ipsc brew  (Miltenyi Biotec)

Journal: Frontiers in Immunology

Article Title: RAG recombinase expression discriminates the development of natural killer cells

doi: 10.3389/fimmu.2025.1607664

Figure Lengend Snippet: RAG expression can be detected in HSPCs and NK progenitor cells differentiated from RAG-fate-mapping reporter hiPSC lines in vitro . (A) Schematic illustration of the reporter construct and target site for the generation of RAG-fate-mapping reporter hiPSC lines. A reporter cassette consisting of an inverted EGFP sequence flanked by two RSS was inserted into the AAVS1 locus using CRISPR/Cas9. Targeting by RAG1 and RAG2 recombinases results in flipping of the EGFP cassette into sense orientation. (B) Expression of GFP, CD45, and CD56 was analyzed using flow cytometry as demonstrated by this representative gating strategy. GFP + and GFP − NK cells were discriminated by CD45 bright /CD45 dim and CD56 bright /CD56 dim expression, respectively. (C) Quantification of GFP expression in HSPCs and NK cells obtained from iPSC with biallelic (RSS–EGFP +/+ ) and monoallelic (RSS–EGFP +/− ) reporter integration, respectively, was assessed by flow cytometry after hematopoietic differentiation (HSPC), and at weeks (w) 1–3 of the NK cell differentiation. Additional RAG1 and RAG2 mRNA was transfected into HSPCs by nucleofection to induce targeting of the reporter construct. Shown are the percentages of cell populations described in the legend and at indicated time points. (D) Distribution of CD45 bright and CD45 dim expression in GFP + and GFP − NK cell populations, respectively, is shown for NK cells obtained from RSS–EGFP +/+ iPSC (CD45 bright + CD45 dim = 100%). (E) Distribution of CD45 bright CD56 bright /CD56 dim and CD45 dim CD56 bright /CD56 dim populations is shown for GFP − and GFP + NK cell populations (RSS–EGFP +/+ ), respectively. Shown are means ± SEM obtained from at least three experiments. Statistical analysis was performed using two-way ANOVA. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: IPSCs were cultured on Vitronectin XFTM (STEMCELL Technologies) in StemMACSTM iPSC Brew XF (Miltenyi) with daily medium change.

Techniques: Expressing, In Vitro, Construct, Sequencing, CRISPR, Flow Cytometry, Cell Differentiation, Transfection

Journal: Frontiers in Immunology

Article Title: RAG recombinase expression discriminates the development of natural killer cells

doi: 10.3389/fimmu.2025.1607664

Figure Lengend Snippet: NK lineage surface marker expression indicates a more mature phenotype in RAG-fate-mapped NK cells. (A) RAG-fate-mapping reporter iPSCs were differentiated into NK cells and studied weekly for NK lineage marker expression using flow cytometry. Expression of indicated surface markers were analyzed in GFP + versus GFP − CD45 + NK cell precursors (RSS–EGFP +/+ ). (B) Mean expression profiles are shown as percentages in heatmaps for CD45 bright , CD45 dim , and total CD45 + NK lymphocytes. (C) Expression of indicated NK lineage markers was studied in GFP + versus GFP − CD56 bright , CD56 dim , and total CD56 + NK cell precursors, respectively. Shown is the gating strategy performed on one representative sample obtained at week 3 of the differentiation protocol gated on CD45 + CD56 + cells. (D) Expression of indicated NK cell marker was studied in GFP + versus GFP − CD56 + NK cell precursors (RSS–EGFP +/+ ). (E) The mean expression levels of NK cell marker analyzed are shown as percentage distributions in a heatmap for both RSS–EGFP +/+ and RSS–EGFP +/− iPSC-derived cells, respectively. Statistical analysis was performed using 2Way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: IPSCs were cultured on Vitronectin XFTM (STEMCELL Technologies) in StemMACSTM iPSC Brew XF (Miltenyi) with daily medium change.

Techniques: Marker, Expressing, Flow Cytometry, Derivative Assay

Journal: Frontiers in Immunology

Article Title: RAG recombinase expression discriminates the development of natural killer cells

doi: 10.3389/fimmu.2025.1607664

Figure Lengend Snippet: RAG-fate-mapped NK cells are characterized by increased cytokine expression and cytotoxic potential, but impaired DNA damage response. iPSCs carrying bi-allelic reporter constructs (RSS–EGFP +/+ ) were differentiated into NK cells over 3 weeks and subsequently harvested for functional studies. Cells were cocultured with K562 target cells labeled with cell tracer dye at indicated effector:target (E:T) ratios for 1 (h) Expression of CD107a (A) , perforin (B) , granzyme B (C) , and interferon gamma (D) was determined in GFP + and GFP − CD56 + CD16 − and CD56 + CD16 + NK cells, respectively, using flow cytometry. Percentual expression is shown. (E) The antibody-dependent cytotoxicity (ADCC) of NK cells obtained at week 3 of differentiation was quantified using the murine mastocytoma cell line P815. Murine P815 target cells were coated with human anti-CD16 or IgG isotype, respectively, labeled with CellTrace™, and cocultured with NK cells at indicated effector:target (E:T) ratios. Uncoated p815 cells served as control. (F) Degranulation capacity of GFP + /GFP − and CD56 + CD16 − /CD56 + CD16 + NK cells, respectively, was measured by CD107a expression in response to coculture with anti-CD16-coated P815 target cells at indicated effector:target (E:T) ratios. Percentage of CD107a-expressing cells is shown on a linear scale. (G) NK cells obtained at weeks 1–3 of differentiation were irradiated with 2 Gy and fixed at indicated time points. Geometric mean fluorescent intensities (MFIs) of γH2AX are shown for GFP + and GFP − NK cell populations at indicated time points after irradiation. (H) The survival responses were analyzed in the respective GFP + and GFP − populations at indicated time points after 2-Gy ionizing radiation. The percentage of vitality was calculated based on survival rates of unirradiated cells. Shown are results obtained from RSS–EGFP +/+ iPSC as means ± SEM from at least three experiments. Statistical analysis was performed using two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

Article Snippet: IPSCs were cultured on Vitronectin XFTM (STEMCELL Technologies) in StemMACSTM iPSC Brew XF (Miltenyi) with daily medium change.

Techniques: Expressing, Construct, Functional Assay, Labeling, Flow Cytometry, Control, Irradiation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Differences in neuronal ciliation rate and ciliary content revealed by systematic imaging-based analysis of hiPSC-derived models across protocols

doi: 10.3389/fcell.2025.1516596

Figure Lengend Snippet: Variability in ciliation rate in different undifferentiated hiPSC lines. (A) Confocal image of primary cilia in hiPSCs as a maximum intensity z-projection (top) and x-projection (bottom) showing that cilia sit on top of cells pointing towards the medium. (B) Scatter plot showing ciliation rate (% PCNT-positive basal bodies colocalizing with ARL13B-positive cilia) of different sub-confluent iPSC lines at day 2 after replating. Each datapoint represents one fields of view (FOV), with the color-coding indicating different samples (individual wells) for each of the four cell lines. Black lines show the mean for each hiPSC line. Statistical analysis was performed with a Tukey’s multiple comparison test using the mean ciliation rate of each sample. Note the variability between lines, individual samples and between the ROIs of each sample. (C) Cilia length measured in 3D with CiliaQ. Each datapoint represents a single cilium. Plots show the mean and standard deviation.

Article Snippet: The hiPSC line HMGU1 subclone 5 was quality controlled and expanded on Geltrex-coated vessels by EDTA passage in different media depending on the differentiation protocol used: for cortical neuron differentiation in Essential8 (E8) medium (Thermo Fisher), for Gibco NSCs and cerebral organoids in StemFlex medium (Thermo Fisher) and for dopaminergic neurons in StemMACS iPSC Brew (Miltenyi Biotec).

Techniques: Comparison, Standard Deviation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Differences in neuronal ciliation rate and ciliary content revealed by systematic imaging-based analysis of hiPSC-derived models across protocols

doi: 10.3389/fcell.2025.1516596

Figure Lengend Snippet: Adenylyl Cyclase 3 (AC3) is expressed on primary cilia of neuronal cells but not in iPSC cilia. (A) Representative images (maximum projection of a confocal stack) of immunofluorescence stainings with anti-AC3 and anti-ARL13B antibody in various neuronal cell types generated through different protocols as indicated. Basal bodies are marked with PCNT (white) and nuclei are counterstained with Hoechst. Each image is shown twice, with (left) and without (right) ARL13B staining, for better assessment of the AC3 staining. Arrowheads point to sparse cilia. White boxes highlight the regions shown in the insets on the right with separate fluorescence channels. (B) Quantification of AC3+ cilia at all the differentiation stages and different protocols in all the lines: we assessed ciliation rate based on ARL13B-positive cilia (ARL13B+/PCNT+) and ciliation rate based on AC3-positive cilia (AC3+/PCNT+) separately and from these numbers calculated the percentage of AC3-positive cilia over ARL13B-positive cilia. Images of additional differentiation stages and protocols can be found in . The proportion of AC3+ cilia increases with increasing maturity of the neurons.

Article Snippet: The hiPSC line HMGU1 subclone 5 was quality controlled and expanded on Geltrex-coated vessels by EDTA passage in different media depending on the differentiation protocol used: for cortical neuron differentiation in Essential8 (E8) medium (Thermo Fisher), for Gibco NSCs and cerebral organoids in StemFlex medium (Thermo Fisher) and for dopaminergic neurons in StemMACS iPSC Brew (Miltenyi Biotec).

Techniques: Immunofluorescence, Generated, Staining, Fluorescence