Gene Correction of Wiskott-Aldrich syndrome iPS Cells Rescues Proplatelet Defects and Improves Platelet Size

Abstract

Wiskott-Aldrich syndrome (WAS) is a severe X-linked disorder caused by loss-of-function mutations in the WAS gene, responsible for encoding WAS protein (WASP), a key regulator of the actin cytoskeleton in all hematopoietic cells, except red blood cells. The mechanism underlying microthrombocytopenia, a distinctive feature of WAS and a major contributor to mortality, remains not fully elucidated. In this study, using different gene-editing strategies, we corrected mutations in patient-derived WAS-induced pluripotent stem cell (iPSC) lines, generating isogeneic WAS-iPSC lines. These included lines with direct mutation-specific correction and lines incorporating a WASP transgene cassette regulated by the MND or WAS1.6 kb promoter integrated at the safe harbor AAV1 site. Our results demonstrated that direct mutation correction successfully restored WASP levels to the equivalent of the wild-type in iPSC-derived megakaryocytes (MKs). In contrast, the AAV1-targeted strategy using the MND and WAS1.6 promoters yielded a lower level of WASP. Notably, only the mutation-specific correction lines exhibited improvements in proplatelet structures and generated larger-sized platelets. Our findings underscore the crucial roles of WASP during human thrombopoiesis and suggest that therapeutic approaches, such as direct gene correction, which can achieve physiologic levels of WASP in MKs, hold promise for ameliorating platelet defects in individuals with WAS.

Fig. 1

WAS isogenic model. (A) Schematic diagram of WASP gene knockout (KO) using CRISPR/Cas9. (B) DNA sequencing analysis of the WASP gene of CRISPR/Cas9-transfected iPSC clones. (C) Flow cytometry analysis of CD34 expression in WT-iPSCs and WAS mutated WT-iPSCs derived from sac. (D) Flow cytometry analysis of CD41 and CD42b expression in WT-iPSC and WAS mutated WT-iPSC derived megakaryocytes. (E) Western blot analysis revealed the absence of WASP expression in WAS-mutated WT iPSC-derived MKs. (F) Immunofluorescence staining of proplatelet formation showed defects in proplatelet structures of WAS-KO iPSC-derived MKs. (G) Immunofluorescence staining of platelets generated from the WT and WAS-KO iPSCs using an anti-α-tubulin antibody (green). (H) Box plot showing platelet diameter of tubulin-stained discoid-shaped platelets generated from the WT and WAS-KO iPSCs. Data are presented as mean ± SEM, n  = 300 (**** p  < 0.0001). iPSC, induced pluripotent stem cell; MK, megakaryocyte; WAS, Wiskott–Aldrich syndrome; WASP, WAS protein; WT, wild-type.

Fig. 2

WASX503R gene correction. (A) Schematic diagram of WASX503R gene correction using ZFN. (B) DNA sequencing showed single base correction at c.1507 position of the WASP gene in cWASX503R iPSCs. (C) Western blot analysis revealed WASP expression in the WT-iPSC and ccWASX503R-derived MKs. (D) Electron microscopic examination of platelets generated from WT-iPSC, WASX503R-iPSC, and ccWASX503R-iPSC-derived megakaryocytes. Bottom panel, immunofluorescence staining of platelets generated from WASX503R and ccWASX503R iPSCs using an anti-α-tubulin antibody (red). (E) Box plot showing platelet diameter of tubulin-stained discoid-shaped platelets generated from WT, WASX503R, cWASX503R, and ccWASX503R iPSCs. Data are presented as mean ± SEM, n  = 120 (**** p  < 0.0001). iPSC, induced pluripotent stem cell; MK, megakaryocyte; WASP, Wiskott–Aldrich syndrome protein; WT, wild-type; ZFN, Zinc finger nuclease.

Fig. 3

WASQ19X gene correction. (A) Schematic diagram of endogenous gene correction at WASQ19X using TALEN with a donor vector and CRISPR/Cas9 with ssODNs. (B) Schematic diagram of WASP gene knock-in at the AAVS1 site using TALEN with a donor vector. (C) DNA sequencing analysis showed mutation correction at c.55 positions of the WASP gene with a silent mutation at positions −1 and −4 of the mutation site. (D) mRNA expression of the WASP gene under the control of the MND promoter and WAS1.6 promoter in the AAVS1 locus compared with normal expression levels using droplet digital PCR. (E) Western blot analysis of WASP expression in iPSCs and iPSC-derived megakaryocytes. The expression of WASP was detected in AAVMNDPW(Q19X) iPSCs and AAVW1.6PW(Q19X) iPSCs but not in WASQ19X iPSCs. In megakaryocytes, WASP was detected in WT and cWASQ19X-derived MKs and rarely detected in AAVW1.6PW(Q19X). (F) Box plot showing platelet diameter of tubulin-stained discoid-shaped platelets generated from WASQ19X, cWASQ19X, AAVW1.6W(Q19X), and AAVMNDW(Q19X) MKs. Data are presented as mean ± SEM, n  = 300 (**** p  < 0.0001). (G) Immunofluorescence staining of platelets generated from WASQ19X, cWASQ19X, AAVW1.6W(Q19X), and AAVMNDW(Q19X)-derived MKs using an anti-α-tubulin antibody (green). (H) Western blot analysis of N-WASP expression in iPSC-derived MKs. iPSC, induced pluripotent stem cell; MK, megakaryocyte; WASP, Wiskott–Aldrich syndrome protein; WT, wild-type.

Fig. 4

Proplatelet defects in WAS-derived MKs. (A) Immunofluorescence staining of proplatelet formation. MKs were cultured on a matrigel-coated coverslip for 24 hours. Cells were fixed and stained with anti-α-tubulin antibody (green) and phalloidin (red). WT, ccWASX503R, and cWASQ19X-derived proplatelets exhibited thicker proplatelet shafts and larger proplatelet tips compared with WASX503R, WASQ19X, AAVW1.6PW(Q19X), and AAVMNDW(Q19X). (B) Time-lapse fluorescence image of proplatelet formation in WASX503R and ccWASX503R-iPSC-derived MKs stained with SiR-tubulin. At the early stage of proplatelet formation, the extension of pseudopodia was observed in both conditions, but with a difference in its size and microtubule concentration. WASX503R showed a rapid proplatelet process. The microtubule bundles in ccWASX503R-derived proplatelet shafts are thickened and form larger proplatelet tips compared with those observed in WASX503R. Box plot showing proplatelet tip diameter (C) and tubulin thickness of proplatelet (D) of WT, WAS-KO, WASX503R, ccWASX503R, WASQ19X, cWASQ19X, AAVW1.6PW(Q19X), and AAVMNDW(Q19X)-iPSC-derived proplatelets. Data are presented as mean ± SEM, n  = 63 and n  = 59, respectively (**** p  < 0.0001, ** p  < 0.005, * p  < 0.05). iPSC, induced pluripotent stem cell; KO, knockout; MK, megakaryocyte; WAS, Wiskott–Aldrich syndrome; WASP, WAS protein; WT, wild-type.

Fig. 5

Podosome formation. (A) Immunofluorescence staining of podosomes. MKs were cultured on a matrigel-coated coverslip for 72 hours. Cells were stained with SPY555-FastAct and fixed. Podosome-forming MKs were detected in WT, ccWASX503R, cWASQ19X, and AAVW1.6PW(Q19X). (B) The bar graph showed the percentage of podosome-positive cells in each condition ( n  = 3; **** p  < 0.0001). (C) Fluorescence image with an orthogonal view of MKs cultured on interleukin (IL)-1β-treated BMEC1 feeder layer (8-μm cell culture insert) in the presence of SDF1α in the bottom well. Twenty-four hours after culture, cells were fixed and stained with CD42b (purple), anti-α-tubulin antibody (green), and phalloidin (red). In WASQ19X condition, only planar contact between MK and BMEC1 was observed without podosome formation. For corrected WASQ19X-D3, white arrowheads showing podosome forming MKs which protruded their cytoplasm and nucleus toward BMEC1 feeder cells. Crossed gray lines indicate the selected orthogonal planes. MK, megakaryocyte; WT, wild-type.