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

Journal: Molecular Oncology

Article Title: Cancer‐associated fibroblasts educate normal fibroblasts to facilitate cancer cell spreading and T‐cell suppression

doi: 10.1002/1878-0261.13077

Figure Lengend Snippet: Localization of CEFs in gastric cancer and continuous generation of CEFs. (A) Paraffin sections of human gastric cancer specimens (case #1 and case #2) were immunostained with anti‐ASPN (red), anti‐α‐SMA (green), and anti‐Cytokeratin19 (white or magenta). Asterisks indicate blood vessels. Scale bar = 500 μm. The distribution of positive cells by each antibody was quantified using zen software. The fluorescence intensity was measured in the direction of invasion (white arrow) and plotted from proximal to distal (right panel). Dotted line indicates border between SM: submucosa and MP: muscle layer. In both cases, α‐SMA was stained in muscle layer. (B–E) Gastric cancer specimens (case#1) were immunostained with indicated antibodies. Confocal microscope images of similar regions in submucosa (SM) were shown. Scale bar = 50 μm. (F) Top: CEF‐3d indicates CEFs 3 days after generation. Bottom, left: mRNA levels of IL‐1β, IL‐33, and CXCL6 in CEFs 3 days after preparation (CEF‐3d) were compared with NFs by qRT‐PCR ( n = 3, ** P < 0.01 and *** P < 0.001). Right: CM of CEF‐3d did not disperse OCUM‐12 cells. The data in bar charts are presented as the mean ± SD. (G) L‐CEF: NF‐54 cells were treated with CAF‐CM for 7 days. L‐CEF‐7d indicates L‐CEFs 7 days after removal of CAF‐CM. Left; Representative image of OCUM‐12 cells incubated with L‐CEF‐7d‐CM. Right; Expression of CEF marker genes in L‐CEF‐7d was examined by western blot. (H) CEF 2 and CEF 3 (CEF‐educated fibroblasts): the second and the third generation of CEFs were prepared as indicated in the scheme. OCUM‐12 cells incubated with the NF‐CM, CEF 2 ‐CM, or CEF 3 ‐CM for 24 h. Representative images are shown. Disp + indicates cell dispersion. Scale bars = 200 μm. Right: Immunoblot analysis of CEF marker genes in CEF 2 and CEF 3 cells with indicated antibodies. (I) Cell dispersion was analyzed using Delaunay triangulation plot. The graph indicates the average size of whole triangles (* P < 0.05). Statistical significance was calculated using a one‐way ANOVA followed by Tukey's post hoc tests. The data in bar charts are presented as the mean ± SD.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): anti‐IDO (sc‐376413), anti‐IGF‐I (sc‐74116), anti‐IGFBP‐4 (sc‐517440), anti‐IGF‐IR (sc‐81464), anti‐Kynurenine (sc‐69890), anti‐Smad2/3 (sc‐6032), anti‐PAPP‐A (for immunoblotting, sc‐365226), from Cell Signaling Technology (Danvers, MA, USA): anti‐phospho‐AKT (9271S), E‐cadherin (3195), anti‐COX2 (12282), anti‐phospho‐ERK (9101S), anti‐ERK (9102), anti‐Phospho‐IGF‐1Rβ (3024), anti‐phospho‐IKBα (2859), anti‐phospho‐IKKa/B (2697), NF‐κB p65 Antibody Sampler Kit (4767), anti‐vimentin (3932S), anti‐phospho‐Smad2 (3108S), anti‐Snail (3879), anti‐phospho‐p38 (9211S), anti‐p38 (9212), from Sigma‐Aldrich (St. Louis, MO, USA): anti‐KYNU (HPA031686), anti‐LIF (HPA018844), anti‐α‐tubulin (T5168), from Abcam (Cambridge, UK): anti‐CXCL5+CXCL6 (EP13083) (ab198505), anti‐IL‐1B (ab9722), or from Proteintech (16806‐1‐AP, Chicago, IL, USA): anti‐CST1 (16025‐1‐AP), anti‐IL‐33 (66235‐1), anti‐KMO (10698‐1‐AP), and anti‐PAPP‐A (66962‐1‐Ig, for immunostaining).

Techniques: Software, Fluorescence, Staining, Microscopy, Quantitative RT-PCR, Incubation, Expressing, Marker, Western Blot

Journal: Molecular Psychiatry

Article Title: Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

doi: 10.1038/s41380-020-00992-0

Figure Lengend Snippet: a , b Micrographs are reverted monochrome for FITC channel of AQP4 staining in the cerebral cortex a or hippocampus b in WT or Il33 −/− mice, revealing significant reduction of AQP4 after 60 weeks in Il33 −/− brains. CA , cornu ammonis; DG , dentate gyrus. c Statistical summary for immunofluorescence-based detection of AQP4 expression level demonstrates a significant reduction of AQP4 in both cerebral cortex and hippocampus at/after 60 weeks as compared with WT mice. IOD , integrated optical density. d Immunofluorescence shows disappearance of n-AQP4 in Il33 −/− brain at or after 60 weeks; note the presence of tubular/linear p-APQ4 (arrows) in Il33 −/− brains at 60 and 75 weeks; numerous age-related pigments in 75-week Il33 −/− brain are indicated by arrowheads. Silver staining (lower panels) demonstrates substantial numbers of neurites in Il33 −/− brain at 60 weeks, and nearly none at 75 weeks. e RT-PCR on cortical RNA shows a reduction of Aqp4 mRNA in Il33 −/− brains after 45 weeks. f Western blot of the cortical proteins shows reduction of AQP4 (green) in Il33 −/− mice at 65 weeks; α-actin (red) was used as internal control and probed simultaneously; its staining saturation is revealed (white) for more accurate evaluation. Bars’ unit = μm.

Article Snippet: Following antibodies were used in this study: biotin goat anti-mouse IL33 (R&D System, Minneapolis, MN, USA), monoclonal rat anti-mouse IL33 (clone 396118, ProSci, Charleston, SC, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (astrocyte marker, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-tubulin β3 (neurons, R&D system), mouse monoclonal antibody AT8, which recognizes phosphor-tau(Ser202/Thr205) (ThermoFisher, Waltham, MA, USA), mouse monoclonal antibodies to PHF1 tau (Sigma-Aldrich), FITC-labeled or non-labeled anti-α-actin (Sigma-Aldrich), and rabbit anti-AQP4 antibody (Millipore).

Techniques: Staining, Immunofluorescence, Expressing, Silver Staining, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Molecular Psychiatry

Article Title: Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

doi: 10.1038/s41380-020-00992-0

Figure Lengend Snippet: a Immunofluorescence shows more PHF1 (green) in glomeruli of WT mice than Il33 −/− littermates at 70 weeks; right panels are enlarged boxed areas of left panels to show PHF1 in cytoplasm of glomerular mesangial cells (arrows). b Electron microscopy shows structural relationship of mesangial cells (MC) with other cells and glomerular basement membrane (GBM). c Western blot shows PHF1 in the cerebral cortex and isolated glomeruli of WT or Il33 −/− mice at 70 weeks; note an additional band of low molecular weight of PHF1 (arrow) among glomerular proteins, which is absent in cortical proteins. d RT-PCR shows a lack of tau mRNA in glomeruli as compared to the cortex in both WT and Il33 −/− mice. e Immunohistochemistry on PHF1 in the cerebral cortical and glomerular tissue from the same representative WT or Il33 −/− mice reveals a reverse correlation between PHF1 in the two tissue locations, i.e., more neuronal PHF1 vs less glomerular PHF1 (arrows) in Il33 −/− mice, and vice versa in WT mice. f Statistical analysis shows a positive regression between glomerular PHF1 and cortical AQP4 among WT and Il33 −/− mice. IOD , integrated optical density. g Immunofluorescence on normal human kidneys reveals the presence of PHF1, but not phospho-tau(Ser202/Thr205) (AT8), in glomeruli; an isotype IgG control is also shown; numbered boxed areas are enlarged to show PHF1 in mesangial cells. Bars’ unit = μm.

Article Snippet: Following antibodies were used in this study: biotin goat anti-mouse IL33 (R&D System, Minneapolis, MN, USA), monoclonal rat anti-mouse IL33 (clone 396118, ProSci, Charleston, SC, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (astrocyte marker, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-tubulin β3 (neurons, R&D system), mouse monoclonal antibody AT8, which recognizes phosphor-tau(Ser202/Thr205) (ThermoFisher, Waltham, MA, USA), mouse monoclonal antibodies to PHF1 tau (Sigma-Aldrich), FITC-labeled or non-labeled anti-α-actin (Sigma-Aldrich), and rabbit anti-AQP4 antibody (Millipore).

Techniques: Immunofluorescence, Electron Microscopy, Membrane, Western Blot, Isolation, Molecular Weight, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Control

Journal: Molecular Psychiatry

Article Title: Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

doi: 10.1038/s41380-020-00992-0

Figure Lengend Snippet: a Immunofluorescence shows a significant increase in p-AQP4 (red) expression in the cortex of Il33 −/− mice after receiving rIL33 injection ( Il33 −/− +rIL33) as compared with PBS controls ( Il33 −/− +PBS); two-color staining (lower panels) reveals high density of p-AQP4 (red) but nearly no granular n-APQ4 around neurons (green, tubulinβ3) in Il33 −/− +rIL33 mice, in contrast to many (arrows) in WT mice. Also note much less tubulinβ3 in both Il33 −/− +rIL33 and Il33 −/− +PBS mice than WT mice. b Immunofluorescence shows an increase in p-AQP4 expression in hippocampus (upper panels) of Il33 −/− +rIL33 mice; note that GFAP + astrocyte (green) population is similar among three groups (lower panel). c Western blot on cortical proteins shows quantity of AQP4 in Il33 −/− +rIL33 mice is comparable to WT mice, and much higher than Il33 −/− +PBS controls. d , e Statistics on total AQP4 expression (% of area) ( d ), and numbers of AQP4 + regions ( e ) in the cortex demonstrates that rIL33 injection enhances total AQP4 expression level but not numbers of AQP4 + regions.

Article Snippet: Following antibodies were used in this study: biotin goat anti-mouse IL33 (R&D System, Minneapolis, MN, USA), monoclonal rat anti-mouse IL33 (clone 396118, ProSci, Charleston, SC, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (astrocyte marker, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-tubulin β3 (neurons, R&D system), mouse monoclonal antibody AT8, which recognizes phosphor-tau(Ser202/Thr205) (ThermoFisher, Waltham, MA, USA), mouse monoclonal antibodies to PHF1 tau (Sigma-Aldrich), FITC-labeled or non-labeled anti-α-actin (Sigma-Aldrich), and rabbit anti-AQP4 antibody (Millipore).

Techniques: Immunofluorescence, Expressing, Injection, Staining, Western Blot

Journal: Molecular Psychiatry

Article Title: Requirement of brain interleukin33 for aquaporin4 expression in astrocytes and glymphatic drainage of abnormal tau

doi: 10.1038/s41380-020-00992-0

Figure Lengend Snippet: a Immunohistochemistry shows neuronal PHF1 in indicated groups; note slightly reduced PHF1 in Il33 −/− +rIL33 mice as compared with Il33 −/− +PBS controls; no PHF1 was detectable in WT mice. Bars’ unit = μm b Regression analysis shows no linear correlation between total cortical AQP4 level (% of area) vs neuronal PHF1 among Il33 −/− +PBS, Il33 −/− +IL33, and WT mice; note that total AQP4 level in Il33 −/− +IL33 mice is not proportional to neuronal PHF1. Neuronal PHF1 was quantitated on digital immunostaining imagines, and expressed as IOD/neurons. c Statistical analysis shows a linear negative regression (black dotted line) between cortical and glomerular PHF1 among WT , Il33 −/− +PBS and Il33 −/− +rIL33 mice at 70 weeks; three mice were used for each group. d ELISA shows a sixfold increase in serum pCol peptide that had been injected into ventricles 24 h ago in Il33 −/− +rIL33 mice, as compared to PBS controls. e Diagram depicts two aqueous flows driven by astrocyte AQP4 (lightly colored arrows): Flow 1, driven by n-AQP4, brings neuronal wastes away from neurons, and Flow 2, convective flow driven by p-AQP4 toward perivenous space, flushes brain wastes further to perivenous space, i.e., glymphatics. Therefore, a lack of n-AQP4 results in ineffective removal of neuronal wastes, e.g., PHF1 from neurons.

Article Snippet: Following antibodies were used in this study: biotin goat anti-mouse IL33 (R&D System, Minneapolis, MN, USA), monoclonal rat anti-mouse IL33 (clone 396118, ProSci, Charleston, SC, USA), rabbit anti-glial fibrillary acidic protein (GFAP) (astrocyte marker, Sigma-Aldrich, St. Louis, MO, USA), mouse monoclonal anti-tubulin β3 (neurons, R&D system), mouse monoclonal antibody AT8, which recognizes phosphor-tau(Ser202/Thr205) (ThermoFisher, Waltham, MA, USA), mouse monoclonal antibodies to PHF1 tau (Sigma-Aldrich), FITC-labeled or non-labeled anti-α-actin (Sigma-Aldrich), and rabbit anti-AQP4 antibody (Millipore).

Techniques: Immunohistochemistry, Immunostaining, Enzyme-linked Immunosorbent Assay, Injection