Fig 1: 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 haemogenic endothelium. Once arterial cells commit to haemogenic endothelium fate, they start to express haematopoietic 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 haematopoietic transition.
Fig 2: 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 haemogenic 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 the absence (CTR) and in the 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. Figure 5—source data 1.Spreadsheet source file containing the source data used for the plots in Figure 5.Each tab is labelled to uniquely refer to a specific panel.
Fig 3: IGFBP2 addition to the in vitro differentiation leads to a higher number of functional haematopoietic 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 doxycycline (DOX). (B) Number of haematopoietic colonies obtained after coculture on OP9 in the 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 embryoid bodies (EBs) (n=4 from independent differentiations, two-way ANOVA, ns = p>0.99). (D) Expansion of haematopoietic 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 aorta-gonad-mesonephros (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. Figure 4—source data 1.Spreadsheet source file containing the source data used for the plots in Figure 4.Each tab is labelled to uniquely refer to a specific panel.
Fig 4: Additional analyses of the effect of IGFBP2 addition during in vitro differentiation.(A) Dot plot showing the expression profile of the genes coding for the enzymes mediating glycolysis in CTR and IGFBP2-treated cells at day 10 and 13. (B) Dot plot showing the expression profile of the genes coding for the checkpoints of the glycolysis in CTR and IGFBP2-treated cells at days 10 and 13. (C) Percentage of cells expressing different markers following OP9 coculture of CD34+ cells (ns for all markers, Kruskall-Wallis one-way ANOVA). (D) OP9 cocultures of CD34+ DLL4+ and CD34+DLLL4- (scale bar indicates 50 μm), and in (E) their colony formation capacities following 1 week of OP9 coculture (mixed-effect analysis, Sidak’s post-test, ****p<0.0001, **p<0.01). (F) Cluster composition in cells treated with IGFBP2 data shows the composition at day 10 and day 13. Figure 5—figure supplement 1—source data 1.Spreadsheet source file containing the source data used for the plots in Figure 5—figure supplement 1.Each tab is labelled to uniquely refer to a specific panel.
Fig 5: IGFBP2 addition to the in vitro differentiation leads to a higher number of functional haematopoietic 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 doxycycline (DOX). (B) Number of haematopoietic colonies obtained after coculture on OP9 in the 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 embryoid bodies (EBs) (n=4 from independent differentiations, two-way ANOVA, ns = p>0.99). (D) Expansion of haematopoietic 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 aorta-gonad-mesonephros (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. Figure 4—source data 1.Spreadsheet source file containing the source data used for the plots in Figure 4.Each tab is labelled to uniquely refer to a specific panel.
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