Figure 4.
The small molecule inhibitor CHIR99021 enhances the maturity of FoP-MKs via wnt pathway activation. (A) Profiles for the expression of downstream Wnt target genes following 24-hour exposure to CHIR99021. Differentiating cells were treated with the CHIR99021 inhibitor between D1 and D2 (shaded panel) during mesoderm induction (indicated by the expression of Brachyury). Quantitative PCR graphs showing expression of CDX4, HoxA9 and Sall4 between day 0 and day 5 postinduction for iDELTA-3.7 CHIR99021 (red), DMSO control (green). All are expressed relative to HMBS, as the endogenous control, and D1 levels; n = 3 biological replicates; mean ±1× SEM. HoxA9 expression remains significantly. **P < .01 upregulated at day 5. (B) MK maturation from iLIPSC-GR1.1.2 and iDELTA-3.7 with CHIR99021. Bar graphs showing the number of viable MKs for iLIPSC-GR1.1.2 on day 20 and iDELTA-3.7 on day 30 expressed relative to AMK. AMK (blue bar), CHIR99021 (red bar) or DMSO (green bar). Viable MKs for iLIPSC-GR1.1.2 peak earlier at D20 in CHIR99021 treated cultures. Mean ±1× SEM; n = 6 biological replicates, AMK vs CHIR99021 and CHIR99021 vs DMSO are not significantly different using Kruskal-Wallis multiple comparison test. Viable MKs at day 30 for iDELTA-3.7 are significantly higher in CHIR99021 treated cultures using Kruskal-Wallis multiple comparison test AMK vs CHIR99021, **P = .007; AMK vs DMSO, P = ns; CHIR99021 vs DMSO, *P = .01. Mean ± 1× SEM; n = 12 biological replicates. (C) Cytospins of iDELTA-3.7 MKs cultured in either AMK alone, with CHIR99021 or with DMSO (control for CHIR99021) stained using Rapid Romanowski. Black arrowsheads indicate multinucleated cells. Multinucleated MKs are more frequent in the CHIR99021 samples although rare large cells are also seen in the DMSO as illustrated here. Scale bars, 50 μm. (D) Transmission electron microscopy of mature iDELTA3.7 MKs at day 27 reveals a much more extensive DMS in the CHIR99021-treated cells. Scale bars, 2 μm, α granules (α), dense granules (δ), multilobular nucleus (N), mitochondria (M). Left image DMSO sample ×1700 (8 images examined), right CHIR99021-treated samples ×1700 (15 images examined). The rectangle indicates the magnified area shown of the cytoplasmic DMS (below) ×3500. (E) Switch from “embryonic” to “adult” phenotype in CHIR99021-treated MKs. Bar graphs showing expression levels of a panel of markers of MK maturity. CHIR99021-treated iDELTA-3.7 derived MKs have levels of messenger RNA expression closer to those seen in peripheral blood (PB)–derived MKs. After normalization to the endogenous control gene HMBS, results are expressed relative to PB-derived MKs calculated using the relative standard curve method. Bars: red, CHIR99021-treated iMKs; green, DMSO control. Mean ± 1× SEM, n = 3. KDR, a marker of fetal identity, is significantly reduced compared with the DMSO control, **P < .01, Mean ± 1× SEM, n = 7, VWF, a marker of “adult” phenotype is significantly increased compared with the DMSO control **P < .01, Mean ± 1× SEM, n = 5 as is STAT5a ***P < .001, Mean ± 1× SEM, n = 3 and DKK1 *P < .05, Mean ± 1× SEM, n = 3. The maturity markers ITGA2 (CD49b) n = 3, HOXA9 n = 6 and GPIbβ (CD42C) n = 3 all Mean ± 1× EM, P = ns. (F) Platelet production from CHIR99021-treated MKs is increased. Bar graph showing the number of viable Calcein-AM+ CD41+ CD42+ (left) platelets produced per MK for platelets derived from iLIPSC-GR1.1.2 (n = 3) and iDELTA-3.7 (n = 8) from MKs cultured in AMK alone (blue bars), CHIR99021 (red), or vehicle control (green). The increase is nonsignificant for iLIPSC-GR1.1.2 for both AMK vs CHIR99021 and CHIR99021 vs DMSO, for iDELTA-3.7 *P = .03 for AMK vCHIR99021 and **P = .08 for CHIR99021 vs DMSO using ANOVA plus Bonferroni’s multiple comparison test. Mean ± 1× SEM. iLIPSC-GR1.1.2, n = 3 and iDELTA-3.7 n = 8. (G) Expression of VWF and CD9 proteins is increased in CHIR99021-treated MKs shown for iDELTA3.7. VWF expression was assessed by flow cytometry. Both VWF and CD9 expression are elevated by CHIR99021 treatment. Top bar chart shows %CD42a+VWF+ and bottom %CD42a+CD9+ both show a significant increase in CHIR99021 treated cultures compared with DMSO control. CHIR99021 vs DMSO for % CD42a+VWF+ **P < .01, CHIR99021vs DMSO for % CD42a+CD9+ ***P < .001, Mean ±1× SEM, n = 5.

The small molecule inhibitor CHIR99021 enhances the maturity of FoP-MKs via wnt pathway activation. (A) Profiles for the expression of downstream Wnt target genes following 24-hour exposure to CHIR99021. Differentiating cells were treated with the CHIR99021 inhibitor between D1 and D2 (shaded panel) during mesoderm induction (indicated by the expression of Brachyury). Quantitative PCR graphs showing expression of CDX4, HoxA9 and Sall4 between day 0 and day 5 postinduction for iDELTA-3.7 CHIR99021 (red), DMSO control (green). All are expressed relative to HMBS, as the endogenous control, and D1 levels; n = 3 biological replicates; mean ±1× SEM. HoxA9 expression remains significantly. **P < .01 upregulated at day 5. (B) MK maturation from iLIPSC-GR1.1.2 and iDELTA-3.7 with CHIR99021. Bar graphs showing the number of viable MKs for iLIPSC-GR1.1.2 on day 20 and iDELTA-3.7 on day 30 expressed relative to AMK. AMK (blue bar), CHIR99021 (red bar) or DMSO (green bar). Viable MKs for iLIPSC-GR1.1.2 peak earlier at D20 in CHIR99021 treated cultures. Mean ±1× SEM; n = 6 biological replicates, AMK vs CHIR99021 and CHIR99021 vs DMSO are not significantly different using Kruskal-Wallis multiple comparison test. Viable MKs at day 30 for iDELTA-3.7 are significantly higher in CHIR99021 treated cultures using Kruskal-Wallis multiple comparison test AMK vs CHIR99021, **P = .007; AMK vs DMSO, P = ns; CHIR99021 vs DMSO, *P = .01. Mean ± 1× SEM; n = 12 biological replicates. (C) Cytospins of iDELTA-3.7 MKs cultured in either AMK alone, with CHIR99021 or with DMSO (control for CHIR99021) stained using Rapid Romanowski. Black arrowsheads indicate multinucleated cells. Multinucleated MKs are more frequent in the CHIR99021 samples although rare large cells are also seen in the DMSO as illustrated here. Scale bars, 50 μm. (D) Transmission electron microscopy of mature iDELTA3.7 MKs at day 27 reveals a much more extensive DMS in the CHIR99021-treated cells. Scale bars, 2 μm, α granules (α), dense granules (δ), multilobular nucleus (N), mitochondria (M). Left image DMSO sample ×1700 (8 images examined), right CHIR99021-treated samples ×1700 (15 images examined). The rectangle indicates the magnified area shown of the cytoplasmic DMS (below) ×3500. (E) Switch from “embryonic” to “adult” phenotype in CHIR99021-treated MKs. Bar graphs showing expression levels of a panel of markers of MK maturity. CHIR99021-treated iDELTA-3.7 derived MKs have levels of messenger RNA expression closer to those seen in peripheral blood (PB)–derived MKs. After normalization to the endogenous control gene HMBS, results are expressed relative to PB-derived MKs calculated using the relative standard curve method. Bars: red, CHIR99021-treated iMKs; green, DMSO control. Mean ± 1× SEM, n = 3. KDR, a marker of fetal identity, is significantly reduced compared with the DMSO control, **P < .01, Mean ± 1× SEM, n = 7, VWF, a marker of “adult” phenotype is significantly increased compared with the DMSO control **P < .01, Mean ± 1× SEM, n = 5 as is STAT5a ***P < .001, Mean ± 1× SEM, n = 3 and DKK1 *P < .05, Mean ± 1× SEM, n = 3. The maturity markers ITGA2 (CD49b) n = 3, HOXA9 n = 6 and GPIbβ (CD42C) n = 3 all Mean ± 1× EM, P = ns. (F) Platelet production from CHIR99021-treated MKs is increased. Bar graph showing the number of viable Calcein-AM+ CD41+ CD42+ (left) platelets produced per MK for platelets derived from iLIPSC-GR1.1.2 (n = 3) and iDELTA-3.7 (n = 8) from MKs cultured in AMK alone (blue bars), CHIR99021 (red), or vehicle control (green). The increase is nonsignificant for iLIPSC-GR1.1.2 for both AMK vs CHIR99021 and CHIR99021 vs DMSO, for iDELTA-3.7 *P = .03 for AMK vCHIR99021 and **P = .08 for CHIR99021 vs DMSO using ANOVA plus Bonferroni’s multiple comparison test. Mean ± 1× SEM. iLIPSC-GR1.1.2, n = 3 and iDELTA-3.7 n = 8. (G) Expression of VWF and CD9 proteins is increased in CHIR99021-treated MKs shown for iDELTA3.7. VWF expression was assessed by flow cytometry. Both VWF and CD9 expression are elevated by CHIR99021 treatment. Top bar chart shows %CD42a+VWF+ and bottom %CD42a+CD9+ both show a significant increase in CHIR99021 treated cultures compared with DMSO control. CHIR99021 vs DMSO for % CD42a+VWF+ **P < .01, CHIR99021vs DMSO for % CD42a+CD9+ ***P < .001, Mean ±1× SEM, n = 5.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal