Comparison of in vitro iPSC-derived and in vivo AGM-derived endothelial cells identifies 9 differentially expressed transcription factors.

A - Schematic of the analytic pipeline used to identify the target genes. B - Integrative analysis of single cell transcriptome of in vitro derived endothelial (IVD_Endo) and hematopoietic cells (IVD_HPCs) with in vivo developed endothelial cells (venous, vEC; arterial, aEC; arterial hemogenic, HECs) from human embryos (CS12-CS14) visualised on UMAP dimensions. C - Target genes expression level showing higher expression in arterial hemogenic endothelium in vivo than in vitro derived cells.

The inducible iSAM cassette successfully mediates activation of endogenous gene expression upon DOX induction.

A - Schematic of the iSAM cassette containing the TET-on system under the control of EF1α and dCAS9-P2A-MS2-p65-HSF1-T2A-mCherry under the rTTA responsive elements, separated by genetic silencer and flanked by AAVS1 specific homology arms. B - RUNX1C gene expression activation after transient transfection of the iSAM plasmid and gRNAs in presence or absence of DOX in human iPSC line (n=3 from independent transfections). C – RUNX1 protein expression upon iSAM activation after transient transfection of the iSAM plasmid and gRNAs with DOX in human iPSC line detected by immunostaining. D Expression of the iSAM cassette reported by mCherry tag during the differentiation protocol, the representative images (bright field – BF, and fluorescence) show embryoid bodies at day 3 of differentiation. E - Schematic of the gRNA 2.1 containing the capture sequence for detection during the scRNAseq pipeline. F - RUNX1C gene activation level obtained using either the gRNA 2.0 or 2.1 backbone (n=3 from independent transfections of the 4 different gRNAs). G - Statistical analysis of the gRNAs activation level showing no significant variation following addition of the capture sequence (n=3 for each of the 4 different gRNAs).

Single Cell RNA sequencing in combination with CRISPR activation identifies arterial cell type and functional hematopoietic expansion in association with activation of the 9 target genes.

A - Gene expression profile of target genes following target genes’ activation, heatmap shows the expression level of the target genes in the iSAM_NT and iSAM_AGM treated with DOX following normalisation on the -DOX control. B - Dimension reduction and clustering analysis of the scRNAseq data following activation, filtered on cells where the gRNA expression was detected. C -Arterial (GJA4, DLL4), venous (NRP2, APLNR) and hemogenic marker (CD44, RUNX1) expression distribution in the clusters indicated by the colour. D - Expression distribution visualised on the UMAP plot showing the location of arterial cells marked by DLL4, and hemogenic endothelium marked by CD44 and RUNX1. E - Heatmap of the top 15 marker genes for each of the clusters. F - Contribution of the different libraries to the clusters showing that arterial cell cluster is overrepresented in the iSAM_AGM treated with DOX, compared to the other libraries. G - Expansion of the arterial population assessed by the membrane marker expression of DLL4+ following targets’ activation, quantified by flow cytometry at day 8 of differentiation (Data are normalised on the iSAM_NT + DOX sample, n=5 independent differentiations, * p = 0.0417 paired t-test). H - Colony forming potential of the suspension progenitor cells derived from the two lines treated with or without DOX following OP9 coculture activation, data show the colony obtained for 104 CD34+ input equivalent (n=3 from independent differentiations * p<0.05, Tukey’s two-way ANOVA).

IGFBP2 addition to the in vitro differentiation leads to a higher number of functional hematopoietic progenitor cells.

A - Violin plot of IGFBP2 expression profile in the arterial cells obtained from the different conditions, in the presence or absence of gRNAs and DOX. B -Number of hematopoietic colonies obtained after coculture on OP9 in presence or absence of IGFBP2 (n=3-4 from independent differentiations, ** p=0.0080, Sidak’s Two way ANOVA). C – Percentage of DLL4+ arterial cells differentiation within the CD34+ compartment analysed by flow cytometry in day 8 EBs (n=4 from independent differentiations, Two-way Anova, ns =p>0.99). D – Expansion of hematopoietic progenitors analysed using markers’ expression on suspension progenitors derived after coculture of CD34+ cells onto OP9 support (data are expressed as fold over the CTR in the absence of IGFBP2 (n=4 from independent differentiations, * p<0.02, Sidak’s Two way ANOVA. E - Single-cell transcriptomic analysis of developing AGM collected from human embryos at Carnegie Stages14 and 15 enriched for CD31+ and CD34+ showing the IGFBP2 expression profile in vivo in the AGM.

IGFBP2 alters cell metabolism by inducing a reduction in glycolytic ATP production.

A – Clustering analysis of the single cell transcriptomic time course analysis of differentiating cells at day 10 and day 13 in the absence (CTR) or presence of IGFBP2. Arrows indicate the difference in the clustering due to the addition of IGFBP2 compared to control. B – Expression profile of arterial markers, GJA4 and DLL4, and hemogenic marker RUNX1 (top - the dashed line shows the location of the shift in gene expression of cells treated with IGFBP2) and their expression profile in the endothelial cells cluster marked by Growth factor binding in absence (CTR) and in presence of IGFBP2 (GJA4 p=1E-54, DLL4 p=1.2E-119, RUNX1 p=8.2E-163). C – KEGG enrichment analysis of the genes upregulated at day 13 upon IGFBP2 treatment. The arrow shows the ranking of the Oxidative Phosphorylation pathway. D - Dot Plot showing the expression profile of the genes coding for the enzyme of the Oxidative Phosphorylation pathway. E -Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) profile in cells at day 13 of differentiation reporting mitochondrial respiration and glycolysis, respectively. F – ATP production rate divided by that deriving from glycolysis and from mitochondrial respiration, in cells treated with IGFBP2 and controls at day 13. G – Ratio of the ATP production between glycolysis and mitochondrial respiration in cells treated with IGFBP2 and controls at day 13.

Model summarising the results.

During development, some endothelial cells undergo arterialisation, as identified by their arterial genes’ expression profile (e.g. DLL4). Arterial cells, characterised by high dependency on glycolysis, are expanded by our CRISPR activation approach, resulting in more blood production since these cells are the cell-of-origin of the hemogenic endothelium. Once arterial cells commit to hemogenic endothelium fate, they start to express hematopoietic genes (e.g. RUNX1); this process is enhanced by IGFBP2 via induction of both RUNX1 expression and increased dependency on Oxidative Phosphorylation, known to be important for the progression of the endothelial to hematopoietic transition.