Figure 1.
Figure 1. Reprogramming identifies stages of clonal evolution in MDS. (A) Experimental scheme. BMMCs are extracted from the iliac crest of patients with MDS and cryopreserved. CD34+ HPCs are isolated and reprogrammed by episomal transfection of OSKM transcription factors. Single reprogrammed cells give rise to iPSC colonies, and individual colonies are genotyped for disease alleles and expanded into clonal iPSC lines. Individual lines are differentiated into CD34+ HPCs and multipotential progenitor lines established by 5F transfection. (B) Molecular abnormalities present in the MDS-RA1 patient. Standard karyotype showing the reciprocal t(4;12)(q31.3;q15) translocation (arrows). EGR1(red)/D5S23 FISH and SNP arrays showing a del(5)(q31.2) deletion; and Sanger sequencing showing heterozygous EZH2 (p.R685H, NM_001203247.1: c.2054G>A) and SF3B1 (p.G742D, NM_012433.2: c.2225G>A) mutations. (C) Percentage of patient BMMCs positive for t(4;12) by karyotyping, del(5q) by FISH, and point mutations (VAF × 2 for heterozygous mutations). (D) Genotyping of iPSC lines generated by nonintegrating Sendai or episomal reprogramming. iPSC lines were genotyped for t(4;12) by karyotyping, del(5q) by FISH, and Sanger sequencing for point mutations. Percent iPSCs of each genotype is indicated by color (total isolated = n), with gray indicating colonies negative for MDS mutations. Genotypes are abbreviated as follows: t(4;12)-only, t(4;12);SF3B1 (t;SF3B1), t(4;12);SF3B1;EZH2 (t;S;EZH2), t(4;12);SF3B1;EZH2;del(5q) (t;S;E;5q). (E) Genotypes of the individual iPSC lines (across) showing successive acquisition of patient-specific abnormalities (down). Only MDS-derived iPSCs are shown. (F) The inferred order of somatic mutations during clonal evolution. Somatic variants were identified by WES in the patient and one or more subclonal MDS-iPSCs but not in normal isogenic iPSCs. Variants are ordered according to iPSC genotype. Complete data are shown in supplemental Table 3. (G) Comparison of clonal representation (percentage of total colonies) by genotyping CFUs (n = 62) and iPSCs derived from patient CD34+ cells. Single colonies were genotyped for each mutation by allele-specific quantitative polymerase chain reaction. (H) Proportion of premalignant (red; partial complement of mutations) and malignant (blue; all mutations) subclones in CFU versus iPSC colonies of 4 patients with MDS. Number indicates fold enrichment in iPSCs compared with CFUs.

Reprogramming identifies stages of clonal evolution in MDS. (A) Experimental scheme. BMMCs are extracted from the iliac crest of patients with MDS and cryopreserved. CD34+ HPCs are isolated and reprogrammed by episomal transfection of OSKM transcription factors. Single reprogrammed cells give rise to iPSC colonies, and individual colonies are genotyped for disease alleles and expanded into clonal iPSC lines. Individual lines are differentiated into CD34+ HPCs and multipotential progenitor lines established by 5F transfection. (B) Molecular abnormalities present in the MDS-RA1 patient. Standard karyotype showing the reciprocal t(4;12)(q31.3;q15) translocation (arrows). EGR1(red)/D5S23 FISH and SNP arrays showing a del(5)(q31.2) deletion; and Sanger sequencing showing heterozygous EZH2 (p.R685H, NM_001203247.1: c.2054G>A) and SF3B1 (p.G742D, NM_012433.2: c.2225G>A) mutations. (C) Percentage of patient BMMCs positive for t(4;12) by karyotyping, del(5q) by FISH, and point mutations (VAF × 2 for heterozygous mutations). (D) Genotyping of iPSC lines generated by nonintegrating Sendai or episomal reprogramming. iPSC lines were genotyped for t(4;12) by karyotyping, del(5q) by FISH, and Sanger sequencing for point mutations. Percent iPSCs of each genotype is indicated by color (total isolated = n), with gray indicating colonies negative for MDS mutations. Genotypes are abbreviated as follows: t(4;12)-only, t(4;12);SF3B1 (t;SF3B1), t(4;12);SF3B1;EZH2 (t;S;EZH2), t(4;12);SF3B1;EZH2;del(5q) (t;S;E;5q). (E) Genotypes of the individual iPSC lines (across) showing successive acquisition of patient-specific abnormalities (down). Only MDS-derived iPSCs are shown. (F) The inferred order of somatic mutations during clonal evolution. Somatic variants were identified by WES in the patient and one or more subclonal MDS-iPSCs but not in normal isogenic iPSCs. Variants are ordered according to iPSC genotype. Complete data are shown in supplemental Table 3. (G) Comparison of clonal representation (percentage of total colonies) by genotyping CFUs (n = 62) and iPSCs derived from patient CD34+ cells. Single colonies were genotyped for each mutation by allele-specific quantitative polymerase chain reaction. (H) Proportion of premalignant (red; partial complement of mutations) and malignant (blue; all mutations) subclones in CFU versus iPSC colonies of 4 patients with MDS. Number indicates fold enrichment in iPSCs compared with CFUs.

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