Page 90 - Haematologica March 2020

M. Nasri et al.
Unaffected phagocytic activity of ELANE KO PMN in zebrafish embryos
To evaluate the phagocytic activity of ELANE KO PMN in vivo, we transplanted fluorescently labeled polymor- phonuclear leukocytes (PMN) generated from ELANE KO HD into zebrafish embryos (Figure 8A). PMN were inject- ed into the duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. Subsequently, Alexa-594-conjugated Staphylococcus aureus BioParticles were injected locally in the tail fin close to the caudal vein. Live imaging showed that human neutrophils migrated into the caudal hematopoietic tissue (CHT), which is equivalent to the fetal liver in mammals and pro- vides a human-compatible environment.24,25 Confocal imaging of this region revealed that most of the ELANE KO neutrophils were found inside the perivascular pocket (Figure 8B) and many of them have engulfed bacteria (white arrows in Figure 8B and 8C, Online Supplementary Movie S1). Time-lapse in vivo imaging of the xenotrans-
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planted embryos also revealed that human ELANE KO PMN have the capability to form surface protrusion with- in the perivascular region (Figure 8D, Online Supplementary Movie S2). We could not detect a difference between trans- planted human ELANE KO and control MOCK PMN in zebrafish embryos (data not shown). These observations indicate that human ELANE KO PMN are able to migrate and phagocyte in vivo.
ELANE KO restores deregulated expression of UPR gene BiP and anti-apoptotic factor Bcl-xl in ELANE KO iPSC derived cells of CN patients
We further evaluated the effects of ELANE KO on the expression of UPR gene BiP and anti-apoptotic factor Bcl- xl (Online Supplementary Figure S11). We analyzed pure ELANE KO HSPC (for Bcl-xl) or neutrophils (for BiP) gen- erated from iPSC of two CN patients. We found that ELANE KO HSPC express elevated mRNA levels of Bcl-xl and BiP expression was markedly reduced in ELANE KO
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Figure 5. Efficient CRISPR/Cas9 RNP-based ELANE knockout in HSPC. (A) Scheme of the generation of ELANE KO HSPC using electropo- ration with ELANE-specific CRISPR/Cas9-gRNA ribonucleoprotein (RNP). (B) TIDE results of edit- ed CD34+ HSPC at day 7 and 14 of liquid culture differentiation. (C and D) Hematopoietic stem and progenitor cells (HSPC) of healthy controls (C), or two CN patients (D) were electroporated with ELANE-specific CRISPR/Cas9 RNP, on day 14 of culture, cells were lysed in Laemmli buffer and Western blotting (WB) analysis using anti- neutrophil elastase (NE) antibody against C-ter- minus of NE was performed, staining with α- tubulin antibody was used as loading control. Representative WB images of cells from two independent experiments are depicted.
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haematologica | 2020; 105(3)