Reprogramming Human Endothelial to Hematopoietic Cells Requires Vascular Induction

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

Manufacture of autologous engraftable hematopoietic stem and progenitor cells (HSPC) offers tremendous therapeutic potential. Using in vitro cultures, human pluripotent stem cells can be differentiated into hematopoietic progenitors, which often have limited expansion potential and do not engraft myeloablated recipients [1][2][3] . Enforced expression of transcription factors (TFs) has also been used to reprogram somatic cells, into hematopoietic lineages [4][5][6] . Employing cellular fusion, we have shown that direct conversion of somatic cells into fetal HSPCs is also feasible 7 . Yet, these prior efforts have been unable to produce human hematopoietic cells capable of long-term multilineage engraftment [4][5][6][7] . We hypothesized that in addition to TF expression, hematopoietic specification and long-term engraftment may require inductive signals from the microenvironment. Indeed, the central instructive role of tissue-specific endothelial cells (EC) 8 in supporting organ regeneration 9,10 , including hematopoietic stem cell (HSC) self-renewal and reconstitution of multilineage hematopoiesis, has recently come to light [11][12][13][14][15][16][17][18] .
In mammals, definitive HSCs originate in the vascular microenvironment of the aortagonad-mesonephros (AGM) [19][20][21][22][23][24] , placenta 25 and arterial vessels 26 . Putative HSCs bud off from hemogenic vascular cells lining the dorsal aorta floor and umbilical arteries, where they are in cellular contact with non-hemogenic ECs 27 . This ontological endothelial to hematopoietic transition (EHT) is mediated in part through expression of the TF RUNX1 21 , its non-DNA binding partner core binding factor-β 28 , GFI1 and GFI1b 29 , 30 . However, the contribution of micro-environmental inductive signals provided by anatomically distinct niches and tissue-specific vascular niches 8 within the AGM, fetal liver and placenta remain poorly defined.
We have identified a minimal set of four TFs-FOSB, GFI1, RUNX1, and SPI1 (FGRS)that reprogram full-term human umbilical vein ECs (HUVECs) and human adult dermal microvascular ECs (hDMEC) into hematopoietic cells with long-term multipotent progenitor cell (MPP) activity (rEC-hMPP). The reprograming was successful only when a unique serum-free vascular niche platform was used. Subsets of rEC-hMPPs were immunophenotypically marked as HSCs and were capable of long-term primary and secondary multilineage engraftment in immunodeficient mice. We demonstrate that enforced or transient expression of FGRS-TFs combined with inductive signals from specialized vascular niche Calls 1,11,31 are essential for efficient conversion of ECs into rEC-hMPPs.
propagated from hematopoietic organs 32,33 balance self-renewal and differentiation of human and mouse long-term HSCs and MPPs by production of physiological levels of Notch-ligands, Kit-ligand, BMPs, Wnts and other angiocrine factors 14 .
To identify TFs that drive EHT, we first identified TFs differentially expressed by Lin − CD34 + umbilical cord HSPCs, but not by HUVECs (Extended Data Figure 1A-D). We then cultivated CD45 − CD133 − cKit − CD31 + HUVECs that were devoid of hemogenic potential 34 ( Figure 1A) and transduced them with lentiviral-vectors expressing various combinations of differentially-expressed TF transcripts using GFP as a marker. Three days after transduction, HUVECs were replated on subconfluent serum-free E4EC-monolayers. Within2 weeks of co-culture with E4ECs, round GFP + CD45 + cells began to bud from transduced HUVECs and morph into grape-like colonies ( Figure 1B). Systematic one-byone dropout of candidate TFs demonstrated that expression of FOSB, GFI1, RUNX1, and SPI1 (FGRS) was necessary and sufficient for hematopoietic reprogramming of HUVECs (Extended Data Figure 1B, C). Co-culture of FGRS transduced-ECs (FGRS-EC) with E4EC monolayers augmented the yield and stability of the hematopoietic-like colonies, which manifested morphological features of hematopoietic progenitors ( Figure 1C). Within 4 weeks of co-culture with E4ECs, FGRS-ECs began to proliferate and form GFP + CD45 + colonies ( Figure 1A,C). Serum suppressed colony formation and naïve HUVECs could not survive without serum and failed to support the emergence of CD45 + cells ( Figure 1D). FGRS-transduction of 5×10 4 HUVECs followed by 3-weeks of serum-free co-culture with E4ECs yielded 32.3±10.5 colonies ( Figure 1D) (efficiency of reprogramming 1.5% see methods), occasionally forming multi-colony structures (Extended Data Figure 2A). Once colonies formed, proliferation of GFP + cells increased and after 5 weeks of co-culture with E4ECs, up to 20×10 6 GFP + CD45 + cells were produced, a ~400-fold expansion of the input FGRS-ECs ( Figure 1D). Since clonal CD45 + cells, but not CD45 − cells, form colonies it is unlikely that E4ECs are mistakenly identified as hematopoietic cells (Extended Data Figure  2B,C). Thus, FGRS-ECs required sustained inductive and supportive signals from E4ECvascular niche for efficient hematopoietic reprogramming.
To date, efforts to differentiate pluripotent stem cells into repopulating hematopoietic cells have had limited success [1][2][3] . We hypothesized that FGRS-TFs could augment hematopoietic differentiation of human embryonic stem cells (hES). To test this, we first differentiated hESCs into ECs (hES-EC) 35,36 and then transduced purified VEGFR2 + CD144 + hES-ECs with FGRS. Although this approach generated CD45 + CD144 − progeny (Extended Data Figure 2D), these cells did not form stable hematopoietic-like colonies and did not proliferate ( Figure 1A black line). Thus, hES-ECs are not as permissive as HUVECs for reprogramming into hematopoietic cells.

rEC-hMPPs are derived from non-hemogenic ECs
To rule out the possibility that rEC-hMPPs are derived from rare contaminating hemogenic cells 25,34 , we cultured naïve ECs in optimal pro-hematopoietic media or performed clonal reprogramming of ECs. Neither serum-withdrawal, nor addition of hematopoietic cytokines induced formation of CD45 + CD34 + cells from naïve HUVECs (Extended Data Figure  3A,B). Furthermore, clonal cultures of mature CD45 − CD144 + CD31 + CD62E(E-selectin) + ECs 32,33 did not form CD45 + rEC-MPPs (Extended Data Figure 3C,D and Extended Data Figure 4A,B,C). Thus, rEC-hMPPs were not derived from scarce population of spontaneously differentiating ECs with preexisting hemogenic potential.
The BM of robustly engrafted recipient NSG mice contained a small population of cells with Lin − CD45RA − CD38 − CD90 − CD34 + immunophenotype of human MPPs 37 ( Figure 3E). To ensure that engrafted cells were derived from FGRS-ECs, we purified hCD45 + cells from recipient BM ( Figure 3F) and seeded single-cells into 96-well plates for whole genome amplification (WGA) and detection of viral vector integration. All hCD45 + cells had been transduced by lentiviral vectors, and 19/21 cells showed integration of all four FGRS-TFs ( Figure 3F, Extended Data Figure 5). To verify that these cells were progeny of rEC-hMPPs, we seeded hCD45 + cells for CFC-assays to examine viral integration in individual colonies ( Figure 3G). We demonstrated that all tested colonies were derived from cells that had integrated the lentiviral vectors expressing FGRS ( Figure 3G). Therefore, engrafted human hematopoietic cells were derived from transplanted rEC-hMPPs.
To test whether FGRS-induced reprogramming triggered expression of endogenous human FGRS genes 24 , we expressed genetically distinct murine TFs (mFGRS) using induciblelentiviral vectors to reprogram HUVECs into rEC-hMPPs and then assessed for endogenous human FGRS-gene expression. Transient expression of mFGRS with E4EC-coculture for 3weeks induced a 100-fold greater expression of endogenous genes than that of switched-off mFGRS transcripts (Extended Data Figure 6A,B,C). Therefore, rEC-hMPPs do not require continuous expression of exogenous FGRS-TFs to sustain their hematopoietic cell fates.
Furthermore, we speculated that enforced SPI1 expression might prevent rEC-hMPPs from differentiating into T-cells 39,40 Therefore, we constitutively expressed FGR-TFs with a Tetinducible SPI1 and E4EC-induction, which resulted in generation of small but significant population of CD3 + T-cells (Extended Data Figure 6D,E). Thus, generation of lymphoid cells from rEC-hMPPs could be optimized by transient expression of specific TFs.
Next, we compared the transcription profiles of rEC-hMPPs, before and after NSG engraftment, to those of naive HUVEC, hDMEC and purified Lin-CD34 + cord blood HSPCs ( Figure 4C). FGRS-transduction plus E4EC-induction activated hematopoietic genes and down-regulated vascular gene signatures ( Figure 4C). Importantly, 22-weeks posttransplantation, CD45 + CD34 + rEC-hMPPs had a transcription pattern similar to normal CD34 + cord blood HSPCs and distinct from the ECs from which they were derived ( Figure  4C). Notably, pluripotency genes were not induced in rEC-hMPPs indicating that reprogramming does not require transition through a destabilizing pluripotent intermediate ( Figure 4D).
Notably, rEC-hMPPs isolated from primary and secondary engrafted mice manifested no evidence of malignant transformation (Extended Data Figure 8; 9; 10A) or genetic abnormalities or (Extended Data Figure 10B).

Discussion
The availability of engraftable autologous human cells offers the potential to cure a wide spectrum of benign and malignant hematologic disorders. Prior efforts using pluripotent stem cells have been obstructed by low efficiency and poor engraftment 2,3,41,42 . Here, we have taken advantage of ontological link between endothelial and hematopoietic development to efficiently reprogram mature, fetal and adult ECs into engraftable selfrenewing hMPPs without transitioning through a potentially destabilizing pluripotent intermediate. Just as support from non-hemogenic vascular cells important for EHT during development, we found the instructive contribution of the vascular niche was central to reprogramming ECs to hematopoietic cells.
Differentiating pluripotent stem cells or expanding AGM-derived cells to engraftable hematopoietic cells has been inefficient, when stromal cells have been used for niche-like support 2,3,41 . This could be due to: 1) poor inductive function of stromal cells in serum-free culture; and/or 2) distinguishing features of ECs that resemble the hematogenic niche cells that support EHT [11][12][13][14][15] . For example, E4ECs produce the proper stoichiometry of inductive angiocrine factors, including Notch, BMP and c-Kit pathways 4 that are important for EHT 43 . Thus, adult organ-specific pro-hematopoietic vascular niches, such as HUVECs [11][12][13][14][15]36 , BM 13 , hepatic and splenic sinusoids 8 may share functional characteristics with EHT-inductive niche cells. The rEC-hMPPs can engraft primary and secondary recipient mice with individual cells capable of differentiating to multiple hematopoietic lineages. However, the influence of recipient microenvironmental signals and temporal aspects of reprogramming influence the outcome of xenograft studies. NSG mice lack the proper niches for T-cell differentiation and we were not able to determine whether engrafted rEC-hMPPs could give rise to T-cells. We found that temporally restricted expression of SPI1, along with sustained FGR, increased the ability of the rEC-hMPP to differentiate to lymphoid lineages suggesting that sustained SPI1 interfered with lymphogenesis. Notably, even transient expression of FGRS is sufficient to activate endogenous TFs. The age of recipient mice was also important because transplantation of neonatal (2-week old) NSG mice enhanced lymphoid engraftment by rEC-MPPs. Therefore, temporal and chronological expression of FGRS-TFs with proper stoichiometry combined with vascular niche signals appears to increase the yield of rEC-hMPPs with authentic multilineage long-term selfrenewing HSPC potential.
Direct reprogramming of ECs into engraftable HSPCs orchestrated by the inductive signals conveyed by tissue-specific vascular niches offers an innovative way to decipher the hierarchy of TFs and micro environmental cues that guide hematopoietic development. Our approach lays a foundation for engineering engraftable autologous rEC-hMPPs and potentially true HSCs for treatment of patients with hematological disorders.

Manufacturing of vascular niche platform
To establish the vascular niche monolayers, HUVECs were purified and transduced with lentiviral vector carrying a cassette of adenoviral E4ORF1 gene (E4ECs) as previously described 31 or obtained as VeraVecs from Angiocrine Bioscience, New York, NY. E4ECs proliferate in serum-free and xenobiotic-free conditions only supplemented with minimal angiogenic factors. All naïve ECs that are non-transduced with E4ORF1 are depleted during passaging in serum-free conditions. Confluent monolayers of E4ECs are contact inhibited, non-transformed and propagate as homogenous monolayers providing ideal instructive niche for reprogramming and sustaining FGRS-transduced ECs into rEC-hMPPs.

Increasing Efficiency of Reprogramming
To increase efficiency of the reprogramming, we developed a strategy to select those subsets of ECs that were most likely transduced with the proper stoichiometry of all four FGRS. We initially focused on generating ECs transduced with GFI1, SPI1 and FOSB TFs because their expression in ECs is negligible (Extended Data Fig. 1). To accomplish this, we transduced 5×10 6 ECs with FGRS lentiviral "cocktail" marked by puromycin resistance (SPI1) or GFP (FOSB and GFI1). We then applied puromycin selection for 2 days to enrich SPI1-expressing cells and sorted them for GFP expression to enrich for SPI1 + GFP + (FOSB/ GFI1) ECs. Subsequently, we transduced these GFP + puromycin resistant cells with RUNX1, seeded into 12-well plates, and expanded them for two days (10 5 cells per plate, n=3). We then re-plated 10 4 of the GFP + puromycin resistant cells on serum-free E4EC vascular niche layer in hematopoietic media and quantified the number of hematopoietic clusters that emerge after ~20 days of co-culture. We found that GFP + puromycin resistant cells yielded 156.0±3.6 (n=3) hematopoietic-like colonies per 10 4 re-plated cells suggesting that the efficiency of reprogramming was at least 1.5% (156/10 4 ). This calculation assumes that each colony originates from a single reprogrammed cell. The efficiency is likely much higher in cells expressing the appropriate stoichiometric quantities of each factor.

Identification of viral integration on a single-cell and single-colony level
To identify the presence of viral integration on a single-cell level, we sorted human CD45 + cells from the marrow of rEC-hMPP engrafted mice into a 96-well plate 1 cell per well into a lysis buffer for the Phi29 (multiple displacement amplification -MDA) based wholegenome amplification (WGA). To do single-cell WGA, we used a commercially available kit REPLI-g (Qiagen, Cat.No. 150343). Each WGA reaction product was followed by a set of PCR reaction with primers specific to the CMV promoter and a transgene (FOSB, GFI1, RUNX1, SPI1). All PCR reactions were conducted separately. We used empty wells (no cells sorted) as controls for non-specific amplification. WGA products of the control wells were used for PCR reactions with primers specific to the CMV promoter and a transgene.
To identify the presence of viral integration on a single-cell level, we captured expanding colonies from the plates for CFC assay. Fourteen days after the start of CFC assay 3 distinct cell aggregations/colonies were detected and analyzed (three upper images). Four PCR reactions were performed for each amplified colony using their genomic DNA as template. Cells from the colonies were re-suspended and washed twice in excessive amounts of PBS (10ml) and transferred into the lysis buffer for the WGA. All following procedures were the same as those described for the single-cell viral integration identification.

Clonal reprogramming of HUVECs to rEC-hMPPs
HUVECs were isolated from umbilical cord and grown in EC-growth medium. After 2 to 3 passages, CD144 + CD31 + CD62E(E-selectin) + CD45 − HUVECs were FACS sorted into 96well plates at 1, 2, 5 and 10 cells per well densities for clonal expansion. We used CD62E (E-selectin) surface marker to sort mature activated ECs. Passaging of HUVECs results in upregulation of E-selectin in 40 to 60% of the HUVECs. Expanding clonal populations of selected cells were subsequently transduced with the FGRS TFs followed by replating onto the E4EC monolayers to reprogram them into rEC-hMPPs. Hematopoietic activity of clonally derived CD45 + CD34 + rEC-hMPPs was assessed using standard methylcellulose-CFC assay.

RNA-Seq processing and analysis
Total RNA was prepared using the Applied Biosystems Arcturus PicoPure RNA isolation kit. The quality of the extracted RNA was checked on an Agilent Technologies 2100 Bioanalyzer. The extracted RNA was used for sequencing using Illumina HiSeq2000. The sequencing output was checked for quality using Illumina pipeline. PE 51×2 and SE 51 reads were mapped to the human genome (hg18) using TopHat (http://tophat.cbcb.umd.edu/) default parameters. RefSeq transcript levels (FPKMs) were then quantified using CuffLinks (http://cufflinks.cbcb.umd.edu/) with upper-quartile normalization and sequence-specific bias correction. For heatmap visualization we determined the maximum FPKM of each transcript across the samples shown. FPKMs were then divided by this number to produce scaled expression values. Heatmaps of gene expression and gene expression clustering were built using GENE-E matrix visualization and analysis platform (http:// www.broadinstitute.org/cancer/software/GENE-E/). Clustering of gene expression in the heat maps was conducted using one minus Pearson correlation as dissimilarity measure between transcription profiles. GEO accession number: GSE57662. The GSE57662 study can be reviewed at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE57662.

Comparative Genomic Hybridization (CGH)
Genomic DNA was extracted from HUVECs, FACS sorted CD45 + rEC-hMPPs, and CD45 + CD34 + rEC-hMPPs sorted from the BM of the NSG mice. Before DNA extraction, CD45 + CD34 + rEC-hMPPs sorted from the BM were expanded for 72 hours in vitro. As a positive control of chromosomal rearrangements we used a CGH array of a leukemic cell line with a duplication of the chromosome 7 and a deletion of the chromosome 10. Extracted DNA was digested, labeled by random priming and hybridized to the Agilent 1M CGH arrays. The arrays were scanned in an Agilent DNA microarray scanner and obtained data was visualized using Feature Extraction software (version 10.7; Agilent).

Differentiation and reprogramming of human embryonic stem cells (hES)
We used a transgenic hES reporter line that specifically identifies differentiated EC derivatives via a fluorescent reporter driven by a fragment of the human VE-cadherin promoter 1,35 . To augment endothelial commitment, hES differentiation was initiated in coculture with E4EC vascular niche cells, described above. One day before plating hES to begin differentiation, MEF conditioned medium was replaced with hES culture medium without FGF-2 and supplemented with 2 ng/ml BMP4. The next day, hES cells were plated directly onto E4EC monolayers in hES culture medium (without FGF-2, plus 2 ng/ml BMP4) and left undisturbed for 48 hours. This point of culture was considered as differentiation day zero. Cells were sequentially stimulated with recombinant cytokines in the following order: day 0 to 7 -supplemented with 10 ng/ml BMP4; day 2 to 14supplemented with 10 ng/ml VEGF-A; day 2 to 14 -supplemented with 5 ng/ml FGF-2; day 7 to 14 -supplemented with 10 µM SB-431542. At day 14 of culture, FACS sorting was used to purify the fraction of hES-derived ECs co-expressing the vascular specific CD144 (VE-cadherin) reporter and CD31. These cells were transduced with the FGRS cocktail and 2-3 days later plated on a layer of serum-free E4EC monolayers. The extent of reprogramming was assessed by flow cytometry.

Phagocytosis assay
The rEC-hMPPs generated from 3 to 4 weeks, were cultured in the presence of M-CSF (10ng/ml), SCF (10ng/ml), Flt-3 (10ng/ml), TPO (10ng/ml), and 10% FBS for additional two weeks with E4EC vascular niche layer. We observed an increase in size and granularity of the cultured cells (data not shown). The culture was washed with PBS twice to remove non-adherent cells. Growth media mixed with green fluorescent beads (GFB) at a low concentration of 1 µl/ml was applied to the attached cells for one hour at 37°C.After the incubation, the cells were washed twice with PBS and live cells were stained with the monocytic CD14 antibody. Cells were fixed and stained with DAPI for nuclear visualization. We visualized GFB inside CD14 + cells, but not in CD144 (VE-cadherin) + endothelial cells (Extended Data Figure 2G). by density gradient using Ficoll-Paque (GE) and enriched for CD34 + HSPC using magnetic separation using anti-CD34 microbeads (Miltenyi) or FACS sorting. Further purification was achieved by negative selection of Lin + cells using Human Progenitor Cell Enrichment Kit (StemCell Technologies) or FACS sorting. RNA from FACS sorted Lin − CD34 + CD45 + cells was isolated by using Arcturus PicoPure RNA isolation kit (Applied Biosystems; This kit was used for all RNA extraction procedures).

Lentiviral vectors
Candidate transcription factors (TFs) were subcloned into either pLVX-IRES-ZsGreen1 lentivector (Clontech), pLOC lentivector (OpenBiosystems), or LV105 lentivector (Genecopoeia). For inducible expression of mouse or human FGRS factors we used Tet-On 3G inducible lentiviral vectors (Clontech). Lentiviral particles were packaged as previously described 7 . In short, human embryonic kidney 293FT (HEK293FT) cells were cotransfected with a lentivector and two helper plasmids psPAX2, and pMD2.G (Trono Lab through Addgene) in the equal molar ratio. Supernatant was collected 48-52 hours posttransfection, filtered and concentrated using Lenti-X concentrator (Clontech). Viral titers were determined in limiting dilution experiments using HUVECs as target cells. We used either the number of GFP + cells, or the number of formed colonies in the presence of selection antibiotics (puromycin) as a read-out for the number of infectious viral particles per volume. We used an average MOI of 5 to 10 for infection of ECs. expression [log 2 (RNA-seq value)] in HUVECs and freshly purified CD34 + Lin − cord blood cells. B. One-by-one elimination of TFs experiment revealed a minimal set of TFs; FOSB, GFI1, RUNX1, and SPI1, (FGRS) capable of generating hematopoietic colonies in the HUVEC culture. A pulled set of 26 TFs minus one TF was evaluated for the ability to evoke formation of hematopoietic clusters (day 21 to 25; n=3). Asterisks show statistically significant (p<0.05) reduction of the number of hematopoietic clusters in the transduced HUVECs compared to the full set of TFs. 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-TFs are necessary and sufficient for generation of hematopoietic colonies (day21 to 25; n=3). "All" in B and C: all TFs are present. "Control" in B and C: all TFs are absent.

D. Schema for reprogramming of ECs 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 replated on subconfluent monolayers of E4EC ECs. The emerging colonies of hematopoietic cells were subjected to 1) multi-variate 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. observed 5-fold expansion of CD45 + cells (56.6 ×10 3 ±7.9 ×10 3 ; n=3) and dramatic reduction of CD45 − cells number (4.6×10 3 ±1.0 ×10 3 ; n=3). C. Clonal expansion of CD45 + cells. CD45 + cells were FACS sorted into 96-well plates at the density of 1 and 2 cells/well. After seven days of culture, we observed CD45 + cell expansion in 6.3±2.1 wells (93.1±14.5 cells/well) of 1-cell sort and 29.0±4.3 wells (112.1±21.2 cells/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 rather due to survival of sorted cells than a reflection of the number of cells sorted into a well.

D. FGRS-induced generation of hematopoietic cells by hES-derived endothelium (hES-EC).
Representative experiment demonstrating that transduced hES-EC (Right 4 panels) generated significantly higher number of CD45 + CD144-cells compared to control nontransduced hES-EC (left 3 panels). E. Lineage-specific surface marker analysis of the GFP + CD45+ population of rEC-hMPPs. GFP + CD45 + population showed that some of these cells expressed lineage specific surface markers such as CD43 + (8.96%±2.3; n=3), CD90 + (Thy-1 + ) (6.15%±1.13; n=3) and CD14 + (40.0%±4.95; n=3) (representative flow cytometry measurements; left four panels, statistics for all experiments is in the right-hand bar-graph, n=3). F. Immunophenotypic analysis of CFU colonies grown in the CFC assay performed in Figure 2C We performed two sets of experiments to exclude the possibility that rEC-hMPPs were derived from spontaneously differentiating HUVECs with hemogenic or hemangioblastic potential 25,34 . A to B: In optimal pro-hematopoietic cultures, naïve non-transduced ECs fail to spontaneously differentiate into rEC-MPPs. Sandler  A. We grew non-FGRS-transduced HUVECs in the serum-free media used for reprogramming. Neither serum withdrawal, nor addition of hematopoietic cytokines induced formation of CD45 + CD34 + cells and HUVECs sustained their vascular identity. Indeed, serum withdrawal increases number of CD34 + HUVECs; SF-serum free, CK-cytokines cocktail (see methods), SB -TGFβ inhibitor SB431542. B. Serum withdrawal suppresses HUVEC proliferation. Inhibition of TGFβ signaling (SB) combined with cytokine cocktail (see methods) restores proliferative potential of HUVECs in serum free media. 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 ECs, but not cytokine-mediated outgrowth of contaminating pre-existing hemogenic ECs. C to E: Clonal reprogramming of non-hemogenic HUVECs into rEC-hMPPs using FGRStransduction and vascular-induction. We performed EC clonal reprogramming experiments to exclude the possibility that rEC-hMPPs were derived from spontaneously differentiating HUVECs with pre-existing hemogenic or hemangioblastic potential 25,34 . C. Since E-selectin is only expressed in ECs, we generated clonal cultures of CD45 − CD144 + CD31 + CD62E(E-selectin) + ECs 32,33 . 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 E4ECs. D. These clonal cultures yielded rEC-hMPPs comparable to bulk HUVEC cultures. The numbers of hematopoietic-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 hematopoietic-like colony derived from a 1-cell clone #2. Therefore, it is unlikely that rEC-hMPPs are derived through spontaneous differentiation of pre-existing ECs with hemogenic or hemangioblastic potential.

Extended Data Figure 4. Clonal reprogramming of non-hemogenic HUVECs into immunophenotypic and functional rEC-hMPPs using FGRS-transduction and vascularinduction
A,B,C: CFC assay of clonally reprogrammed rEC-hMPPs. CD45 + CD34 + rEC-hMPPs were sorted out (red gate in FACS plots; upper left) and plated for CFC assay. Typical hematopoietic colonies arose in the assay (middle column microphotographs; x4 magnification). 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 3 independent clones are shown. Thus, given the high efficiency of clonal reprogramming of mature authentic ECs into rEC-hMPPs, it is unlikely that rEC-hMPPs are spontaneously derived from a very rare population of a pre-existing hemogenic or hemangioblastic HUVECs. off the expression of mouse FGRS and cells were cultured for 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 hematopoietic colonies with cells expressing human CD235, CD11b, CD83, and CD14. B. Comparison of transcriptional profiles of the human FGRS expression in human HUVECs, hCD45 + rEC-hMPPs programmed using inducible mouse FGRS, CD45 + CD34 + rEC-hMPPs, 22 weeks posttransplantation, hDMEC-derived CD45 + CD34 + rEC − hMPPs after 15 weeks post-secondary engraftment and naïve CD34 + Lin + cells purified from cord blood. 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. RNA-Seq reads that align to human FGRS sequences -"Map to Human", RNA-Seq reads that align to mouse FGRS sequences -"Map to Mouse", and RNA-Seq reads that align to mouse FGRS sequences without a possibility to align to human sequences -"Map to mouse Only". D to E: Optimizing differentiation of rEC-hMPPs into lymphoid progeny D. The number of T-lymphoid progeny of engrafted rEC-hMPP was negligibly small, raising the possibility that constitutive SPI1 expression prevents rEC-hMPP from differentiating into T-cells 39,40 . To test this, HUVECs were transduced with lentiviral vectors expressing GFP and that constitutively express FGR-TFs with a Tet-inducible SPI1 (FGR+SPI1-Tet-On construct) for 3 days followed by replating for E4EC-induction. After 27 days of FGR and doxycycline-induced SPI1 expression on E4ECs, GFP + CD45 + hematopoietic-like colonies emerged. Then, doxycycline was withdrawn and the reprogrammed cells were cultured serum-free 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 + CD45 + cells emerging during reprogramming of HUVECs by FGR+SPI1-Tet-On construct and E4EC-induction. E. rEC-hMPPs differentiate into CD3 + , CD19 + and CD14 + hematopoietic cells in the absence of exogenous expression of SPI1. After 3 weeks, the numbers of the 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 TFs.

Extended Data Figure 8. Analysis of bone marrow (BM) and liver of primary transplanted mice for signs of malignant transformation
Analysis of BM (A) and liver (B) of mice 10 months after primary transplantation (from Figure 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 BM was determined by sequential multi-cross sectional Wright Giemsa and Hematoxylin-Eosin (H&E) staining and compared to age control non-transplanted NSG mice. We did not observe any evidence of fibrosis or alteration of the geometry of the bone  Analysis of spleen of mice 10 months after primary transplantation (from Figure 3B) of HUVEC derived rEC-hMPPs as well as BM (n=2), spleen-and liver-(n=2, Also Extended Data Figure 10A) of mice that were engrafted with secondary transplanted hDMEC-derived rEC-hMPP cells 15 weeks post-transplantation (from Figure 5B)  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 leukemias, lymphomas or myeloproliferative neoplasm (MPN): lymphadenopathy, splenomegaly/ organomegaly, illness or hemorrhage. Circulating hCD45 + cells in PB displayed no evidence of lympho/myeloproliferation or dysplasia. Furthermore, microscopic architecture of BM, spleen and liver was normal and without fibrotic remodeling or aberrant deposition of collagen or desmin. All images were acquired using color CCD camera. In primary transplants the scale bar is 200µm 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 center 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 shown images (Primary Transplant). All images in Secondary Transplant are acquired at 60x magnification. All images are acquired at 60x magnification. Top rows of images for each organ are secondary transplants; bottom rows of images for each organ are controls. control 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 leukemias, lymphomas or myeloproliferative neoplasm (MPN): lymphadenopathy, splenomegaly/ organomegaly, illness or hemorrhage. Circulating hCD45 + cells in PB displayed no evidence of lympho/myeloproliferation or dysplasia. Furthermore, microscopic architecture of BM, spleen and liver was normal and without fibrotic remodeling or aberrant deposition of collagen or desmin. All images are acquired at 60x magnification. 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-hMPP sorted from the engrafted NSG BM (24-weeks post-transplantation) and expanded for 72 hours in vitro.
A human tumor sample was used as positive control of chromosome rearrangement. Extracted DNA was digested, labeled by random priming and hybridized to the Agilent 1M CGH arrays. The arrays were scanned in an Agilent DNA microarray scanner and obtained data was 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 BM. Hence, rEC-hMPPs remain genetically stable in vitro and in vivo and are not transformed.  C. Clonal CFC assay of BM hCD45 + hCD34 + cells(n=3; left plot). Emerging colonies were counted and classified (middle table). CFC colonies derived from single plated hCD45 + hCD34 + cell comprise of mixed-lineage erythroid and myeloid progenies(right plots). D. Reprogrammed cells isolated from the host retained their multi-lineage potential in vitro; secondary CFC assay. E. Schematic representation of steps of reprogramming of ECs into rEC-hMPP by FGRS.