Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Stem cell transcriptome of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst MSC, hESC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Dendrogram showing hierarchical clustering of MSC, hESC and MSC-hiPSC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing MSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC (d) and MSC-hiPSC (e) is reported in the indicated tables [Fisher's exact test].

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test

Journal: EBioMedicine

Article Title: A circular RNA map for human induced pluripotent stem cells of foetal origin

doi: 10.1016/j.ebiom.2020.102848

Figure Lengend Snippet: Mesenchymal potential of MSC-hiPSC. a) Heatmap showing differentially expressed genes amongst different F-hiPSC and MSC-hiPSC. Gene expression values are represented by the colour key. b) Principal Component Analysis (PCA) showing 3D visualization of Principal Component (PC) 1, PC2 and PC3 of differentially expressed genes for different F-hiPSC, MSC-hiPSC and hESC. c) Volcano plot showing p-value and fold change (FC) of gene expression data comparing F-hiPSC to MSC-hiPSC. Vertical dashed lines delimitate FC below and above 2, the horizontal dashed line shows p-value=0.05 [two-tailed t -test]. Colour code: FC greater than 2 reaching (green) or not (yellow) statistical significance; FC smaller than 2 reaching (red) or not (black) statistical significance. Enrichment in gene ontology terms of the biological processes category (GOTERM_BP_DIRECT) for upregulated genes of MSC-hiPSC (d) and F-hiPSC (e) is reported in the indicated tables [Fisher's exact test]. f) Schematic of the differentiation protocol toward MSC-like cells. g) Representative images of adipogenic (A, scale bar is 50 µm), osteogenic (O, scale bar is 50 µm) and chondrogenic (C, scale bar is 400 µm) mesenchymal derivatives. h) Left panel: representative density plot showing CD45 + hematopoietic cells (P3) gated from total cells of the cobblestone area-forming cell assay; FSC-A, forward scatter area, a.u., arbitrary units. Right panel: representative histograms showing CD34 + hematopoietic progenitor subpopulation (purple) of CD45 + cells compared to unstained control (grey); the vertical axis represents event percentage count (Count%).

Article Snippet: The same day, CD34 + hematopoietic progenitor cells were isolated from cord blood by magnetic labelling using the Indirect CD34 MicroBead Kit (130–046–701; Miltenyi) on MS MACS separation columns (Miltenyi) following manufacturer's instructions, and seeded at 5000 cells/cm 2 on top of MSC-like cells in myelocult H5100 (05,150; STEMCELL Technologies) supplemented with 10 −6 hydrocortisone (H0888; Sigma-Aldrich).

Techniques: Gene Expression, Two Tailed Test, Control

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: A millifluidic bioreactor allows the long term culture of primary lymphocytes or CD34 + hematopoietic cells while allowing the detection of tumorigenic expansion

doi: 10.3389/fbioe.2024.1388312

Figure Lengend Snippet: Prospective model for in vitro safety assessment of gene-edited human hematopoietic stem and progenitor cells (hHSPCs). The MOAB system aims to be used to test the expansion of cells bearing an unwanted protumorigenic mutation in gene-editing procedures. In case the test does not detect a significative expansion of cells, the autologous transplantation can be performed (top right), conversely if the test highlights a malignant phenotype, the cells cannot be employed for clinical applications and the autologous transplantation cannot be performed (Bottom, right). Created in BioRender.com .

Article Snippet: CD34 + HSPCs were isolated through magnetic cell separation using the CD34 MicroBead Kit UltraPure (Miltenyi Biotec, Italy), MS Columns (Miltenyi Biotec, Italy), and a MACS Separator (Miltenyi Biotec, Italy).

Techniques: In Vitro, Mutagenesis, Transplantation Assay

Journal: Journal of medical genetics

Article Title: Germline mutations in the DNA damage response genes BRCA1, BRCA2, BARD1 and TP53 in patients with therapy related myeloid neoplasms.

doi: 10.1136/jmedgenet-2011-100674

Figure Lengend Snippet: Figure 1 (A) Pedigree and mutational analysis of index patient UPN 5869. The pedigree indicates a hereditary breast and ovarian cancer syndrome. Electropherograms illustrate the BRCA1 c.3112G/T mutation (arrow) as well as a nearby single nucleotide polymorphism (SNP) in DNA from fibroblasts and CD34+ leukaemic cells. Both mutation and SNP became homozygous in the leukaemic clone. The SNP array reveals a copy number state 1 and loss of heterozygosity (LOH) at the BRCA1 locus on chromosome 17q of CD34+ leukaemic blasts. (B) Pedigree and mutational analysis of index patient UPN 6371. The pedigree is not specific for a certain syndrome. Electropherograms illustrate the TP53 c.845_848dupGGCG mutation in germline as well as somatic DNA. SNP array shows copy number state 1 and LOH at the TP53 locus on chromosome 17p of CD34+ leukaemic cells. Filled symbols, subjects with malignancies; open symbols, asymptomatic subjects; the arrow indicates the index patient; number within symbol corresponds to the number of asymptomatic individuals. Red regions represent losses and violet regions LOH. BlaCa, bladder cancer; BrCa, breast cancer; Ca, cancer of unknown origin; GaCa, gastric cancer; Leuk, leukaemia of unknown type; Pheo, pheochromocytoma; Plas, plasmacytoma; ProCa, prostate cancer; UtCa, uterine cancer; y, age in years at diagnosis.

Article Snippet: CD34+ cells were separated from diagnostic blood or bone marrow specimens using the magnetic activated cell sorting CD34 MicroBead Kit together with MS Columns and MiniMACS Separator (all Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Mutagenesis, Biomarker Discovery

Journal: bioRxiv

Article Title: Structural surfaceomics reveals an AML-specific conformation of Integrin-β2 as a CAR-T therapy target

doi: 10.1101/2022.10.10.511511

Figure Lengend Snippet: a. Identified cross-linked peptides mapped on to the crystal structure of integrin-αL/integrin-β2 heterodimer (PDB: 5E6R). b. Flow cytometry histogram plot showing expression of total and activated integrin-β2 on AML and B-cell lines (BV and Namlwa). The y-axis represents percent count normalized to mode. Gating strategy shown in . Representative plots from n = 3 independent experiments. c. Flow cytometry plot showing absence of active Integrin-β2 on CD34+ HSPCs from GM-CSF mobilized peripheral blood. Gating strategy shown in . Deidentified patient samples were used for this analysis ( n = 5 independent donors). Representative of 1-2 independent experiments. d. Representative flow cytometry histogram plots showing expression of active Integrin-β2 on primary AML cells. The y-axis represents percent count normalized to mode. Gating strategy shown in . (Representative of n = 10 total deidentified samples, performed in single assay each). e. Heat map showing inverse expression pattern of ITGB2 against other AML targets in publicly available primary AML RNA-seq data. Color bar represents maximum expression in each row based on normalized read counts. Sample size of BEAT AML (adult), TARGET (pediatric) and TCGA were 510, 255 and 150 respectively. f. Aggregated single cell RNA-seq data showing essentially exclusive expression of ITGB2 in hematopoietic tissue, obtained from the Human Protein Atlas .

Article Snippet: Fully de-identified human CD34 cells enriched blood samples were obtained from Bone Marrow and Transplantation Laboratory at UCSF and were MACS sorted using CD34 MicroBead Kit (Miltenyi Biotec, 130-046-702) and incubated with CD3 antibody (Biolegend, 317302, clone – OKT3) for T cell depletion10 minutes prior to injection in mice, to limit any possible development of graft-vs-host disease.

Techniques: Flow Cytometry, Expressing, RNA Sequencing

Journal: bioRxiv

Article Title: Structural surfaceomics reveals an AML-specific conformation of Integrin-β2 as a CAR-T therapy target

doi: 10.1101/2022.10.10.511511

Figure Lengend Snippet: a. Cartoon diagram showing proposed inactive and active conformations of ITGB2. b. Flow cytometry gating strategy for ( , , ). c. Flow cytometry plot showing presence of total Integrin-β2 on CD34+ HSPCs from GM-CSF mobilized peripheral blood. Cells were gated on singlet cells for analysis. Deidentified patient samples were used for this analysis ( n = 5, independent donors). Representative of 1-2 independent experiments. d. Flow cytometry gating strategy for (c) and . e. Flow cytometry gating strategy for . f. Flow cytometry analysis showing expression of active ITGB2 in PDX models of AML (PDX-A and PDX-B). The y-axis represents percent count normalized to mode. Cells were gated on human CD45+ population cells for analysis. Representative plot from n = 2 separate PDX models of AML. g. Flow cytometry gating strategy for (f).

Article Snippet: Fully de-identified human CD34 cells enriched blood samples were obtained from Bone Marrow and Transplantation Laboratory at UCSF and were MACS sorted using CD34 MicroBead Kit (Miltenyi Biotec, 130-046-702) and incubated with CD3 antibody (Biolegend, 317302, clone – OKT3) for T cell depletion10 minutes prior to injection in mice, to limit any possible development of graft-vs-host disease.

Techniques: Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Structural surfaceomics reveals an AML-specific conformation of Integrin-β2 as a CAR-T therapy target

doi: 10.1101/2022.10.10.511511

Figure Lengend Snippet: a. Representative flow cytometry-based cytotoxicity assay showing specificity of aITGB2 CAR-T against activated peripheral blood T cells which harbors activated integrin-β2 (focus on lower right quadrant, with CAR-negative, CD3-positive T-cells). Both resting and activated conditions performed in overnight co-culture assays with aITGB2 CAR-T cells. (Gating strategy similar to shown .) b. Representative flow cytometry analysis showing successful activation of T cells and partial abundance of activated integrin-β2 on activated T cells. (Gating strategy similar to shown in .) c. Quantitative analysis of active T-cell depletion data in (a); n = 3 technical replicates. d. Representative flow cytometry analysis showing no discernible impact of aITGB2 CAR-T against CD34+ HSPCs from GM-CSF mobilized peripheral blood. The y-axis represents percent count normalized to mode. (Gating strategy similar to that shown in ) (n = 1 donor) and similarly for e. T cells and B cells. (Gating strategy similar to shown in .) (n = 3 technical replicates and representative of 2 independent experiments). Also see . f. Schematic flow diagram for generation of humanized immune system (HIS) mice. g. Representative flow cytometry data from HIS mice data showing apparent non-toxicity of aITGB2 CAR-T against myeloid cells (CD14+). All events were used for gating and analysis. (Representative plot from n = 4 – 6 mice and 6 days post CAR-T treatment). h. Quantification of hCD45+ data in (g). Gating strategy similar to shown . p -value by two-tail t -test. *** p < 0.005. i. Complete blood count profiling of HIS mice treated with aITGB2 CAR-T at day 5 (data from n = 4 mice). All the statistical data in this figure are represented as mean ± SEM. For all the in vitro cytotoxicity assays, E:T ratio was 1:1 with overnight incubation time.

Article Snippet: Fully de-identified human CD34 cells enriched blood samples were obtained from Bone Marrow and Transplantation Laboratory at UCSF and were MACS sorted using CD34 MicroBead Kit (Miltenyi Biotec, 130-046-702) and incubated with CD3 antibody (Biolegend, 317302, clone – OKT3) for T cell depletion10 minutes prior to injection in mice, to limit any possible development of graft-vs-host disease.

Techniques: Flow Cytometry, Cytotoxicity Assay, Co-Culture Assay, Activation Assay, In Vitro, Incubation

Journal: STAR protocols

Article Title: Protocol for cell surface biotinylation of magnetic labeled and captured human peripheral blood mononuclear cells.

doi: 10.1016/j.xpro.2022.101863

Figure Lengend Snippet: Figure 1. Schematic representation of the biotin labeling of the CD34+ and CD45+ cells (A) Labelling of target cells by magnetic bead. (B) Biotinylation in MACS column. (C) Termination of biotinilation with TBS. (D) PBS washing step (optional). (E) Elution.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Biological samples Peripheral blood sample (25-year-old, male human subjects) Erciyes University Hospitals NA Chemicals, peptides, and recombinant proteins Potassium phosphate monobasic Sigma-Aldrich P0662-500G Potassium phosphate dibasic Sigma-Aldrich P3786-500G Potassium chloride Sigma-Aldrich P9541 Iodoacetamide (IAA) Aldrich Chemistry C0267-500G Trypsin Gold Promega V5280 BSA Sigma A-4503 10G Tris Sigma-Aldrich T1503-500G EDTA 0.5M pH:8.0 UltraPure Invitrogen 15575-038 NaCl Sigma Life Science S5886-500G RIPA Sigma Life Science R0278-50ML Protease Inhibitor Cocktail Tablet Roche 11 873 580 001 RapiGest Waters 186001861-1MG TCEP Aldrich Chemistry C4706-2G TEAB Sigma-Aldrich T7408-100ML DMSO Sigma-Aldrich D2650 (Continued on next page) 2 STAR Protocols 3, 101863, December 16, 2022 Continued REAGENT or RESOURCE SOURCE IDENTIFIER Streptavidin-FITC Bio-Rad STAR2B 10223 Lys-C Promega V5073 Trypsin Gold Promega V5280 Trifluoroacetic acid (TFA) Sigma-Aldrich 302031-100ML B-Galactosidase SCIEX 4465867 7-AAD BD 344563 Ficoll -Paque Sigma-Aldrich GE17-5442-02 SepMate -50 (IVD) STEMCELL Technologies 85450 Methanol LC-MS/MS Grade Sigma-Aldrich 1.15333.2500 Acetonitrile (ACN) LC-MS/MS grade Fisher Chemical A955-1 1L Formic acid (FA) Fisher Chemical A117-50 50ML Water LC-MS/MS grade Sigma-Aldrich 1.1 Critical commercial assays CD34 microbead Miltenyi Biotech 130-050-301 2ML CD45 microbead Miltenyi Biotec 130-045-801 EZ-LinkTM Sulfo-NHS-SS-biotin Thermo Fisher Scientific 21331 Software and algorithms FACS Diva 8.0.1 BD NA Analyst TF v 1.6 SCIEX NA Peak View (1.2) SCIEX NA ProteinPilot 4.5 Beta SCIEX NA CytoScape 3.9.1, ClueGo 2.5.9 CytoScape Team NA RStudio RStudio, PBC NA Other T75 Cell Culture flask BD 353118 LS MACS column Miltenyi Biotec 130-042-401 Cell strainer BD Falcon 352350 FACS tube BD Falcon 352235 C18 Disk Supelco 66883-U Magnetic separator Miltenyi Biotec 130-098-308 MACSmix Tube Rotator Miltenyi Biotec 130-090-753 MACS Column LS Miltenyi Biotec 130-042-401 Vacuum concentrator Eppendorf NA Ultrasonic water bath ISOLAB 62105001 Thin glasses VWR 548-0020 Thin glasses cap VWR 548-0834 MS tubes VWR 548-0018 C18 High Resolution 2000 GL Sciences 5020-20E0111522 C18 Trap Column GL Sciences 5020-10028 Hemocytometer Thermo NA Flow cytometry (FACSAria III) BD NA Eksigent expert nanoLC 425 integrated with ABSciex TripleTOF 5600+ ABSciex NA ll OPEN ACCESSProtocol

Techniques: Labeling

Journal: Oncotarget

Article Title: Differentiation inducing factor 3 mediates its anti-leukemic effect through ROS-dependent DRP1-mediated mitochondrial fission and induction of caspase-independent cell death

doi: 10.18632/oncotarget.8319

Figure Lengend Snippet: A. DIF-3, ranging from 2.5-20 μM, was added to K562 CML cell lines growing in semi-solid methyl cellulose medium (0.5 × 10 3 cells/ml). Colonies were detected after 10 days of culture by addition of 1 mg/ml of the MTT reagent and were scored using ImageJ quantification software. The results are expressed as the number of colony-forming cells per well after drug treatment. The results are the means ± SD of 3 different measurements performed in triplicate. Error bars = 95% confidence intervals. B. Various concentrations of DIF-3 were added to sorted CD34+ or CD34- primary cells from one CML patient collected at diagnosis. Cells (10 3 cells/ml) were grown in a semi-solid methylcellulose medium. After ten days in culture, MTT reagent (1 mg/ml) was added to the cell culture and the number of cell colonies was determined using ImageJ quantification software.

Article Snippet: CML cells were labelled with CD34 microbeads isolated by magnetic positive selection (StemSepTM Human CD34 Selection Kit; StemCell, Vancouver, BC, Canada).

Techniques: Software, Cell Culture