stemspan tm cd34+ expansion supplement  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: Efficient Methods for Target Gene Manipulation in Haematopoietic Stem Cell Derived Human Neutrophils

doi: 10.1101/2023.06.17.545406

Figure Lengend Snippet: A Representative images of differentiating CD34 + cells at the progenitor stage (day 6) and as segmented cells (day 14) during directed differentiation with G-CSF. The rightmost image shows isolated mature human neutrophils. Cells were deposited onto glass slides, fixed, and stained with a modified Romaowsky stain. B, C Expression of neutrophil surface markers (B) and apoptotic cells (C) during directed differentiation with G-CSF. Cells were stained for 30 minutes on ice and analysed on a BD LSR Fortessa flow cytometer. D Percentage of cells in culture after directed differentiation. Total PMN is the sum of band and segmented neutrophils. Cytospins were imaged and counted manually using ImageJ software. At least 1000 cells from 10 different images were counted to determine percentages. Data for B-D are shown as individual data from six distinct donors (dots), with horizontal bars indicating the median of each group with the interquartile range depicted by error bars.

Article Snippet: Cells were plated at a minimum density of 5 × 10 5 cells/mL in SFEM II medium (StemCell Technologies) with StemSpan TM CD34+ Expansion Supplement (StemCell Technologies).

Techniques: Isolation, Staining, Modification, Expressing, Flow Cytometry, Software

Journal: bioRxiv

Article Title: Efficient Methods for Target Gene Manipulation in Haematopoietic Stem Cell Derived Human Neutrophils

doi: 10.1101/2023.06.17.545406

Figure Lengend Snippet: A fMLP-stimulated respiratory burst measured by chemiluminescence in differen-tiated CD34 + cells (left) and neutrophils (PMN, right). 4.5 × 10 5 cells were primed with PAF (orange) or mock-primed vehicle (blue) for 5 minutes. Primed or mock-primed cells were incubated with luminol and horseradish peroxidase (HRP) and stimulated with fMLP. Luminescence measured at 5 second intervals after stimulation. Data are in relative light units (RLU) presented as mean and standard deviation of three distinct donors. B 4.5 × 10 5 cells were incubated for 30 minutes with 25 µ g/mL of serum opsonised Staphylococcus aureus (SA) or Escherichia coli (EC) bioparticles in the presence of 3 µ M dihydrorhodamine and analysed on an LSR Fortessa flow cytometer. Experiments incubated at 37°C are shown in orange, and identical cold controls at 4°C are shown in blue. Events shown are of all live cells (Fig. S1B). C-D Differentiated CD34 + cells and human neutrophil phagocytosis (C) and oxidative responses (D) from experiments depicted in B. Data from six distinct donors are shown as dots, with horizontal bars indicating the median of each group with the interquar-tile range depicted by error bars. Data were analysed with the Kruskal-Wallis test between PMN and differentiated CD34 + cells in each condition, where ns denotes a nonsignificant result. E Neutrophil extracellular traps. Differentiated CD34 + cells were incubated with (right) or without (left) 50 nM PMA for 2 hours, stained with 0.2 µ M Sytox Green. Images are representative NETs from one of three biological replicates. Scale bars in these images show a distance of 10 µ m. F Neutrophil granule proteins. Differentiated CD34 + cells were deposited on glass slides, air dried, stained with antibodies to neutrophil elastase (NE, green) and myeloperoxidase (MPO, red), and nuclei counterstained with DAPI. Images are rep-resentative of stained cells from three distinct donors. Scale bars in these images show a distance of 2 µ m. All images were obtained on a Zeiss LSM 980 confocal microscope.

Article Snippet: Cells were plated at a minimum density of 5 × 10 5 cells/mL in SFEM II medium (StemCell Technologies) with StemSpan TM CD34+ Expansion Supplement (StemCell Technologies).

Techniques: Incubation, Standard Deviation, Flow Cytometry, Staining, Microscopy

Journal: bioRxiv

Article Title: Efficient Methods for Target Gene Manipulation in Haematopoietic Stem Cell Derived Human Neutrophils

doi: 10.1101/2023.06.17.545406

Figure Lengend Snippet: A Map of the lentiviral payload used in initial transduction optimisation exper-iments. The puro-T2A-tagBFP protein here is driven by a constitutive PGK promoter. Lentiviral elements up-and downstream of the payload are not shown. B Timeline of stag-gered lentiviral transduction of differentiating CD34 + HSPC. Progenitors were expanded in StemSpan for six days (teal), and then in G-CSF for 14 days (blue). Spinfection was car-ried out every five days from day three as shown in orange. C Representative histograms of tagBFP expression during differentiation at days six, 13, and 20. Experiments show live single cells whose gating is shown in Fig. S1C. D tagBFP expression in transduced cells during G-CSF directed differentiation. Live single cells were classed as tagBFP + by drawing a gate on the untransduced population to a positivity of 0.5% (Fig. S1C). Data are shown as median (dot) and interquartile range (error bars) of twelve biological replicates. All data was obtained on an Attune NxT flow cytometer.

Article Snippet: Cells were plated at a minimum density of 5 × 10 5 cells/mL in SFEM II medium (StemCell Technologies) with StemSpan TM CD34+ Expansion Supplement (StemCell Technologies).

Techniques: Transduction, Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: Efficient Methods for Target Gene Manipulation in Haematopoietic Stem Cell Derived Human Neutrophils

doi: 10.1101/2023.06.17.545406

Figure Lengend Snippet: A Differentiation timeline with nucleofection (red) after 3 days of StemSpan preconditioning. Nucleofected cells were rested in media containing StemSpan and 5 µ M Z-VAD-FMK, a pan-caspase inhibitor, to mitigate cell losses after nucleofection. Cells were then differentiated G-CSF at 10ng/mL for 14 days. B,C CD11b expression by flow cytom-etry at day 20. MFI (B), and total CD11b - the product of percent positive and MFI of the positive cells (C) are shown. D Genomic indel profiles obtained by Sanger sequencing and Tracking of Indels by DEcomposition (TIDE) analysis comparing ITGAM guide RNA 1 compared to donor-specific nontargeting controls. The Cas9 cut site is shown as 0nt, and insertions (right, positive) and deletions (left, negative) are expressed as a percentage of total sequences. Data at 0nt are indicate no sequence change. Total efficacy is shown at the left of the graph and is the sum of percentage indels. E,F Functional capacities of CRISPR-edited neutrophils. Phagocytosis of serum opsonised pHrodo red conjugated bioparticles of S. aureus (SA) and E. coli (EC) are shown in (E), and oxidative response by DHR fluores-cence are shown in (F). NT, Nontargeting guide; ITGAM KO, ITGAM gRNA1. For and 5E-F, individual data from six distinct donors are shown as dots, with horizontal bars indicating the median of each group with the interquartile range depicted by error bars. Asterisks indicate statistical significance (Friedman’s test) as detailed by bars between rel-evant groups: *p ≤ 0.05;**p ≤ 0.01; ***p ≤ 0.001. Gating strategies are shown in Fig. S1D and all flow data was obtained on the Attune NxT flow cytometer. Example outputs of TIDE analysis are shown in Fig.S2.

Article Snippet: Cells were plated at a minimum density of 5 × 10 5 cells/mL in SFEM II medium (StemCell Technologies) with StemSpan TM CD34+ Expansion Supplement (StemCell Technologies).

Techniques: Expressing, Sequencing, Functional Assay, CRISPR, Flow Cytometry