Page 194 - 2019_05-HaematologicaMondo-web

T.K. Bariana et al.
Forward programming to MK
Induced pluripotent stem cells were reprogrammed to MK (named iMK hereafter) using a protocol for generating MK described by Moreau et al.17 Kresc and Kev iMK were generated in three independent experiments. Details of iMK reprogramming, immunophenotyping, the proplatelet assay, and RNA sequenc- ing are given in the Online Supplementary Methods.
AB
Results
C
D
E
Figure 4. Kdsr morpholino knockdown is associated with reduced thrombopoiesis in zebrafish. (A) Tg(cd41:EGF) embryos were injected with a kdsr ATG-MO (1000 mM) or with buffer (control). Embryos were lysed 72 hpf and used for immunoblotting. GFP and Kdsr proteins were reduced in the kdsr knockdown condition. Equal amounts (50 mg) were loaded (5 randomly selected embryos for each of the 4 conditions). Staining of Gapdh was used as loading control. (B) Quantification of immunoblot after normalization for Gapdh. Mean values are plotted and error bars show the standard deviation, analyzed by one-way ANOVA. (C) Quantification by flow cytom- etry of the number of GFP-labeled thrombocytes in Tg(cd41:EGFP) Danio rerio embryos at 72 hpf for kdsr-MO (800 or 1000 mM) or buffer (control) injected fish. Values are means and Standard Deviations as quantified for 10 randomly selected embryos for each condition, performed in trip- licate. Results were analyzed by one-way ANOVA. (D) Grayscale stereo-microscope images (x20 original magnification) in the tail region at 72 hpf showed a reduced number of GFP-labeled thrombocytes (in white). (E) KDS levels in lysates from 72 hpf embryos (20/condition) for kdsr- MO (800 mM) or buffer (control) injected fish. KDS was detected in the lysates from the MO-inject- ed embryos but not in the control-injected embryos.
Clinical characteristics
The 8-year old male propositus was born to healthy, unrelated parents of European ancestry (Figure 2A). At 4 months of age he presented with a viral infection and was found to have a platelet count of 65x109/L and mild nor-
1040
haematologica | 2019; 104(5)