Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 1 | Identification of CD177+ and CD275+ ADE subpopulations. a, Schematic representation of hESC differentiation toward DE. b,c, Representative FACS plots of apparently homogeneous FOXA2+/SOX17+ DE (b) showing a heterogenous population marked by CXCR4+/CD117+ cells (c) (n = 3 (b), n = 6 (c) biologically independent experiments). d–g, Gene expression profiles of CXCR4+/CD117−, CXCR4high/CD117high, CXCR4mid/CD117mid and CXCR4low/CD117low cells for FOXA2 (d), SOX17 (e), CER1 (f) and HHEX (g) (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h, Summary of the antibody screen identifying and isolating CD177 and CD275 as markers of ADE subpopulations. CXCR4 and FOXA2 are used as controls to identify the whole DE. i, hPSCs and hPSC-derived DE stained for CXCR4, CD177 and CD275 as shown by live-cell FACS (n = 10 biologically independent experiments). AA, activin A; D, day.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Gene Expression, Derivative Assay, Staining

Journal: Nature biotechnology

Article Title: Generation of pancreatic β cells from CD177 + anterior definitive endoderm.

doi: 10.1038/s41587-020-0492-5

Figure Lengend Snippet: Fig. 2 | Molecular profiling of CD177+, CD275+ and CXCR4+ DE subpopulations reveals distinct signatures. a, Summary of differentiation protocol toward DE/ADE followed by MACS to enrich for CD177, CD275 and CXCR4 populations. b, Principal component analysis showing that mRNA-derived transcriptome profiles are characteristic of different DE/ADE subpopulations (n = 3 biologically independent experiments). c–e, Bar graphs of selected and significantly enriched gene ontology terms in CD275+ versus CXCR4+ (c), CD177+ versus CD275+ (d) and CD177+ versus CXCR4+ (e) DE populations (n = 3 biologically independent experiments). Enrichment P values are calculated by HOMER findGO.pl based on the cumulative hypergeometric distribution. f,g, Validation of the microarray analysis by qPCR for noncanonical WNT/PCP components and ligands (f) and canonical WNT components and ligands (g). Data were normalized to 18S (ANOVA, n = 3 biologically independent experiments). Data are represented as mean ± s.e.m.; P < 0.05 and P < 0.01. Statistically nonsignificant results are not indicated in the figure. h,i, Western blot analysis (h) and quantification (i) of WNT/PCP components such as p-JNK and DVL2 in ADE subpopulations (n = 3 biologically independent experiments). GAPDH is used as a loading control. Data are represented as mean ± s.e.m. j, Immunofluorescence analysis validated the exclusive localization of β-catenin in the membrane in CD177+ ADE cells and in the cytoplasm and nucleus in CD275+ ADE and CXCR4+ DE cells (n = 3 biologically independent experiments). FOXA2 is used as a nuclear marker. Scale bars, 20 µm and 10 µm in inset. PC1/2, principal component 1/2.

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Human CXCR4-PE,Miltenyi Biotech,130-098-354, dilution 1:40; Human CXCR4-APC,Miltenyi Biotech, 120-010-802, dilution 1:40; Human CD117-APC, Miltenyi Biotech, 130-091-733, dilution 1:40; Human CD117-PE, Miltenyi Biotech, 130-091-734, dilution 1:40; FOXA2-Alexa Fluor® 488, R and D, IC2400G; dilution 1:10 SOX17-APC, R and D, IC1924A; dilution 1:10 Human CD177-APC, Miltenyi Biotech, 120-017-498; dilution 1:20 Human CD275-APC, Miltenyi Biotech, 120-012-112; dilution 1:20 PE Mouse anti-PDX1, BD PharmingenTM, 562161; dilution 1:40 4 nature research | reporting sum m ary O ctober 2018 Alexa Fluor® 647 Mouse anti-Nkx6.1, BD PharmingenTM, 563338; dilution 1:40 Alexa Fluor® 647 Mouse IgG1 κ Isotype Control, BD PharmingenTM, 563023; dilution 1:40 Rabbit FOXA2, Cell signalling, 8186; dilution 1:1000 Goat SOX17 Acris/Novus GT15094, dilution 1:1000 Goat CER1 R&D Systems AF1075, dilution 1:1000 Mouse β-catenin BD 610154, dilution 1:1000 Guinea pig INSULIN Thermo Schientific PA1-26938, dilution 1:100 Guinea pig C-Peptide Abcam ab30477, dilution 1:300 Rabbit MAFA Betalogics LP9872, dilution 1:100 Rabbit MAFA ,Novus Biologicals, NB400-137, dilution 1:100 Rabbit GLUT1 Thermo Fisher PA1-37782, dilution 1:100 Goat GATA6 R&D Systems AF1700, dilution 1:1000 Mouse SOX2 Abgent / Bio Cat AM2048, dilution 1:1000 Rabbit CDX2 Santa Cruz sc-134468, dilution 1:1000 Mouse GCG Sigma G2654-.2ML, dilution 1:300 Goat PDX1 R&D Systems AF2419, dilution 1:500 Rabbit NKX6.1Novus biologicalsNBP1-49672, dilution 1:500 Goat NKX6.1R&D systemsAF5857, dilution 1:300 Rabbit p-JNK Cell signalling 4668, dilution 1:1000 Rabbit DVL2 Cell signalling 3216, dilution 1:1000 Mouse GAPDH Merck Biosciences CB1001, dilution 1:6000 Validation All primary antibodies were validated for their expression on undifferentiated cells and/or pancreatic human sections/islets.

Techniques: Derivative Assay, Biomarker Discovery, Microarray, Western Blot, Control, Immunofluorescence, Membrane, Marker

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 1. Rapid testing microfluidic platform for early thrombosis recapitulates SARS-CoV-2 and Spike-mediated technologies thrombotic effect. a) Schematic of SARS-CoV-2 infection-mediated thrombus formation in the microcirculation. In response to viral infection, inflammatory cells are recruited to activate the extrinsic and intrinsic coagulation pathways, leading to thrombin production. Thrombin cleaves fibrinogen to fibrin, which in turn, pro- motes platelet aggregation and fibrin deposition to form blood clots in the microcirculation. b) Similarly, SARS-CoV-2 vaccination therapies Spike-based technologies have the potential role to increment blood coagulation. c) SARS-CoV-2, Spike protein, and Spike variant for mimicking microcirculation environment were assessed for their thrombotic phenotypes in multiple endothelialized microfluidic channels (2 cm x 400 mm x 100 mm). Antibody anti-IL6 and decoy nanoliposome-hACE2 were also tested together with the aforementioned conditions. SARS-CoV-2, Spike protein, and Spike variant expressed using viral vectors were incubated in the PDMS-based microfluidic channels for 12 h at 37 °C, followed by a thrombosis assay in the presence of human subject-specific whole blood at wall shear stress of 25 dyne cm−2. Thrombus formation was quantified in terms of fibrin and platelet deposition.

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Infection, Coagulation, Variant Assay, Incubation, Shear

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 3. SARS-CoV-2 and Spike-mediated inflammatory cytokines regulates coagulation cascade. a) Tissue factor (TF) binds to coagulation factor VII (FVII) to initiate the thrombosis. SARS-CoV-2 infection also induces endothelial release of cytokines (such as IL-1, IL-6, and TNF-𝛼) that mediate platelet activation and coagulation cascades. SARS-COV-2 treatment for 48 h significantly upregulated HAEC mRNA expression of TNF-𝛼, IL-1, IL-6, and IL-15 as demonstrated by the heatmap (*p < 0.05 CTRL vs SARS-COV-2, n = 4 by qRT-PCR). The heat map was constructed by using Euclidean distance with average linkage. The Z-score centered log2-transformed gene in each sample is presented by a color scale, and gene upregulation is denoted in blue, and downregulation in red. b) Spike mutation D614G inflammatory effect was tested HAECs. A microarray heatmap represents 22 genes and selected control genes in HAECs in response to Spike D614G. Hierarchical clustering heatmap reveal differentially expressed genes in response to Lenti-S mutation (in the presence or absence of Lipo-hACE2), normalized to the lenti-CTRL respectively. The heatmap was constructed as previously described, and the Z-score centered log2-transformed gene in each sample was presented as a color scale. Each condition was performed in triplicate (n = 3). In addition to increased cytokines and chemokines mRNA expression level, higher mRNA expression level of endothelial marker of thrombosis, such as vWF and PAI-1, was also observed. Furthermore, the activation of the Toll-like receptor signaling pathway suggests its crucial role in enhancing downstream inflammation and thrombosis (see Figure S5, Supporting Information) (*p < 0.05: n = 3). c) Immunocytochemical analysis showed Lenti-S D614G increasing protein level of IL-6 (in red). Nuclei were stained with DAPI (scale bar: 50 μm).

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Coagulation, Infection, Activation Assay, Expressing, Quantitative RT-PCR, Construct, Transformation Assay, Mutagenesis, Microarray, Control, Marker, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Rapid Detection and Inhibition of SARS-CoV-2-Spike Mutation-Mediated Microthrombosis.

doi: 10.1002/advs.202103266

Figure Lengend Snippet: Figure 4. Lipo-hACE2 and anti-IL-6 attenuate SARS-CoV-2-mediated inflammation and thrombosis. a) In the endothelialized microfluidic platform, HAECs were exposed to SARS-CoV-2, Lenti-S D614G, or Lipo-S in the presence or absence of Lipo-hACE2 or anti-IL-6 (scale bar = 100 μm). Lipo-hACE2 and anti-IL-6 attenuated SARS-CoV-2, Lenti-S, or Lipo-S-mediated fibrin deposition. b) Both Lipo-hACE2 and anti-IL-6 reduced SARS-CoV-2, Lipo-S, and Lenti-S mediated increases in surface coverage of fibrin and platelet deposition (*p < 0.05 vs Lipo-S or Lenti-S mutation; ***p < 0.001 vs SARS-CoV-2, n = 4).

Article Snippet: (Asp 17 614→Gly) and pLenti-CMV-MCS-hACE2-IRES-sfGFP-SV-Puro Vectors The mature polypeptide of human ACE2 (GenBank NM_021804.3) was cloned in to the XbaI-BamHI site of pLenti-CMV-MCS-green fluorescent protein (GFP)-stomatitis virus (SV)-puro (Addgene #73582).

Techniques: Mutagenesis