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Reprogramming human endothelial cells to haematopoietic cells requires vascular induction

Nature volume 511, pages 312–318 (2014)Cite this article

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Abstract

Generating engraftable human haematopoietic cells from autologous tissues is a potential route to new therapies for blood diseases. However, directed differentiation of pluripotent stem cells yields haematopoietic cells that engraft poorly. Here, we have devised a method to phenocopy the vascular-niche microenvironment of haemogenic cells, thereby enabling reprogramming of human endothelial cells into engraftable haematopoietic cells without transition through a pluripotent intermediate. Highly purified non-haemogenic human umbilical vein endothelial cells or adult dermal microvascular endothelial cells were transduced with the transcription factors FOSB, GFI1, RUNX1 and SPI1 (hereafter referred to as FGRS), and then propagated on serum-free instructive vascular niche monolayers to induce outgrowth of haematopoietic colonies containing cells with functional and immunophenotypic features of multipotent progenitor cells (MPPs). These endothelial cells that have been reprogrammed into human MPPs (rEC-hMPPs) acquire colony-forming-cell potential and durably engraft into immune-deficient mice after primary and secondary transplantation, producing long-term rEC-hMPP-derived myeloid (granulocytic/monocytic, erythroid, megakaryocytic) and lymphoid (natural killer and B cell) progenies. Conditional expression of FGRS transgenes, combined with vascular induction, activates endogenous FGRS genes, endowing rEC-hMPPs with a transcriptional and functional profile similar to that of self-renewing MPPs. Our approach underscores the role of inductive cues from the vascular niche in coordinating and sustaining haematopoietic specification and may prove useful for engineering autologous haematopoietic grafts to treat inherited and acquired blood disorders.

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Figure 1: Reprogramming of HUVECs and hES-ECs into haematopoietic cells by FGRS transcription-factor transduction and vascular induction.
Figure 2: rEC-hMPPs phenotypically and functionally resemble multilineage HSPCs.
Figure 3: rEC-hMPPs are capable of in vivo erythro-myeloid-megakaryocytic multilineage engraftment.
Figure 4: Functional and transcriptional analysis of adult hDMEC-derived rEC-hMPPs.
Figure 5: Adult human hDMEC-derived rEC-hMPPs are capable of in vivo primary and secondary multilineage engraftment.

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Gene Expression Omnibus

Data deposits

Data have been deposited in the GEO database under accession number GSE57662.

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Acknowledgements

V.M.S. is supported by Empire State Stem Cell Board (ESSCB) and New York State Department of Health (NYSDH C026878). S.R. is supported by Ansary Stem Cell Institute (ASCI), HHMI, ESSCB/NYSDH (C024180, C026438, C026878, C028117), NHLBI (R01HL097797, R01HL119872, U01 HL099997), NIDDK (R01DK095039) , NCI (U54CA163167), Qatar National Priorities Research Foundation grant NPRP08-663-3-140 and the Qatar Foundation BioMedical Research Program. J.M.S. is supported by grants from NCI (CA159175 and CA163167), NHLBI (HL119872 and HL055748), Starr Foundation and a Leukemia & Lymphoma Society Scholar award. J.M.B. is supported by an American Society of Hematology Scholar Award, NHLBI U01-HL099997 and Angiocrine Bioscience and ASCI. We acknowledge the contribution of J. Z. Xiang and Agnes J. Viale for enabling and executing molecular profiling, and E. Gars for technical support. We appreciate W. Schachterle for recommendations and edits of the manuscript.

Author information

Authors and Affiliations

  1. Department of Genetic Medicine, Ansary Stem Cell Institute, and Howard Hughes Medical Institute, Weill Cornell Medical College, New York, 10065, New York, USA

    Vladislav M. Sandler, Raphael Lis, Ying Liu, Alon Kedem, Daylon James, Jason M. Butler & Shahin Rafii

  2. Ronald O. Perelman and Claudia Cohen Center for Reproductive Medicine, Weill Cornell Medical College, New York, 10065, New York, USA

    Raphael Lis, Alon Kedem & Daylon James

  3. HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine, Weill Cornell Medical College, New York, 10065, New York, USA

    Olivier Elemento

  4. Department of Medicine, Hematology-Oncology, Weill Cornell Medical College and the New York Presbyterian Hospital, New York, 10065, New York, USA

    Joseph M. Scandura

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  1. Vladislav M. Sandler
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Contributions

V.M.S. and S.R. conceived and designed the project, performed experiments, analysed the data and wrote the manuscript. R.L., Y.L. and J.M.B. performed experiments, interpreted and analysed data. J.M.S. interpreted, analysed data and wrote the manuscript. D.J., O.E. and A.K. performed the experiments and analysed the data. All authors commented on the paper.

Corresponding author

Correspondence to Shahin Rafii.

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Competing interests

S.R. is the founder of and consultant to Angiocrine Bioscience New York, NY. J.M.B. received sponsored research funding from Angiocrine Bioscience New York, NY.

Extended data figures and tables

Extended Data Figure 1 Screening strategy for identification of a minimal set of transcription factors for reprogramming endothelial cells into haematopoietic cells.

a, Candidate genes tested for reprogramming of HUVECs into haematopoietic colonies. To identify transcription factors that drive EHT transition, we performed RNA-seq of HUVECs and Lin−CD34+ umbilical cord HSPCs to select transcription factors differentially expressed by HSPCs, but not by HUVECs. We then screened various combinations of differentially expressed transcription factors to identify a minimal set capable of reprogramming endothelial cells to haematopoietic cells. Levels of expression (log2[RNA-seq value]) in HUVECs and freshly purified Lin−CD34+ cord blood cells are shown. b, One-by-one elimination of transcription factor experiment revealed a minimal set of transcription factors—FOSB, GFI1, RUNX1 and SPI1 (FGRS)—capable of generating haematopoietic colonies in the HUVEC culture. A pooled set of 26 transcription factors minus one transcription factor was evaluated for the ability to evoke formation of haematopoietic clusters (day 21–25; n = 3). Asterisks show statistically significant (P < 0.05) reduction of the number of haematopoietic clusters in the transduced HUVECs compared to the full set of transcription factors. Control represents non-transduced HUVECs. Transduced cells were cultured on a layer of serum-free E4EC monolayers. c, One-by-one elimination of the FGRS factors shows that all four FGRS transcription factors are necessary and sufficient for generation of haematopoietic colonies (day 21–25; n = 3). ‘All’ in b and c indicates that all transcription factors are present. ‘Not transduced’ in b and c indicates that all transcription factors are absent. b, c, Error bars are average ± s.d. d, Schema for reprogramming of endothelial cells into human multipotent progenitor cells (rEC-hMPPs). Clonal or bulk populations of HUVECs or hDMECs were transduced with the FGRS and after 3 days were re-plated on subconfluent monolayers of E4EC endothelial cells (VeraVecs). The emerging colonies of haematopoietic cells were subjected to (1) multivariate immunophenotypic analyses; (2) clonal and oligo-clonal CFC assay; and (3) molecular profiling (RNA-seq). Tissues of the engrafted mice were processed for histological examination to rule out any malignant transformation.

Extended Data Figure 2 FGRS transduction and vascular induction reprogram HUVECs, but not hES-ECs, to proliferating functional rEC-hMPPs.

a, Multi-colony niche-like structure that physically separates developing haematopoietic colonies from surrounding E4EC vascular niche. The emerging multi-colony sinusoidal-like structures create a unique cellular interface between E4EC monolayers and transduced endothelial cells giving rise to haematopoietic clusters (n = 4, scale bar is 1,000 μm). b, Expansion potential of reprogrammed hCD45+ haematopoietic cells. hCD45+ (12 × 103) and hCD45− (60 × 103) cells were sorted into separate wells and expanded for 2 days. We observed fivefold expansion of hCD45+ cells (56.6 × 103 ± 7.9 × 103; n = 3) and marked reduction of hCD45− cell number (4.6 × 103 ± 1.0 × 103; n = 3). c, Clonal expansion of hCD45+ cells. hCD45+ cells were FACS sorted into 96-well plates at the density of 1 and 2 cells per well. After 7 days of culture, we observed hCD45+ cell expansion in 6.3 ± 2.1 wells (93.1 ± 14.5 cells per well) of 1-cell sort and 29.0 ± 4.3 wells (112.1 ± 21.2 cells per well) of the 2-cell sort (n = 3). The difference between cell number in 1- and 2-cell sort was statistically not significant (P = 0.78), suggesting that the difference in the number of wells with detected cell expansion was due to survival of sorted cells rather than a reflection of the number of cells sorted into a well. d, Reprogramming of hES-derived endothelial cells (hES-ECs) into haematopoietic cells. Representative experiment demonstrating that transduction of hES-ECs with FGRS (F and G lentivector constructs containing IRES-GFP cassette) and E4EC vascular induction generated significantly higher numbers of GFP+hCD45+hCD144 − cells (four panels on the right) compared to control non-transduced hES-ECs (three panels on the left). To accurately detect the expression of CD144 in the hES-ECs being reprogrammed into putative rEC-hMPPs, we used fluorescent monoclonal antibodies to human CD144. e, Lineage-specific surface marker analysis of the hGFP+CD45+ population of rEC-hMPPs. hGFP+CD45+ population showed that some of these cells expressed lineage-specific surface markers, such as hCD43+ (8.96 ± 2.3%; n = 3), hCD90+ (Thy-1+) (6.15 ± 1.13%; n = 3) and hCD14+ (40.0 ± 4.95%; n = 3) (representative flow cytometry measurements; top four panels, statistics for all experiments is in the bottom bar graph, n = 3). f, Immunophenotypic analysis of CFC colonies grown in the CFC assay performed in Fig. 2c, d. g, Macrophages differentiated from rEC-hMPPs are functionally capable of phagocytosis. The images (upper row and lower left) show groups of firmly plastic-adherent hCD14+ cells (red staining) with clearly visible phagocytosed green fluorescent beads (GFB; green). Endothelial CD144+(VE-cadherin) cells (white staining) were not co-localized with beads. Most (85.1 ± 15.1%) GFBs were localized inside hCD14+ cells (bottom-right graph, 1). A smaller population of GFBs was distributed outside hCD14+ and CD144 (VE-cadherin)+ cells (14.8 ± 7.43%; bottom-right graph, 2). The percentage of GFBs co-localized with endothelial cells was negligible (4.8 ± 0.83%; bottom-right graph, 3), n = 9. Scale bars are 25 μm. Error bars are average ± s.d.

Extended Data Figure 3 Naive HUVECs are devoid of haemogenic potential capable of spontaneous differentiation into MPPs.

We performed two sets of experiments to exclude the possibility that rEC-hMPPs were derived from spontaneously differentiating HUVECs with haemogenic or haemangioblastic potential25,34. a, b, In optimal pro-haematopoietic cultures, naive non-transduced endothelial cells fail to spontaneously differentiate into rEC-hMPPs. a, We grew non-FGRS-transduced HUVECs in the serum-free media used for reprogramming. Neither serum withdrawal nor addition of haematopoietic cytokines induced formation of hCD45+hCD34+ cells and HUVECs sustained their vascular identity. Indeed, serum withdrawal increases the number of CD34+ HUVECs. CK, cytokine cocktail (see Methods); SB, TGF-β inhibitor SB431542; SF, serum free. b, Serum withdrawal suppresses HUVEC proliferation. Inhibition of TGF-β signalling (SB) combined with cytokine cocktail (see Methods) restores proliferative potential of HUVECs in serum free media. The difference between proliferation of HUVECs in serum free media and all other conditions is statistically significant (asterisk; P < 0.005). Statistical significance between pairs of different conditions is shown with blue arrows and P values, where P < 0.005 is statistically significant. Therefore, human rEC-hMPPs originate from reprogrammed endothelial cells, but not cytokine-mediated outgrowth of contaminating pre-existing haemogenic endothelial cells. c–e, Clonal reprogramming of non-haemogenic HUVECs into rEC-hMPPs using FGRS transduction and vascular induction. We performed endothelial cell clonal reprogramming experiments to exclude the possibility that rEC-hMPPs were derived from spontaneously differentiating HUVECs with pre-existing haemogenic or haemangioblastic potential25,34. c, Because E-selectin is only expressed in activated endothelial cells, we generated clonal cultures of CD45−CD144+CD31+CD62E (E-selectin)+ endothelial cells32,33. To this end, CD144+CD31+CD62E+CD45−HUVECs were sorted into 96-well plates at 1, 2, 5 and 10 cells per well densities for clonal expansion. Proliferating clones were transduced with FGRS and induced with serum-free E4EC monolayers. d, These clonal cultures yielded rEC-hMPPs comparable to bulk HUVEC cultures. The numbers of haematopoietic-like colonies emerging from 1-cell, 2-cell, 5-cell and 10-cell clones are not statistically different (P > 0.05). e, An example of a haematopoietic-like colony derived from a 1-cell clone number 2. It is unlikely that rEC-hMPPs are derived through spontaneous differentiation of pre-existing endothelial cells with haemogenic or haemangioblastic potential. Error bars are average ± s.d. Scale bars, 400 μm.

Extended Data Figure 4 Clonal reprogramming of non-haemogenic HUVECs into immunophenotypic and functional rEC-hMPPs using FGRS transduction and vascular induction.

a–c, CFC assay of reprogrammed hCD45+hCD34+ rEC-hMPPs generated from clonally selected CD45−CD144+CD31+CD62E (E-selectin)+ mature HUVECs, as shown in Extended Data Fig. 3d, e. CD45−CD144+CD31+CD62E+endothelial cells were sorted as 1 cell per well, 2 cells per well, and 5–10 cells per well. Expanding clones of the endothelial cells were transduced with FGRS and then induced with vascular niche. After 3 to 4 weeks, emerging hCD45+hCD34+ rEC-hMPPs were sorted out (red gate in FACS plots; upper left) and plated for CFC assay. Typical haematopoietic colonies arose in the assay (middle column, microphotographs; original magnification, ×4). FACS plots on the right show immunophenotypic analysis of the cells that arose in the CFC assay, demonstrating differentiation into human CD45−CD235+ erythroid, CD11b+ macrophage CD14+ monocytic, CD41a+ megakaryocytic and CD83+ dendritic progenies. The graph in the left lower corner shows quantification of the CFC assay (n = 3). Identical panels are shown for two 2-cell clones and one 5-cell clone. A total of three independent clones is shown. Thus, given the high efficiency of clonal reprogramming of mature authentic endothelial cells into rEC-hMPPs, it is unlikely that rEC-hMPPs are spontaneously derived from a very rare population of a pre-existing haemogenic or haemangioblastic HUVECs. Scale bars, 400 μm.

Extended Data Figure 5 Single-cell analysis of lentiviral integration into engrafted rEC-hMPPs.

Engrafted hCD45+ rEC-hMPPs purified from the bone marrow of primary NSG recipient mouse (Fig. 3e, f) were sorted into a 96-well plate (1 cell per well), lysed in corresponding well for whole genome amplification (WGA) using Phi29 enzyme (see Methods). Amplified DNA is shown for all 21 cells in the top two gels. Amplified DNA was used as a template for PCR reactions with a forward primer specific for the CMV promoter and reverse primer specific for the coding sequence of a reprogramming factor. t-test PCR with a lentiviral vector. EW indicates empty well (no template DNA). Red asterisks show failed PCR amplification of viral integration. PCR products are visible as low molecular mass bands labelled as 1, FOSB; 2, GFI1; 3, RUNX1; 4, SPI1.

Extended Data Figure 6 Conditional expression of FGRS is sufficient for optimal generation of rEC-hMPPs with multilineage potential, including T-cell lymphoid cells.

a–c, Conditional expression of mouse inducible FGRS (mFGRS) factors activates endogenous human FGRS in HUVECs sustaining functional haematopoietic cell fate of rEC-hMPPs. a, To test whether FGRS-induced reprogramming triggered expression of endogenous FGRS genes24, HUVECs were transduced with lentiviral vectors expressing mFGRS-Tet-On and a trans-activator, and grown on E4EC vascular niche for 18–22 days (n = 4) in the presence of doxycycline. Doxycycline was removed from the culture medium after 18–22 days to shut off the expression of mouse FGRS and cells were cultured for an additional 7–10 days. Human CD45+CD34+ cells were FACS isolated for CFC assay and whole-transcriptome deep sequencing (RNA-seq). CFC assay revealed emergence of haematopoietic colonies with cells expressing human CD235, CD11b, CD83 and CD14. b, Comparison of transcriptional gene profiles of human FGRS in: primary HUVECs; rEC-hMPPs reprogrammed from hDMECs by transduction with inducible Tet-On mouse FGRS (mFGRS) and vascular induction for 3–4 weeks (tet-CD45+hDMEC-rEC-hMPPs); engrafted human CD45+CD34+ cells purified from the bone marrow of NSG mice 22 weeks post-primary transplantation with HUVEC-reprogrammed rEC-hMPPs (HUVEC CD45+CD34+ in vivo); engrafted human CD45+CD34+ cells purified from the bone marrow of NSG mice 15 weeks post-secondary engraftment with hDMEC-reprogrammed rEC-hMPPs (hDMEC CD45+CD34+ in vivo); and naive purified Lin−CD34+ cells from cord blood (CB). c, Analysis of whole-transcriptome RNA-seq of rEC-hMPPs derived using inducible mouse FGRS (n = 3). All RNA-seq reads were aligned against human and mouse FGRS sequences. ‘Map to human’ indicates RNA-seq reads that align to human FGRS sequences; ‘Map to mouse’ indicates RNA-seq reads that align to mouse FGRS sequences; and ‘Map to mouse only’ indicates RNA-seq reads that align to mouse FGRS sequences without a possibility to align to human sequences. d, e, Optimizing differentiation of rEC-hMPPs into lymphoid progeny. d, The number of T-lymphoid progeny of engrafted rEC-hMPPs was negligibly small, raising the possibility that constitutive SPI1 expression prevents rEC-hMPPs from differentiating into T cells39,40. To test this, HUVECs were transduced with lentiviral vectors expressing GFP and that constitutively express FGR transcription factors with a Tet-inducible SPI1 (FGR+SPI1-Tet-On construct) for 3 days followed by re-plating for E4EC induction. After 27 days of FGR and doxycycline-induced SPI1 expression on E4ECs, GFP+hCD45+ haematopoietic-like colonies emerged. Then, doxycycline was withdrawn and the reprogrammed cells were cultured serum-free haematopoietic media (SFHM) with Delta-like-4 expressing OP9-stroma (OP9-DLL4) supplemented with IL-7, IL-11 and IL-2. There is an increase of the number of GFP+hCD45+ cells emerging during reprogramming of HUVECs by FGR+SPI1-Tet-On construct and E4EC induction. e, rEC-hMPPs differentiate into CD3+, CD19+ and CD14+ haematopoietic cells in the absence of exogenous expression of SPI1. After 3 weeks, the numbers of myeloid and lymphoid cells were quantified by flow cytometry. We were able to reliably detect a small fraction of CD3+ cells (0.16 ± 0.01%; n = 3), a larger number of CD19+ (1.17 ± 0.13%; n = 3) and CD14+ (16.46 ± 1.02%; n = 3) cells. Thus, generation of lymphoid cells from rEC-hMPPs could be optimized by transient expression of transcription factors. Error bars are average ± s.d.

Extended Data Figure 7 Adult human hDMEC-derived rEC-hMPPs are capable of in vivo primary multilineage engraftment.

a, Immunophenotypic analysis of cells grown in the CFC assay (from Fig. 4b). These panels show quantification of surface marker expression in the cells isolated from colonies in the CFC assay (n = 3). hDMECs differentiated into hCD45−CD235+ erythroid, CD11b+CD14+ monocyte/macrophage and CD83+ dendritic cell progenies. Minimal CD144 (VE-cadherin) was detected. b, Analysis of peripheral blood (PB) of mice at 4, 6 and 12 weeks after primary transplantation (Fig. 5a) revealed circulating hCD45+ and their hCD33+, hCD14+ myeloid and hCD45−hCD235+ erythroid progenies (n = 6). Mouse CD45 (mCD45+) cells were excluded from analyses. Mouse cells, blue; human cells, red. c, Analysis of spleen of mice at 14 weeks after primary transplantation (Fig. 5a) revealed the presence of hCD45+ (red gate) and their lymphoid (hCD19+, hCD56+) and myeloid (hCD11b+, hCD41a+) progenies (n = 3). Mouse CD45 (mCD45+ cells, blue populations) is shown. d, Analysis of mouse bone marrow at 14 weeks after primary transplantation (Fig. 5a). Lin−CD45RA− cells (blue gate) were analysed for CD38 and CD90 expression (green and red gates) and subsequently examined for human CD45 and CD34 expression. This analysis revealed the presence of hCD34+ cells with small populations of both Lin−CD45RA−CD38−CD90+CD34+ and Lin−CD45RA−CD38−CD90−CD34+ cells satisfying phenotypic definition of human HSCs and MPP, respectively (n = 3).

Extended Data Figure 8 Analysis of bone marrow and liver of primary transplanted mice for signs of malignant transformation.

Analysis of bone marrow (a) and liver (b) of mice 10 months after primary transplantation (from Fig. 3b) of HUVEC-derived rEC-hMPPs for signs of malignant transformation. The level of fibrosis was determined using Masson and PicroSirus staining. The architectonic geometry of the bone marrow was determined by sequential multi-cross-sectional Wright–Giemsa and haematoxylin and eosin (H&E) staining and compared to age-controlled, non-transplanted NSG mice. We did not observe any evidence of fibrosis or alteration of the geometry of the bone marrow or liver of the transplanted mice. Furthermore, no recipient mouse manifested any anatomical or symptomatic evidence of leukaemias, lymphomas or myeloproliferative neoplasm (MPN) (that is, lymphadenopathy, organomegaly, illness or haemorrhage). Circulating hCD45+ cells in peripheral blood displayed no evidence of lympho/myeloproliferation or dysplasia. Furthermore, microscopic architecture of bone marrow and liver was normal and without fibrotic remodelling or aberrant deposition of collagen or desmin. All images were acquired using a colour CCD camera. The scale bar is 200 μm for low-resolution images in the left columns and 50 μm for high-resolution images in the right columns. Upper-left image (Giemsa, control) shows a white square in the centre that corresponds to the portion of the image shown at high resolution on the right (the same Giemsa control sample). This rule applies to all images shown.

Extended Data Figure 9 Analysis of spleen of primary transplanted mice and bone marrow, spleen and liver of secondary transplanted mice for signs of malignant transformation.

a, b, Analysis of spleen of mice 10 months after primary transplantation (from Fig. 3b) of HUVEC-derived rEC-hMPPs as well as bone marrow (n = 2), spleen and liver (n = 2, also Extended Data Fig. 10a) of mice that were engrafted with secondary transplanted hDMEC-derived rEC-hMPP cells 15 weeks after transplantation (from Fig. 5b) for signs of malignant transformation. The level of fibrosis was determined using Masson and PicroSirus stainings. The architecture of the bone marrow was determined by sequential multi-cross-sectional Wright–Giemsa and haematoxylin and eosin (H&E) staining and compared to age-controlled non-transplanted NSG mice. We did not observe any evidence of fibrosis or alteration of the geometry of the bone marrow, spleen or liver of the transplanted mice. Furthermore, no recipient mouse manifested any anatomical or symptomatic evidence of leukaemias, lymphomas or myeloproliferative neoplasm (MPN) (that is, lymphadenopathy, splenomegaly/organomegaly, illness or haemorrhage). Circulating hCD45+ cells in peripheral blood displayed no evidence of lympho/myeloproliferation or dysplasia. Furthermore, microscopic architecture of bone marrow, spleen and liver was normal and without fibrotic remodelling or aberrant deposition of collagen or desmin. All images were acquired using a colour CCD camera. In primary transplants the scale bar is 200 μm for low-resolution images in the left columns and 50 μm for high-resolution images in the right columns. The upper-left image (Giemsa, control) shows a white square in the centre that corresponds to the portion of the image shown at high resolution on the right (the same Giemsa control sample). This rule applies to all images shown (primary transplant). All images in secondary transplant are acquired at an original magnification of ×60. All images are acquired at original magnification of ×60. The top rows of images for each organ are secondary transplants; bottom rows of images for each organ are controls.

Extended Data Figure 10 Analysis of liver and spleen of secondary transplanted mice for signs of malignant transformation and analyses of rEC-hMPPs for genetic stability.

a, Analysis of liver and spleen of secondary transplanted mice for signs of malignant transformation. Repeat analysis of spleen and liver of mice that were engrafted with secondary transplanted hDMEC-derived rEC-hMPP cells 15 weeks post-transplantation for signs of malignant transformation (from Fig. 5b). The level of fibrosis was determined by Masson and PicroSirus stainings. The architecture of the bone marrow was determined by sequential multi-cross-sectional haematoxylin and eosin (H&E) staining and compared to age-controlled non-transplanted NSG mice. We did not observe any evidence of fibrosis or alteration of the geometry of the spleen or liver of the transplanted mice. Furthermore, no recipient mouse manifested any anatomical or symptomatic evidence of leukaemias, lymphomas or myeloproliferative neoplasm (MPN) (that is, lymphadenopathy, splenomegaly/organomegaly, illness or haemorrhage). Circulating hCD45+ cells in peripheral blood displayed no evidence of lympho/myeloproliferation or dysplasia. Furthermore, microscopic architecture of bone marrow, spleen and liver was normal and without fibrotic remodelling or aberrant deposition of collagen or desmin. All images are acquired at original magnification of ×60. Top rows of images for each organ are secondary transplants; bottom rows of images for each organ are controls. b, Comparative genomic hybridization analysis (CGH) shows that rEC-hMPPs are genetically stable both in vitro and in vivo. Genomic DNA was extracted from HUVECs, CD45+ rEC-hMPPs (35 days post-transduction) or in CD45+CD34+ rEC-hMPPs sorted from the engrafted NSG bone marrow (24 weeks post-transplantation) and expanded for 72 h in vitro. A human tumour sample was used as positive control of chromosome rearrangement. Extracted DNA was digested, labelled by random priming and hybridized to the Agilent 1M CGH arrays. The arrays were scanned in an Agilent DNA microarray scanner and obtained data were visualized using Feature Extraction software (version 10.7; Agilent). No genomic abnormalities were identified in CD45+ rEC-hMPPs (or in CD45+CD34+ rEC-hMPPs) engrafted in NSG bone marrow. Hence, rEC-hMPPs remain genetically stable in vitro and in vivo and are not transformed.

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Sandler, V., Lis, R., Liu, Y. et al. Reprogramming human endothelial cells to haematopoietic cells requires vascular induction. Nature 511, 312–318 (2014). https://doi.org/10.1038/nature13547

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