Figure 5.
Impaired in vitro hematopoietic differentiation by TET2-deficient iPSCs. (A) Schematic presentation of experimental strategy to assess the hematopoietic differentiation capacity from iPSCs in vitro24 with representative microphotographs (original magnification, ×100) of cell culture at major differentiation stages, starting with embryoid bodies at days 0 to 3, followed by the presence of hematopoietic progenitors (HP) and their budding at days 5 to 12, and culminating in proliferation and maturation at days 13 to 30. (B) Scatter box plots demonstrating the percentage of positive cells detected by flow cytometry at individual time points during differentiation: HP (CD34+/−CD43+), erythro-megakaryocytic progenitors (CD43+CD235a+CD41a+), erythroid progenitors (CD43+CD235a+CD41a−), megakaryocytic progenitors (CD43+CD235a−CD41a+), and myeloid progenitors (CD43+CD235a−CD41a−CD45+). Horizontal lines represent median values from a minimum 6 independent experiments. **P < .01, ***P < .001, nonparametric Kruskal-Wallis test. (C) Quantitative and qualitative results of CFU assay of HP, plated at individual time points of differentiation into semisolid medium and incubated for 2 weeks, showing skewed differentiation toward the myeloid lineage at the expense of erythroid and megakaryocytic (Meg) colonies. Data are mean ± SD from a minimum of 3 independent experiments. (D) Representative microphotographs (original magnification, ×100) of individual CFU classified according to characteristic morphologic features: (i) BFU-E: burst-forming unit-erythoid, (ii) CFU-E: erythroid, (iii) CFU-GM: granulomonocytic, (iv) CFU-Meg: megakaryocytic, (v) above, CFU-E and below, CFU-G: granulocytic, (vi) left, BFU-E and right, CFU-Meg. (E) 5mC/5hmC ratio determined by DNA methylation assay in fibroblasts, iPSCs, and HP from healthy control (C) and patients (P) at day 13 (D13) and day 20 (D20) of hematopoietic differentiation in vitro. Data are mean ± SD from 2 biological repeats. *P < .05 vs healthy control, unpaired Student t test. D9, day 9; D13, day 13; D16, day 16; D20, day 20; D30, day 30; ns, not significant.

Impaired in vitro hematopoietic differentiation by TET2-deficient iPSCs. (A) Schematic presentation of experimental strategy to assess the hematopoietic differentiation capacity from iPSCs in vitro24  with representative microphotographs (original magnification, ×100) of cell culture at major differentiation stages, starting with embryoid bodies at days 0 to 3, followed by the presence of hematopoietic progenitors (HP) and their budding at days 5 to 12, and culminating in proliferation and maturation at days 13 to 30. (B) Scatter box plots demonstrating the percentage of positive cells detected by flow cytometry at individual time points during differentiation: HP (CD34+/−CD43+), erythro-megakaryocytic progenitors (CD43+CD235a+CD41a+), erythroid progenitors (CD43+CD235a+CD41a−), megakaryocytic progenitors (CD43+CD235a−CD41a+), and myeloid progenitors (CD43+CD235a−CD41a−CD45+). Horizontal lines represent median values from a minimum 6 independent experiments. **P < .01, ***P < .001, nonparametric Kruskal-Wallis test. (C) Quantitative and qualitative results of CFU assay of HP, plated at individual time points of differentiation into semisolid medium and incubated for 2 weeks, showing skewed differentiation toward the myeloid lineage at the expense of erythroid and megakaryocytic (Meg) colonies. Data are mean ± SD from a minimum of 3 independent experiments. (D) Representative microphotographs (original magnification, ×100) of individual CFU classified according to characteristic morphologic features: (i) BFU-E: burst-forming unit-erythoid, (ii) CFU-E: erythroid, (iii) CFU-GM: granulomonocytic, (iv) CFU-Meg: megakaryocytic, (v) above, CFU-E and below, CFU-G: granulocytic, (vi) left, BFU-E and right, CFU-Meg. (E) 5mC/5hmC ratio determined by DNA methylation assay in fibroblasts, iPSCs, and HP from healthy control (C) and patients (P) at day 13 (D13) and day 20 (D20) of hematopoietic differentiation in vitro. Data are mean ± SD from 2 biological repeats. *P < .05 vs healthy control, unpaired Student t test. D9, day 9; D13, day 13; D16, day 16; D20, day 20; D30, day 30; ns, not significant.

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