cb aphp  (Miltenyi Biotec)

Journal: Journal of Human Immunity

Article Title: A heterozygous USB1 variant linked to immunodeficiency

doi: 10.70962/jhi.20250110

Figure Lengend Snippet: USB1 P44L expression does not block in vitro neutrophil differentiation of CD34 + human cells. (A) In vitro liquid culture differentiation (SCF, G-CSF) of CD34 + representative images at day 8. CD15 + /CD11b + plots are gated on GFP + (up) and GFP − (down) CD14 − cells for the indicated USB1 variants. (B) Percentage of CD14 − CD15 + CD11b + cells either GFP + (left) or GFP − (right). Bars and error bars are the averages of the percentage of the indicated populations and SD from at least two independent experiments ( n ≥ 2). (C) Representative MGG staining images of sorted GFP + cells showing the characteristic morphology of neutrophils (day 14, n = 3). Scale bar = 20 µm. (D) CFU potential of myeloid differentiation in GFP + sorted cells at 2.5 days after transduction ( n = 3). Ordinary one-way ANOVA statistical analysis was performed. *P < 0.05. Nonsignificant differences were not annotated. CFU, colony-forming unit; EV, empty vector.

Article Snippet: CD34 + cells were isolated using an indirect CD34 microbead kit and a separator (VarioMACS; Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: Expressing, Blocking Assay, In Vitro, Staining, Transduction, Plasmid Preparation

Journal: Journal of Human Immunity

Article Title: A heterozygous USB1 variant linked to immunodeficiency

doi: 10.70962/jhi.20250110

Figure Lengend Snippet: The USB1 P44L variant impacts myeloid differentiation in vitro and in vivo . (A) Percentage of GFP + cells in human CD34 + cells at day 2.5 following the transduction step ( n = 7). Bars and error bars are the averages of the percentage of alive GFP + cells and SD from seven independent experiments. (B) Percentage of GFP + cells at days 8 and 14 normalized to the GFP + population at day 2.5. Error bars are SEM from a minimum of two independent experiments ( n = 2). (C) Colony-forming unit (CFU) potential of myeloid differentiation ( n = 3). (D) CFU potential of myeloid (white bars) and erythroid (brown bars) differentiation ( n = 3). (D) were evaluated after an 8-day culture. Bars and error bars are the averages of the percentage of the indicated populations and SEM from three independent experiments. Ordinary one-way ANOVA statistical analysis was performed. *P < 0.05. Statistically nonsignificant differences were not annotated. (E) Neutrophil count at 2 dpf in zebrafish overexpressing indicated USB1 variant RNAs ( n = 4 biological replicates, in orange mean ± SEM). Ordinary one-way ANOVA statistical analysis was performed. **P < 0.01 and ****P < 0.0001. Statistically nonsignificant differences were not annotated. (F) SMO-A morphants injected with indicated USB1 variants ( n = 4 biological replicates, in orange mean ± SEM). Ordinary one-way ANOVA statistical analysis was performed on log 2 -transformed data. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Statistically nonsignificant differences were not annotated. (G) Classification of Alcian blue staining highlights morphological alterations at 5 dpf ( n = 3). From left to right: ventral view of a control embryo (left) and representative pictures of the mild (middle) and severe (right). Scale bar = 250 μm.

Article Snippet: CD34 + cells were isolated using an indirect CD34 microbead kit and a separator (VarioMACS; Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: Variant Assay, In Vitro, In Vivo, Transduction, Injection, Transformation Assay, Staining, Control

Journal: bioRxiv

Article Title: Systems level analysis of B-cell development identifies BDNF as a driver for human B lymphopoiesis

doi: 10.1101/2025.08.29.673055

Figure Lengend Snippet: a, A schematic depicting the study workflow for identification and prioritization of putative B-cell drivers toward enhancing the development of B-cells. The workflow consists of three major steps: (i) the identification of intercellular interactions in the BM; (ii) the prioritization by selection of factors playing a role in B-cell development upon aging; and (iii) validation of findings using in-vitro, biological systems. BM, Bone Marrow. b, UMAP dimensionality reduction derived from analysis of publicly-available scRNA-seq data (GSE144938). 2127 progenitor B-cells, marked by dashed lines, were utilized for inferring a pseudotime developmental trajectory where each dot represents a cell coloured based on high-resolution cell-typing. c ; Left - A trajectory of 2127 B cell progenitors initiating at HSPCs through LMPP, CLP, proB, preB, immB, regB, memB, immB, naiB and plasma cells. Right - pseudotime scores ranging from 0 (HSPC) to 1 (Plasma cells) (right). d , Scaled expression of three hallmark B-cell developmental genes: CD34, CD19 and CD27, as a function of pseudotime. For each marker, low and high expressions are denoted as blue and red respectively, and an associated graph displays the scaled expression pattern along the trajectory. e , An illustration depicting our stepwise strategy to characterize and short-list putative B-cell drivers by integrating the genes of nascent B-cells and MSCs with R-L pair lists. f , A bar plot summarizing the distribution of significant biological pathways associated with the 52 identified putative B cell drivers, as inferred from Gene Ontology annotations. MSC, Mesenchymal stromal cell; R:L, Receptor:Ligand; UMAP, Uniform Manifold Approximation and Projection; HSPC, Haematopoietic Stem and Progenitor cells; LMPP,; CLP, Common Lymphoid Progenitors; immB, Immature B; regB, regulatory B; memB, memory B; immB, immature B; naiB, naive B.

Article Snippet: We isolated CD34 + BMMC cells from healthy donors with a CD34 Microbead Magnetic Separation Kit with MS Columns (Miltenyi Biotec, cat # 130-046-701).

Techniques: Selection, Biomarker Discovery, In Vitro, Derivative Assay, Clinical Proteomics, Expressing, Marker

Journal: bioRxiv

Article Title: Systems level analysis of B-cell development identifies BDNF as a driver for human B lymphopoiesis

doi: 10.1101/2025.08.29.673055

Figure Lengend Snippet: a , A schematic depicting the experimental design for testing stimulation effects of putative B-cell developmental drivers. Healthy, human CD34 + enriched cells were incubated for 24 hours in the presence of both cytokines, required for survival and differentiation of B-cells, and each of the indicated putative drivers, followed by scRNA-seq. b , Candidates differentially affect biological pathways in progenitor B-cells. A radar chart showing the enrichment score of a gene set comprising 122 genes associated with cell growth, cell proliferation and transcriptional regulation (See Methods) following simulation with the putative drivers. The thick black line represents a neutral effect on the pathway (0). Filled and empty circles represent statistically significant and non-significant results, respectively. c , A heatmap showing Jaccard similarity levels between significant pathways upregulated (left) and downregulated (right) relative to control samples, following stimulation with the annotated putative drivers. d , Summary of three driver groups clustered by their post-stimulation behaviors, as assessed by Jaccard similarity analysis. Each group demonstrates distinct dynamics in their paired receptors along the trajectory. e , A heatmap demonstrating the enrichment score of developmental, cell cycle and interferon related pathways within HSPCs, progenitor B and myeloid subpopulations, as obtained from ssGSEA analysis.

Article Snippet: We isolated CD34 + BMMC cells from healthy donors with a CD34 Microbead Magnetic Separation Kit with MS Columns (Miltenyi Biotec, cat # 130-046-701).

Techniques: Incubation, Control

Journal: bioRxiv

Article Title: Systems level analysis of B-cell development identifies BDNF as a driver for human B lymphopoiesis

doi: 10.1101/2025.08.29.673055

Figure Lengend Snippet: a , A schematic depicting the experimental design aiming to test the impact of BDNF on HSPCs in an in-vitro biological system. CD34 + cells were isolated from healthy human bone marrow samples and co-cultured on SF-5 cells layers with the B cell related cytokines, IL3, IL7, FLT3 and SCF, as well as BDNF. After 12 days, cells were harvested and analyzed by flow cytometry. Representative plots of CD19 + cells out of CD34 + cells at baseline and following 12 days of co-culture with and without BDNF are shown. b , Bar plot showing the % of CD19 + cells out of CD34 + cells 12 days post co-culturing with BDNF

Article Snippet: We isolated CD34 + BMMC cells from healthy donors with a CD34 Microbead Magnetic Separation Kit with MS Columns (Miltenyi Biotec, cat # 130-046-701).

Techniques: In Vitro, Isolation, Cell Culture, Flow Cytometry, Co-Culture Assay