Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: CD16 + cultured neutrophils were similar to PMNs in mobility, ROS production, phagocytosis, and microbial killing (A) Boxplots and violin plots showing the relative expression of genes and proteins involved in neutrophil effector functions . (B) CD11b/CD18-mediated adhesion assay of PMNs (blue) and SL-II CD16 + neutrophils (pink) to uncoated plastic plates ( n = 4). (C) Chemotactic potential of fluorescently labeled PMNs (blue) and SL-II CD16 + neutrophils (pink) based on movement through filters with a pore size of 3 micron ( n = 5). (D) NADPH oxidase assay to determine the production of extracellular peroxide after the addition of opsonized E. coli , zymosan, STZ, PMA, and PAF/fMLP of PMNs (blue) and SL-II CD16 + neutrophils (pink) ( n = 4 for SL-II CD16 + neutrophils and n = 8 for PMNs). (E) Phagocytosis of either unopsonized or opsonized zymosan by PMNs (blue) versus SL-II CD16 + neutrophils (pink) measured by flow cytometry ( n = 3 for SL-II CD16 + neutrophils and n = 4 for PMNs). (F) Representative images of phagocytosis of either unopsonized or opsonized zymosan labeled with FITC solution (green) by PMNs labeled with calcein red-orange (orange) versus SL-II CD16 + neutrophils labeled with calcein red-orange (orange) at the latest timepoint as visualized by imaging flow cytometric analysis. Scale bar was set at 10 μm ( n = 3). (G and H) Killing of opsonized E. coli and S. aureus, and unopsonized and opsonized C. albicans, respectively, shown for PMNs (blue) ( n = 3) versus SL-II CD16 + neutrophils (pink) ( n = 3). Killing was quantified as the inverse of colony-forming units (CFU) as a percentage relative to the CFU at the start of the assay. Negative values, signifying an increase in the number of colonies were considered to be 0. Data shown in (A–D, H) is represented as median and interquartile range, and (E) and (G) is represented as mean ± SD. p values were calculated using Mann-Whitney U tests and labeled as ∗ p < 0.05 and ∗∗ p < 0.01. n values represent the number of individual donor samples.

Article Snippet: Mouse Anti-Human CD66b Monoclonal antibody, FITC Conjugated (Clone 80H3) , Bio-Rad , Cat #MCA216F; RRID: AB_2077860.

Techniques: Cell Culture, Expressing, Cell Adhesion Assay, Labeling, Pore Size, Flow Cytometry, Imaging, MANN-WHITNEY

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: CD34 + HSPC-derived neutrophils cultured in SL-II medium showed higher similarity to PMNs compared to CD34 + HSPC-derived neutrophils cultured in IMDM (A) Representative cytospins of PMNs, CD34 + HSPC-derived neutrophils cultured in SL-II (Bulk) and IMDM medium (Bulk) after Giemsa-May staining. Scalebars set at 10 μm, n = 14. (B) Gating strategy for comparing PMNs versus CD34 + HSPC-derived neutrophils cultured in SL-II or IMDM based on CD11b and CD16 expression as measured by flow cytometry. (C) Representative flow cytometric analysis of surface markers CD66b, CD29 and CD14 expressed on Bulk and CD16 high CD34 + HSPC-derived neutrophils cultured in either SL-II (pink) or IMDM medium (orange) and PMNs (blue). Gray histograms show unstained controls. n = 8 for CD66b, n = 4 for CD29 and CD14. (D) PCA plot showing transcriptomes and non-imputed proteomes for CD34 + HSPCs (yellow), SL-II Bulk obtained at days 14 (light pink) and 17 (black), IMDM Bulk obtained at day 14 (red) and 17 (purple), SL-II fractions MACS sorted for CD16 + obtained at day 14 (gray) and 17 (brown), and PMNs (blue). For SL-II Bulk day 17 transcriptomics n = 4, otherwise n = 6. (E) Pearson correlation between day 17 SL-II CD16 + neutrophils compared to CD34 + HSPCs and PMNs. Correlation values range between 0.6 and 1. (F) Heatmap of z-scores for transcriptome and proteome samples obtained from day 17 SL-II Bulk neutrophils, day 17 SL-II CD16 + neutrophils, PMNs, and CD34 + HSPCs. All transcripts or proteins that were considered differentially abundance between day 0 SL-II Bulk, day 10 SL-II Bulk, day 14 SL-II CD16 + neutrophils and day 17 SL-II CD16 + neutrophils when compared to PMNs were included. (G) Volcano plots of differentially expressed transcripts and proteins between day 17 SL-II CD16 + neutrophils compared to PMNs. (H) Functional enrichment showing the normalized enrichment score (NES) of molecular functions that were enriched within up- or downregulated transcripts and proteins in day 17 SL-II CD16 + neutrophils compared to PMNs. Molecular mechanisms were obtained from different databases, including Wiki-Pathways (∗), Gene Onthology (∗∗), or Reactome (∗∗∗). Neutrophil degranulation is highlighted in red. (I) Scatterplot comparing transcriptome and proteome effect size estimates for all transcript/protein pairs that were identified when comparing day 17 SL-II CD16 + neutrophils and PMNs. Gene/protein pairs highlighted in black were considered statistically significant after multiple testing correction FDR <0.05 and |log2 fold change| > 1 for proteome and 2 for transcriptome. n values represent the number of individual donor samples.

Article Snippet: Mouse Anti-Human CD66b Monoclonal antibody, FITC Conjugated (Clone 80H3) , Bio-Rad , Cat #MCA216F; RRID: AB_2077860.

Techniques: Derivative Assay, Cell Culture, Staining, Expressing, Flow Cytometry, Functional Assay

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: CD34 + HSPC-derived neutrophils gain neutrophil characteristics over time (A) Representative cytospins of CD34 + HSPC-derived neutrophils cultured in SL-II medium (Bulk) during differentiation on day 0, 10, 14, and 17. Cells were stained with Giemsa-May staining and scalebars were set at 10 μm ( n = 14). (B) Representative flow cytometric analysis of surface markers CD34, HLA-DR, CD66b, SIGLEC9, CD177, and CD10 expressed on CD34 + HSPC-derived neutrophils cultured in SL-II medium (pink) on days 0, 10, and the most mature (CD11b/CD16 + ) fraction on days 14 and 17 compared to PMNs (blue). Histograms of unstained controls are shown in gray ( n = 6 for CD34, n = 4 for HLA-DR, n = 8 for CD66b, SIGLEC9, CD177, and CD10). (C) Sankey plot shows the relative proportion of transcripts and proteins that were different when comparing HSPCs on day 0, SL-II Bulk day 10, CD16 + SL-II day 14, and CD16 + SL-II day 17 to PMNs. Transcripts and proteins that were upregulated are shown in red, downregulated in blue, and non-differentially expressed are shown in gray (FDR <0.05 and log2 fold change| > 1 for proteome and 2 for transcriptome). (D) WGCNA network constructed using z-scores of transcripts and proteins from HSPCs on day 0, SL-II Bulk day 10, CD16 + SL-II day 14, and CD16 + SL-II day 17 samples. The 14 modules were clustered based on Pearson’s correlation into four different groups (group A, B, C, and D). Blue edges represent Pearson correlation <0.7, and red edges indicate Pearson’s correlations >0.7. Modules that were enriched for neutrophil degranulation are highlighted in yellow. (E) Enrichment heatmap for granule, mitochondrial, and metabolic databases per module. FDR values were obtained from a chi-square test between transcript/protein pairs in modules and each database separately. (F) Scatterplot of the z-scores for transcriptome and proteome along the four stages of differentiation of SL-II in contrast to PMNs for all modules collapsed to the four groups (A–D). Data in (F) is represented as mean ± SD for all transcript/protein pairs included in the modules for PMNs and each stage of SL-II neutrophil differentiation.

Article Snippet: Mouse Anti-Human CD66b Monoclonal antibody, FITC Conjugated (Clone 80H3) , Bio-Rad , Cat #MCA216F; RRID: AB_2077860.

Techniques: Derivative Assay, Cell Culture, Staining, Construct

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: SL-II CD16 + neutrophils were able to release granules but showed granular content distinct from PMNs (A) Representative FSC/SSC plots measured by flow cytometry for PMNs and SL-II CD16 + neutrophils ( n = 14). (B) Release of serine-proteases ELANE and CTSG as measured by the maximal combined degradation of DQ-BSA after stimulation with CytoB and fMLP. Lysis of cells with Tx-100 was used as a control for maximal release. ( n = 4). (C) Geometric mean florescence intensity of CD63 (azurophilic granules) ( n = 10 for PMNs and n = 4 for SL-II CD16 + neutrophils), CD66b (specific granules) ( n = 9 for PMNs and n = 4 for SL-II CD16 + neutrophils) and LOX-1 (specific granules) ( n = 6 for PMNs and n = 4 for SL-II CD16 + neutrophils) on PMNs (blue) and SL-II CD16 + neutrophils (pink) in unstimulated conditions, after stimulation with PAF/fMLP or CytoB/fMLP measured by flow cytometry. Negative values were considered to be 0. (D) Representative histograms for degranulation (gMFI shown in C) by PMNs (blue) and SL-II CD16 + neutrophils (pink) under different conditions. Histograms of unstained controls are shown in gray. (E) Proportion of valid values for granule proteins for CD34 + HSPCs, CD16 + SL-II neutrophils, and PMNs. (F) Boxplots showing the log2 normalized read counts and log2 LFQ for signature granule proteins (i.e., MPO, ELANE, CTSG, LTF, and MMP9) for CD34 + HSPCs, CD16 + SL-II neutrophils, and PMNs. (G) Scatterplot of the effect size estimates for granule proteins when comparing CD16 + SL-II neutrophils to PMNs. Granule proteins were separated into azurophilic granules, specific granules, gelatinase granules, and secretory vesicles. Dots highlighted in red are considered statistically significant after multiple testing correction (FDR <0.05) and |log2 fold change| > 1. Data in (B), (C), and (F) is represented as median and interquartile range. p values were calculated using Mann-Whitney U tests and labeled as ∗ p < 0.05, ∗∗ p < 0.01. n values represent the number of individual donor samples.

Article Snippet: Mouse Anti-Human CD66b Monoclonal antibody, FITC Conjugated (Clone 80H3) , Bio-Rad , Cat #MCA216F; RRID: AB_2077860.

Techniques: Flow Cytometry, Lysis, Control, MANN-WHITNEY, Labeling