Figure 2.
Characterization of iPSC-derived neutrophil granulocytes. (A) Flow cytometric analysis of control iPSC-derived neutrophil granulocytes at day 29. Experiment performed in triplicate. (B) Quantification of live floating cells per 6 iPSC colonies (per well), determined at indicated time points during differentiation. For control, SRPRA+/MUT, and SRP19MUT/MUT, 2 biological replicates (2 different clones) are presented as mean of 3 independent experiments. For HAX1-knockout (KO) the mean of 1 clone of 3 independent experiments is presented. Statistical analysis using 2-way analysis of variance (ANOVA) followed by Dunnett multiple comparisons test. ∗∗P < .0032 and ∗∗∗∗P < .0001. (C) Light microscopy of control, SRPRA+/MUT, SRP19MUT/MUT, and HAX1-KO iPSC-derived myeloid cells stained with May-Grünwald Giemsa stain at day 29. (D) Quantification of the distribution of precursor populations in iPSC-derived myeloid cells for the indicated genotypes. Floating cells were harvested and stained with May-Grünwald Giemsa stain at day 29 and classified by light microscopy. The quantification was performed for 2 independent experiments; a total of 200 cells per genotype were classified. (E) Statistical analysis of the quantification shown in panel D using 2-way ANOVA followed by Dunnett multiple comparisons test. ∗P < .03, ∗∗∗P < .0009, and ∗∗∗∗P < .0001. Error bars represent mean with standard deviation. Quantification of 2 independent experiments are shown. (F) Immunoblot analysis of XBP-1s (spliced form of XBP-1) expression in iPSC-derived myeloid cells at day14. Experiment performed in triplicate. (G) Quantification of annexin V+ cells in sorted (CD33 high/CD49d high) immature iPSC-derived neutrophil granulocytes. Cells were analyzed on day 26 after differentiation. (n = 3 wells per each clone, multiple t test). For control, SRPRA+/MUT and SRP19MUT/MUT, 2 biological replicates (2 different clones) are presented as mean of 3 independent experiments. (H) Immunoblot analysis of apoptosis-specific markers (cleaved PARP and cleaved caspase 3) in iPSC-derived myeloid cells at day14. Experiment performed in triplicate. ns, not significant.

Characterization of iPSC-derived neutrophil granulocytes. (A) Flow cytometric analysis of control iPSC-derived neutrophil granulocytes at day 29. Experiment performed in triplicate. (B) Quantification of live floating cells per 6 iPSC colonies (per well), determined at indicated time points during differentiation. For control, SRPRA+/MUT, and SRP19MUT/MUT, 2 biological replicates (2 different clones) are presented as mean of 3 independent experiments. For HAX1-knockout (KO) the mean of 1 clone of 3 independent experiments is presented. Statistical analysis using 2-way analysis of variance (ANOVA) followed by Dunnett multiple comparisons test. ∗∗P < .0032 and ∗∗∗∗P < .0001. (C) Light microscopy of control, SRPRA+/MUT, SRP19MUT/MUT, and HAX1-KO iPSC-derived myeloid cells stained with May-Grünwald Giemsa stain at day 29. (D) Quantification of the distribution of precursor populations in iPSC-derived myeloid cells for the indicated genotypes. Floating cells were harvested and stained with May-Grünwald Giemsa stain at day 29 and classified by light microscopy. The quantification was performed for 2 independent experiments; a total of 200 cells per genotype were classified. (E) Statistical analysis of the quantification shown in panel D using 2-way ANOVA followed by Dunnett multiple comparisons test. ∗P < .03, ∗∗∗P < .0009, and ∗∗∗∗P < .0001. Error bars represent mean with standard deviation. Quantification of 2 independent experiments are shown. (F) Immunoblot analysis of XBP-1s (spliced form of XBP-1) expression in iPSC-derived myeloid cells at day14. Experiment performed in triplicate. (G) Quantification of annexin V+ cells in sorted (CD33 high/CD49d high) immature iPSC-derived neutrophil granulocytes. Cells were analyzed on day 26 after differentiation. (n = 3 wells per each clone, multiple t test). For control, SRPRA+/MUT and SRP19MUT/MUT, 2 biological replicates (2 different clones) are presented as mean of 3 independent experiments. (H) Immunoblot analysis of apoptosis-specific markers (cleaved PARP and cleaved caspase 3) in iPSC-derived myeloid cells at day14. Experiment performed in triplicate. ns, not significant.

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