Journal: Nature

Article Title: MAPPING HUMAN HAEMATOPOIETIC STEM CELLS FROM HAEMOGENIC ENDOTHELIUM TO BIRTH

doi: 10.1038/s41586-022-04571-x

Figure Lengend Snippet: (a) UMAP analysis showing hemato-vascular populations (CDH5+ endothelium, RUNX1+HLF+ HSC and RUNX1+HLF- other hematopoietic cells) from CS14–15 AGM tissues (n=3 biologically independent samples). The contribution of cells in venous EC and non-HSC clusters was balanced. (b) UMAP feature plots displaying the expression of landmark genes for HSC emergence. HE was selected based on co-expression of RUNX1 and CDH5 and absence of PTPRC/CD45 (bottom, right; 66 cells). (c) “EHT scorecard” dot plot showing EHT landmark genes and genes co-regulated at different stages of EHT in each cluster (cl 0–8) and HE. Selected genes significantly enriched in HE compared to other populations, or up- or downregulated during transition to/from HE, were selected. (d) UMAP feature plots displaying pre-HE (IL33 and ALDH1A1) and HE (ALDH1A1 and KCNK17) markers. (e) Spatial transcriptomics of CS15d (5 weeks) embryo transverse sections. Upper panels, H&E staining of two sections between vitelline and umbilical arteries, focused on the dorsal aorta and surrounding region (red arrow: IAHC; green arrows: red blood cells, D=dorsal, V=ventral, bars=500μm). Lower panels showing the spatial expression of EHT genes, with the default color scale from Loupe browser, which represents the log2 expression from 0 to the maximum value in the spots. Each dot is 55 μm and shows combined expression of 1–10 cells. (f) H&E section (section #240) of CS15c (5 weeks) aorta at the intersection with vitelline artery (arrow: IAHC). Immunofluorescence staining of aorta for IL33, ALDH1A1, CD31 and DAPI (section #251), CXCR4, KCNK17 and DAPI (section #254) and SPINK2, PTPRC/CD45 and CD31/PECAM and DAPI (section #239). Bars=20μm. Individual antibody stainings were performed minimum three times in independent embryos with comparable staining pattern. (g) Schematic summarizing the model for EHT involving the specification of pre-HE and HE from arterial EC and HSC emergence. Stage-specific markers and signaling switches are shown. Created with BioRender.com.

Article Snippet: Primary antibodies rabbit anti-human PECAM-1/CD31 (Novus Biologicals, NB100–2284, 1:200, Lot# 2), mouse anti-human ALDH1A1 (SantaCruz Biotechnology, sc-374149, 1:200, Lot# L1719), mouse anti-human IL33 (Santa Cruz Biotechnology, sc-517600, 1:50, Lot# H2720), CXCR4 (Novus Biologicals, NB100–715, 1:100, Lot# MCX4–1019, MCX4–0920), mouse anti-human KCNK17 (SantaCruz Biotechnology, sc-390435, 1:100, Lot# D0113), rabbit anti-human SPINK2 (Sigma Aldrich, HPA026813, 1:100, Lot# B118804), mouse anti-human CD45 (Vector Laboratories, VP-V354CE, 1:50, Lot# 6009341) were diluted in antibody dilution buffer (PBS containing 1% BSA, 0.1% TritonX-100, 0.1% cold-water Fish Skin Gelatin, 0.05% Tween20) and samples were incubated overnight at 4°C.

Techniques: Expressing, Staining, Immunofluorescence

Journal: Nature

Article Title: MAPPING HUMAN HAEMATOPOIETIC STEM CELLS FROM HAEMOGENIC ENDOTHELIUM TO BIRTH

doi: 10.1038/s41586-022-04571-x

Figure Lengend Snippet: (a) tSNE plot documenting the main cell types in CS14–15 (4.5–5 weeks) AGM tissues (top, n=3 biologically independent samples). Feature plots displaying the expression of arterial (GJA5), pre-HE (IL33, ALDH1A1), HE (ALDH1A1, KCNK17), HSC (KCNK17 and SPINK2) and liver SPINK2 progenitor (SPINK, IL7R) markers in CS14–15 AGM samples (bottom). (b) First row, H&E staining of seven transverse sections, featuring dorsal aorta. Red arrows indicate intra-aortic hematopoietic cluster (IAHC) and green arrows red blood cells. Spatial sequencing plots showing the expression of arterial (GJA5), pre-HE (IL33, ALDH1A1), HE (ALDH1A1, KCNK17), HSC (KCNK17 and SPINK2) and liver SPINK2 progenitor (SPINK, IL7R) markers. The default color scale from Loupe browser was applied, which represents the log2 expression from 0 to the maximum value in the spots. Each dot is 55 μm and shows combined expression of 1–10 cells. White bars=250μm, black bars=1mm. (c) Immunofluorescence staining of CS15c (5 weeks) aorta for IL33, ALDH1A1, CD31/PECAM and DAPI (Section #251), CXCR4, KCNK17, CD31/PECAM and DAPI (section #254) and SPINK2, PTPRC/CD45, CD31/PECAM and DAPI (Section) #239. White bars=200μm, black bar=20μm. Individual antibody staining was performed minimum three times in independent embryos with comparable staining pattern.

Article Snippet: Primary antibodies rabbit anti-human PECAM-1/CD31 (Novus Biologicals, NB100–2284, 1:200, Lot# 2), mouse anti-human ALDH1A1 (SantaCruz Biotechnology, sc-374149, 1:200, Lot# L1719), mouse anti-human IL33 (Santa Cruz Biotechnology, sc-517600, 1:50, Lot# H2720), CXCR4 (Novus Biologicals, NB100–715, 1:100, Lot# MCX4–1019, MCX4–0920), mouse anti-human KCNK17 (SantaCruz Biotechnology, sc-390435, 1:100, Lot# D0113), rabbit anti-human SPINK2 (Sigma Aldrich, HPA026813, 1:100, Lot# B118804), mouse anti-human CD45 (Vector Laboratories, VP-V354CE, 1:50, Lot# 6009341) were diluted in antibody dilution buffer (PBS containing 1% BSA, 0.1% TritonX-100, 0.1% cold-water Fish Skin Gelatin, 0.05% Tween20) and samples were incubated overnight at 4°C.

Techniques: Gene Expression, Expressing, Staining, Sequencing, Immunofluorescence

Journal: Nature

Article Title: MAPPING HUMAN HAEMATOPOIETIC STEM CELLS FROM HAEMOGENIC ENDOTHELIUM TO BIRTH

doi: 10.1038/s41586-022-04571-x

Figure Lengend Snippet: (a) UMAP showing the contribution of each embryo/AGM (CS10-CS17) or YS (CS11) to CDH5+/RUNX1+ hemato-vascular cells (n=8 biologically independent samples). (b) Feature plots displaying EC, pre-HE, HE and HSC landmark genes. (c) UMAP plots highlighting HSPC (HLF+SPINK2+), HE (CDH5+RUNX1+and/orKCNK17+PTPRC-SPN-SPINK2-), pre-HE (CDH5+RUNX1-PTPRC-SPN-SPINK2-IL33+and/orALDH1A1+) and EC (remaining cells in HE-containing clusters) from early (CS10–11, blue) and HSC-forming (CS13–17, red) waves. (d) “Nascent HSC scorecard” genes in CS10 embryo HPC, CS11 YS HPC and CS13–17 AGM HSCs. (e) “HSPC waves scorecard” dot plot showing genes co-regulated in EC, pre-HE, HE and HSPC from distinct waves. Genes shown are identified through differential expression analysis and GO term enrichment between early HPC (CS10 embryo and CS11YS) vs HSCs. (f) “Endo waves scorecard” dot plot showing selected genes co-regulated in EC, pre-HE, HE and HSPC populations from the early and HSC-forming waves. Genes shown are identified through differential expression analysis and GO term enrichment between early HE (embryo CS10 and CS11, YS CS11) vs. HSC-forming HE (CS13–15). (g) UMAP feature plots displaying the expression of stage-specific markers.

Article Snippet: Primary antibodies rabbit anti-human PECAM-1/CD31 (Novus Biologicals, NB100–2284, 1:200, Lot# 2), mouse anti-human ALDH1A1 (SantaCruz Biotechnology, sc-374149, 1:200, Lot# L1719), mouse anti-human IL33 (Santa Cruz Biotechnology, sc-517600, 1:50, Lot# H2720), CXCR4 (Novus Biologicals, NB100–715, 1:100, Lot# MCX4–1019, MCX4–0920), mouse anti-human KCNK17 (SantaCruz Biotechnology, sc-390435, 1:100, Lot# D0113), rabbit anti-human SPINK2 (Sigma Aldrich, HPA026813, 1:100, Lot# B118804), mouse anti-human CD45 (Vector Laboratories, VP-V354CE, 1:50, Lot# 6009341) were diluted in antibody dilution buffer (PBS containing 1% BSA, 0.1% TritonX-100, 0.1% cold-water Fish Skin Gelatin, 0.05% Tween20) and samples were incubated overnight at 4°C.

Techniques: Activity Assay, Quantitative Proteomics, Expressing