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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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.
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.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
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 lin−c-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).
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).
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.
(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).
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.
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 (lin−Sca-1+Mac1loCD201+), E14.5 fetal liver HSCs (lin−CD45+CD150+CD48−CD201−), 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 (b–e, 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.
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.
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).
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.
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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