Figure 1.
Hematopoietic differentiation of patient iPSC is grossly intact. (A) Hematopoietic differentiation scheme. Spin EB derived from iPSC underwent a multistep hematopoietic differentiation process (top). Mesodermal precursors were evaluated at day 4. HEP were assessed at day 9. At day 16 of hematopoietic differentiation, CD34+CD45+ cells were sorted for CFU assay and gene expression analysis. FB/MSC-ZF includes iPSC from subject 1 (GATA2 R361H) and subject 2 (GATA2 R337X). FB/MSC-Int5 includes iPSC from subject 3 (intron 5 proband). Patient PBMC-derived iPSC efficiency was compared with healthy volunteer PBMC-derived iPSC controls. PBMC-ZF includes 3 clones from subject 1 (R361H). PBMC-Int5F includes 3 clones from subject 3 (proband). PBMC-Int5M includes 3 clones from subject 4 (asymptomatic carrier). A minimum of 2 independent experiments were performed unless otherwise specified. (B) Percentage of mesodermal precursor cells on day 4 of differentiation. (FB/MSC-control: n = 10; ZF, n = 7; Int5, n = 3; PBMC-control: n = 4; ZF, n = 3; Int5, n = 6; Int5M, n = 3). One ZF2 clone (R337X-FB-1) and 3 Int5 clones among the FB/MSC-iPSC group and 3 R361H clones and 3 Int5M clones among the PBMC-iPSC group were tested in 1 experiment. (C) Fraction of HEP at day 9 derived from various control, ZF, or intron 5 GATA2-mutant iPSC (control: n = 10; ZF, n = 6; Int5, n = 3). Three Int5 clones were tested in 1 experiment. (D) Fraction of CD34+CD45+ cells and CD45+ cells at day 16 of hematopoietic differentiation (FB/MSC-control: n = 14; ZF, n = 11; Int5, n = 6; PBMC-control: n = 6; ZF, n = 3; Int5, n = 9; Int5M, n = 6). One ZF clone (R337X-FB-1) among the FB/MSC-iPSC group and 3 R361H clones among the PBMC-iPSC group were tested in 1 experiment. (E) Functional properties of iPSC-derived CD34+CD45+ cells as evaluated by CFU assay. The number and type of CFU colonies from 1000 sorted CD34+CD45+ cells were scored. Total number of colonies per 1000 sorted CD34+CD45+ (FB/MSC-control: n = 20; ZF, n = 10; Int5, n = 6; PBMC-control: n = 6, Int5F, n = 12; Int5M, n = 6). (F) Fraction of CFU subtype per total number of colonies, respective of mutation types. P values were determined by 1-way ANOVA followed by Dunnett multiple comparison test. Int5F and Int5M were compared independently by unpaired Student t test. *P < .05, **P < .01, and ***P < .005; ****P < .0001. (F) Targeted myeloid neoplasm panel next-generation sequencing was performed on patient’s primary cells (dermal fibroblasts, bone marrow stromal cells, or peripheral blood) and iPSC clones derived from those primary cells. *Two separate variants (supplemental Table 9). #GATA2 intronic variants were examined by Sanger sequencing because intronic regions were not covered in the targeted next-generation sequencing.

Hematopoietic differentiation of patient iPSC is grossly intact. (A) Hematopoietic differentiation scheme. Spin EB derived from iPSC underwent a multistep hematopoietic differentiation process (top). Mesodermal precursors were evaluated at day 4. HEP were assessed at day 9. At day 16 of hematopoietic differentiation, CD34+CD45+ cells were sorted for CFU assay and gene expression analysis. FB/MSC-ZF includes iPSC from subject 1 (GATA2 R361H) and subject 2 (GATA2 R337X). FB/MSC-Int5 includes iPSC from subject 3 (intron 5 proband). Patient PBMC-derived iPSC efficiency was compared with healthy volunteer PBMC-derived iPSC controls. PBMC-ZF includes 3 clones from subject 1 (R361H). PBMC-Int5F includes 3 clones from subject 3 (proband). PBMC-Int5M includes 3 clones from subject 4 (asymptomatic carrier). A minimum of 2 independent experiments were performed unless otherwise specified. (B) Percentage of mesodermal precursor cells on day 4 of differentiation. (FB/MSC-control: n = 10; ZF, n = 7; Int5, n = 3; PBMC-control: n = 4; ZF, n = 3; Int5, n = 6; Int5M, n = 3). One ZF2 clone (R337X-FB-1) and 3 Int5 clones among the FB/MSC-iPSC group and 3 R361H clones and 3 Int5M clones among the PBMC-iPSC group were tested in 1 experiment. (C) Fraction of HEP at day 9 derived from various control, ZF, or intron 5 GATA2-mutant iPSC (control: n = 10; ZF, n = 6; Int5, n = 3). Three Int5 clones were tested in 1 experiment. (D) Fraction of CD34+CD45+ cells and CD45+ cells at day 16 of hematopoietic differentiation (FB/MSC-control: n = 14; ZF, n = 11; Int5, n = 6; PBMC-control: n = 6; ZF, n = 3; Int5, n = 9; Int5M, n = 6). One ZF clone (R337X-FB-1) among the FB/MSC-iPSC group and 3 R361H clones among the PBMC-iPSC group were tested in 1 experiment. (E) Functional properties of iPSC-derived CD34+CD45+ cells as evaluated by CFU assay. The number and type of CFU colonies from 1000 sorted CD34+CD45+ cells were scored. Total number of colonies per 1000 sorted CD34+CD45+ (FB/MSC-control: n = 20; ZF, n = 10; Int5, n = 6; PBMC-control: n = 6, Int5F, n = 12; Int5M, n = 6). (F) Fraction of CFU subtype per total number of colonies, respective of mutation types. P values were determined by 1-way ANOVA followed by Dunnett multiple comparison test. Int5F and Int5M were compared independently by unpaired Student t test. *P < .05, **P < .01, and ***P < .005; ****P < .0001. (F) Targeted myeloid neoplasm panel next-generation sequencing was performed on patient’s primary cells (dermal fibroblasts, bone marrow stromal cells, or peripheral blood) and iPSC clones derived from those primary cells. *Two separate variants (supplemental Table 9). #GATA2 intronic variants were examined by Sanger sequencing because intronic regions were not covered in the targeted next-generation sequencing.

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