Figure 4.
Figure 4. hESCs differentiated in the presence of SR-1 promote the development of functional NK cells. (A) Schema of hESC differentiation into lymphoid cells as spin EBs. hESCs are made into spin EBs at day 0 and cultured in stage 1 conditions with defined cytokines to promote mesoderm development for 11 days. At day 11, spin EBs are transferred onto OP9-DL1 in the presence of NKDM to promote lymphoid differentiation. Cells are treated beginning at day 11+0 with either 1 μM SR-1, 10 nM TCDD, or DMSO vehicle control with media exchanges and/or harvesting performed every week for up to 4 weeks. (B) At day 11, differentiated spin EBs (original magnification ×40) are phenotyped for CD34+CD45+ expression and transferred to OP9-DL1 stroma in NKDM. Nonadherent hematopoietic cells cultured either in the presence of DMSO, SR-1, or TCDD were assessed for developing NK-cell immunophenotype based on CD56+CD45+ expression at days 11+21, and 11+28; representative flow cytometry plots from 1 differentiation are shown. (C) Quantification of fold change in total percentage of CD56+CD45+ cells at both day 11+21 and day 11+28 when treated with DMSO, SR-1, or TCDD. SR-1 and TCDD treatments for each differentiation are normalized to DMSO controls; n = 3 independent differentiation experiments, error bars represent SEM. *P < .05 as assessed with 2-way ANOVA + Tukey-Kramer multiple comparisons post hoc test. (D) Nonadherent hematopoietic progenitor cells derived from hESCs differentiated in the presence of SR-1, TCDD, or DMSO controls were harvested at day 11+28 and probed for gene expression by qRT-PCR. For each gene, Ct values were normalized to GAPDH at each time point and data are presented as relative fold change to DMSO-treated controls; n = 3, error bars represent SEM. *P < .05; #P < .01 using the Student t test. (E) Nonadherent hematopoietic progenitor cells derived from hESCs differentiated in the presence of SR-1, TCDD, or DMSO controls were harvested at day 11+28 and assessed for CD107a expression following 4 hours of coculture with K562 target cells at a 2:1 effector:target ratio. Representative flow cytometry plots are shown from 1 experiment. (F) Quantification of percentage of CD107a+ cells when treated with DMSO, SR-1, or TCDD at day 11+28; n = 2-3 replicates. SSC, side scatter.

hESCs differentiated in the presence of SR-1 promote the development of functional NK cells. (A) Schema of hESC differentiation into lymphoid cells as spin EBs. hESCs are made into spin EBs at day 0 and cultured in stage 1 conditions with defined cytokines to promote mesoderm development for 11 days. At day 11, spin EBs are transferred onto OP9-DL1 in the presence of NKDM to promote lymphoid differentiation. Cells are treated beginning at day 11+0 with either 1 μM SR-1, 10 nM TCDD, or DMSO vehicle control with media exchanges and/or harvesting performed every week for up to 4 weeks. (B) At day 11, differentiated spin EBs (original magnification ×40) are phenotyped for CD34+CD45+ expression and transferred to OP9-DL1 stroma in NKDM. Nonadherent hematopoietic cells cultured either in the presence of DMSO, SR-1, or TCDD were assessed for developing NK-cell immunophenotype based on CD56+CD45+ expression at days 11+21, and 11+28; representative flow cytometry plots from 1 differentiation are shown. (C) Quantification of fold change in total percentage of CD56+CD45+ cells at both day 11+21 and day 11+28 when treated with DMSO, SR-1, or TCDD. SR-1 and TCDD treatments for each differentiation are normalized to DMSO controls; n = 3 independent differentiation experiments, error bars represent SEM. *P < .05 as assessed with 2-way ANOVA + Tukey-Kramer multiple comparisons post hoc test. (D) Nonadherent hematopoietic progenitor cells derived from hESCs differentiated in the presence of SR-1, TCDD, or DMSO controls were harvested at day 11+28 and probed for gene expression by qRT-PCR. For each gene, Ct values were normalized to GAPDH at each time point and data are presented as relative fold change to DMSO-treated controls; n = 3, error bars represent SEM. *P < .05; #P < .01 using the Student t test. (E) Nonadherent hematopoietic progenitor cells derived from hESCs differentiated in the presence of SR-1, TCDD, or DMSO controls were harvested at day 11+28 and assessed for CD107a expression following 4 hours of coculture with K562 target cells at a 2:1 effector:target ratio. Representative flow cytometry plots are shown from 1 experiment. (F) Quantification of percentage of CD107a+ cells when treated with DMSO, SR-1, or TCDD at day 11+28; n = 2-3 replicates. SSC, side scatter.

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