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T.K. Bariana et al.
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Figure 6. KDSR reference allele expression rescues inef- fective proplatelet formation. Proplatelet formation by induced pluripotent stem cells reprogrammed to MK (iMK). One hundred percent of live MK plated for the proplatelet assay were CD41 positive and 75% were dual positive for CD41 and CD42 by flow cytom- etry (Online Supplementary Figure S11A). Cytoskeletal marker a-tubulin was stained with antibodies in green and nuclei were stained with DAPI in blue. Proplatelet formation was counted manually. Values shown were analyzed using the paired, two-tailed Student t-test plotted as means and standard deviations. *P<0.05 was considered statistically significant. (A) The number of proplatelets formed at 4 hours (h) per proplatelet-forming MK (PPFMK) by the rescued and non-rescued iMK. The differ- ences were significant at 4 h (P=0.047) but not at 24 h (P=0.20). (B) There was no sig- nificant difference in the num- ber of PPFMK- at 4 h between the two groups (7∙1% vs. 42∙4%; P=0.10), but at 24 h the rescued iMK showed less PPFMK (P=0.03). (C) Representative images from the proplatelet formation assay at 4 and 24 h. Proplatelets are indicated by white arrows. (Top left and right) Results at 4 h for res- cued iMK show increased pro- platelet formation. White scale bars indicate 10 mm. (Bottom left and right) Results at 24 h show little proplatelet forma- tion for the rescued iMK, and residual cells are either frag- mented into platelet-like parti- cles, or consist of bare nuclei. (D) Metabolon mass spectrom- etry results for non-rescued and rescued iMK. The ion counts for KDS detection dif- fered significantly between the non-rescued and non-rescued iMK (P=0.02). There was no
difference in
between the groups of sam- ples but the levels of sphingo- sine and ceramide were lower and higher in the non-rescued versus rescued iMK, respec- tively. ns: not significant.
DHS level
1042
haematologica | 2019; 104(5)