Journal: Stem Cell Reports

Article Title: Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency

doi: 10.1016/j.stemcr.2016.06.011

Figure Lengend Snippet: Disease Modeling of herPAP Using miPAP iPSCs (A) Number of CD41 + progenitor-derived colonies in methylcellulose-based clonogenic assays employing complete (IL-6, erythropoietin, SCF, IL-3, and supplemented with 20 ng/ml human G-CSF + 20 ng/ml murine GM-CSF) or basic medium (50 ng/ml murine GM-CSF only; independent experiments, n = 3 miPAP1 and 2, n = 2 CD45.1 iPSC, mean ± SD) and representative pictures of colonies. Scale bars, 500 μm. (B) GM-CSF clearance assay comparing miPAP-Mφ with CD45.1(10.3) iPSC-Mφ, BMlin − -Mφ, and no cells incubated with 2 ng/ml GM-CSF: at indicated time points (0, 4, 10, 24, and 30 hr) GM-CSF concentrations in supernatants were analyzed by ELISA, normalized to 0 hr (independent experiments, n = 3 BMlin − and CD45.1 iPSC, n = 2 miPAP1 and 2, mean ± SD). (C and D) Flow cytometry plots of STAT5 phosphorylation levels upon stimulation with mGM-CSF (C) and (D) summary of mean fluorescence intensity (MFI) data (independent experiments, n = 3 BMlin − and CD45.1 iPSC, n = 2 miPAP1 and 2, mean ± SD). ns, not significant; ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, two-way ANOVA.

Article Snippet: Hematopoietic CD41 + progenitor cells obtained from iPSC differentiation (8 days after start of EB culture) were seeded at a concentration of 20,000–50,000/ml in basic and complete methylcellulose medium (HSC006 with no cytokines added and HSC007 containing IL-6, erythropoietin, SCF, and IL-3, respectively; R&D Systems).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Phospho-proteomics, Fluorescence

Journal: Frontiers in Oncology

Article Title: Replication stress increases mitochondrial metabolism and mitophagy in FANCD2 deficient fetal liver hematopoietic stem cells

doi: 10.3389/fonc.2023.1108430

Figure Lengend Snippet: Mitophagy activity was decreased in Fancd2-deficient BM HSC. (A) Relative MFI (left panel) and representative flow cytometric plots (right panel) showing MMP in adult (8-12 weeks) HSCs (Fancd2 WT n=6 mice, HET n=2 mice, KO n=8 mice, left panel). (B) Relative MFI of mtROS in adult HSCs (Fancd2 WT n=5 mice, HET n=2 mice, KO n=5 mice). (C) Mitochondrial area, mitochondrial concentration and mitochondrial polarity in HSCs. HSCs were stained with anti-Tomm20 and analyzed by IFM (Fancd2 WT n=559 cells, KO n=431 cells). (D) Relative MFI (left panel) and representative flow cytometric plots (right panel) of mitophagy in BM HSCs (Fancd2 WT n=5 mice, HET n=2 mice, KO n=4 mice). (E) Relative MFI (left panel) and representative FACS plots (right panel) of LA in BM HSCs (Fancd2 WT n=6 mice, KO n=5 mice, left panel). All relative data are divided with average of controls (WT or HET) and show mean ± SD. *P<0.05, ****P<0.0001, ns, not significant.

Article Snippet: HSCs were sorted by FACS Aria II or III (BD) and 500 cells are seeded onto 3ml of mouse methylcellulose media (R&D Systems, HSC007) with APH (50-100ng/ml) or imTOR of Rapamycin (Thermo PHZ1235, diluted with DMSO for 10mM and used as 1:1000, final concentration 10μM) or iTgf-β of SD-208 (CAYMAN 16619, diluted with DMSO for 10mM and used as 1:1000, final concentration 10μM).

Techniques: Activity Assay, Concentration Assay, Staining