American Society of Hematology

Figure 3.

$Figure 3. CRISPR/Cas9-engineered hESCs with AHR deletion demonstrate increased early hematoendothelial cell development. (A) gRNA cassette design targeting AHR. gRNA exon 1 indicates 22-nt gRNA specific to AHR exon 1. (B) Gel electrophoresis of PCR products produced from clonally derived hESC-RUNx1c-tdTomato cells nucleofected with AHR gRNA cassette. Genomic DNA was harvested and primers flanking the AHR exon 1 locus were used to generate a PCR product with predicted full-length of 718 bp. WT indicates negatively selected nucleofected hESC-RUNX1c-tdTomato hESCs; +/− indicates individual clones with AHR heterozygous deletion (AHR+/−); −/− indicates individual clones with AHR homozygous deletion (AHR−/−). (C) Immunoblot of protein lysate harvested from K562 cells (positive control), NK92 NK cells (positive control), WT hESC-RUNX1c-tdTomato (hESC-R1c-tdTom), heterozygous AHR-deleted hESC-RUNX1c-tdTomato (+/−), and homozygous AHR deleted hESC-RUNX1c-tdTomato (−/−). (D) Representative flow cytometry plots at day 6+3, day 6+6, and day 6+9 from 1 differentiation of WT hESC-RUNX1c-tdTomato (WT), heterozygous AHR hESC-RUNX1c-tdTomato deletion (AHR+/−), and homozygous AHR hESC-RUNX1c-tdTomato deletion (AHR−/−). Both adherent and nonadherent cell fractions are harvested at day 6+3, day 6+6, and day 6+9 and assessed for endothelial (CD31, CD144), and hematopoietic (CD33, CD41a, CD43, CD45) phenotype. (E) Representative flow cytometry plots at day 6+3 and day 6+6 from 1 differentiation assessing for RUNX1c expression based on tdTomato fluorescent reporter protein. (F) Nonadherent hematopoietic progenitor cells derived from WT hESC-RUNX1c-tdTomato, AHR+/− hESC-RUNX1c-tdTomato, or AHR−/− hESC-RUNX1c-tdTomato were harvested at day 6+5 and seeded at 50 000 cells per dish in a standard methylcellulose CFU assay (CFU). Colonies were counted for each treatment group following 2 weeks of culture and scored for the following morphological subsets, as previously described; n = 3, error bars represent SEM of the total number of colonies per 50 000 cells seeded. *P < .05 as assessed with 1-way ANOVA + Tukey-Kramer multiple comparisons post hoc test. IS, insertion sequence; Term, termination sequence. *718-bp amplicon; ^571-bp amplicon.$

CRISPR/Cas9-engineered hESCs with AHR deletion demonstrate increased early hematoendothelial cell development. (A) gRNA cassette design targeting AHR. gRNA exon 1 indicates 22-nt gRNA specific to AHR exon 1. (B) Gel electrophoresis of PCR products produced from clonally derived hESC-RUNx1c-tdTomato cells nucleofected with AHR gRNA cassette. Genomic DNA was harvested and primers flanking the AHR exon 1 locus were used to generate a PCR product with predicted full-length of 718 bp. WT indicates negatively selected nucleofected hESC-RUNX1c-tdTomato hESCs; +/− indicates individual clones with AHR heterozygous deletion (AHR^+/−); −/− indicates individual clones with AHR homozygous deletion (AHR^−/−). (C) Immunoblot of protein lysate harvested from K562 cells (positive control), NK92 NK cells (positive control), WT hESC-RUNX1c-tdTomato (hESC-R1c-tdTom), heterozygous AHR-deleted hESC-RUNX1c-tdTomato (+/−), and homozygous AHR deleted hESC-RUNX1c-tdTomato (−/−). (D) Representative flow cytometry plots at day 6+3, day 6+6, and day 6+9 from 1 differentiation of WT hESC-RUNX1c-tdTomato (WT), heterozygous AHR hESC-RUNX1c-tdTomato deletion (AHR^+/−), and homozygous AHR hESC-RUNX1c-tdTomato deletion (AHR^−/−). Both adherent and nonadherent cell fractions are harvested at day 6+3, day 6+6, and day 6+9 and assessed for endothelial (CD31, CD144), and hematopoietic (CD33, CD41a, CD43, CD45) phenotype. (E) Representative flow cytometry plots at day 6+3 and day 6+6 from 1 differentiation assessing for RUNX1c expression based on tdTomato fluorescent reporter protein. (F) Nonadherent hematopoietic progenitor cells derived from WT hESC-RUNX1c-tdTomato, AHR^+/− hESC-RUNX1c-tdTomato, or AHR^−/− hESC-RUNX1c-tdTomato were harvested at day 6+5 and seeded at 50 000 cells per dish in a standard methylcellulose CFU assay (CFU). Colonies were counted for each treatment group following 2 weeks of culture and scored for the following morphological subsets, as previously described; n = 3, error bars represent SEM of the total number of colonies per 50 000 cells seeded. *P < .05 as assessed with 1-way ANOVA + Tukey-Kramer multiple comparisons post hoc test. IS, insertion sequence; Term, termination sequence. *718-bp amplicon; ^571-bp amplicon.

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