted mice. Furthermore, microscopic architecture of bone marrow, spleen and liver was normal and without fibrotic remodelling or abnormal deposition of collagen or desmin. All images were acquired using a colour CCD camera. n = 3 independent primary or secondary transplant experiments. Representative experiments are shown.

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Endothelial to hematopoietic capture by live microscopy

Adult lung ECs were isolated from Runx1-IRES-GFP. Then, Runx1-IRES-GFP adult lung ECs were transduced with FGRS and co-cultured with VN-ECs (HUVEC-E4ORF1). VN-ECs were discriminated from FGRS-transduced Runx1-IRES-GFP adult lung ECs by anti-human CD31 (hCD31) live staining (red). Live confocal images were acquired every 45 min for the duration of the experiment (original magnification 10x). (MOV 4710 kb)

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Lis, R., Karrasch, C., Poulos, M. et al. Conversion of adult endothelium to immunocompetent haematopoietic stem cells. Nature 545, 439–445 (2017). https://doi.org/10.1038/nature22326

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