A hypomorphic PIGA gene mutation causes severe defects in neuron development and susceptibility to complement-mediated toxicity in a human iPSC model
Fig 3
The PIGAc.1234C>T mutation does not impair terminal hematopoietic differentiation during mesoderm induction.
(A). Quantitation of EB-derived blood-like cells (EB-BLCs). A total of 100 hiPSC-derived EBs were assessed for each hiPSC derived cell line and the percentage of EB-BLCs present was determined. The percentage of EB-BLCs derived from PIGAwt hiPSCs was significantly higher than EB-BLCs from PIGAc.1234C>T hiPSCs (**P<0.01, Mann-Whitney test). Results shown are average and standard deviation (mean ± SD) based on three independent experiments. (B). Expression of hematopoietic markers CD33, CD34 &CD45 in EB-BLCs from PIGAwt and PIGAc.1234C>T hiPSCs. The cells were stained with anti-human CD33, anti-human CD34 and anti-human CD45 for flow cytometry analysis. Antibodies and analyzed by flow cytometry. The values shown are from three independent experiments. All values represent average and standard error (mean ± SE). (C). Representative example of FACS analysis of hematopoietic phenotypes in the EB-BLCs from PIGAwt and PIGAc.1234C>T. The zebra plot shows expression of CD59 (X-axis) and CD45 (Y-axis) after three and eight days of hematopoietic differentiation. Unstained PIGAwt cells were used as a control. (D). Enumeration of colony forming units (CFU) from the BLCs derived from PIGAwt and PIGAc.1234C>T. There was no significant difference in CFU colony formation between PIGAwt and PIGAc.1234C>T hematopoietic cells (p>0.05, NS, one-tailed, Unpaired T test). All values represent mean ± SE. Abbreviations: CFU-Macrophage (M); CFU-Granulocyte-Macrophage (GM); committed erythroid BFU-E (BFU) and CFU-E (CFU) progenitors; multipotent progenitor cells CFU-GEMM (GEMM).