Optimized Generation of Functional Neutrophils and Macrophages from Patient-Specific Induced Pluripotent Stem Cells:Ex VivoModels of X⁰-Linked, AR22⁰- and AR47⁰- Chronic Granulomatous Diseases

Abstract

Chronic granulomatous disease (CGD) is an inherited orphan disorder caused by mutations in one of the five genes encoding reduced nicotinamide-adenine-dinucleotide-phosphate oxidase subunits, which subsequently lead to impairment in the production of microbicidal reactive oxygen species (ROS). In order to offer several cell line models of CGD and therefore support research on pathophysiology and new therapeutic approaches, we optimized protocols to differentiate induced pluripotent stem cells (iPSCs) from wild-type, X0-, AR220- and AR470-CGD patient's fibroblasts into neutrophils and into macrophages. Aberrant genetic clones were discarded after chromosome karyotyping and array-comparative genomic hybridization analysis. All remaining iPSC lines showed human embryonic stem cell–like morphology, expressed all tested pluripotency markers and formed embryoid bodies that contained cells originating from all three primary germ layers. Furthermore, each CGD patient-specific iPSC line retained the gp91phox, p47phox, and p22phox mutations found in the corresponding patient's neutrophils. The average production of CD34+ progenitors was of 1.5×106 cells after 10 days of differentiation of 10×106 iPSCs. They were terminally differentiated into about 3×105 neutrophils or into 3×107 macrophages. Based on morphological, phenotypical, and functional criteria both phagocyte types were mature and indistinguishable from the native human neutrophils and macrophages. However, neutrophils and macrophages derived from X0-, AR220-, and AR470-CGD patient-specific iPSC lines lacked ROS production and the corresponding mutated proteins. To simplify the phagocytes' production upon request, progenitors can be cryopreserved. In conclusion, we describe a reproducible, simple, and efficient way to generate neutrophils and macrophages from iPSCs and provide a new cellular model for the AR220-CGD genetic form that has not been described before.

Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

“…35 iPsc culture and hematopoietic differentiation into mature macrophages iPSCs were reprogrammed using a retroviral method (pMXsbased retroviruses encoding the human transcription factors Oct3/4, Sox2, Klf4 and cMyc from Addgene) from human dermal fibroblasts (hDFs) isolated from the skin biopsy of a healthy control (WT) and a X 0 -CGD patient as previously described. 23,36 Skin biopsies were obtained after informed consent (Ethical permission from the south-east V Person Protection Committee [CPP: 09-CHUG-36, FibroCGD, N° AFSSAPS: 2009-A00944-53]). IPSCs were maintained in an undifferentiated state on irradiated mouse embryonic fibroblasts and fully characterized ( Figure S1).…”

“…Then, hematopoietic differentiation of iPSCs into mature macrophages was performed ( Figure S2). 23 Macrophages adhered on a 96-well plate for viability assay, on a 24-well plate for flow cytometry and in cellulo activity tests, and on a four-well Nunc™ Lab-Tek™ II Chamber Slide™ (Thermo Fisher Scientific, Courtaboeuf, France) for the phagocytosis experiment and confocal microscopy, as described in the following sections. Cell culture images were acquired using a Nikon Eclipse TS1000 microscope with an Elipse TS100 microscope with a digital slight DS-U3 camera (Nikon, Champigny-sur-Marne, France).…”

“…phox (clone 44.1) antibodies as previously described. 23 Control staining with appropriate isotype-matched control was included to establish thresholds for positive staining. WT iPSC-derived macrophages were used as positive control.…”

“…19 CGD patient-specific iPSCs were successfully differentiated into neutrophils and/or macrophages recapitulating the ROS-deficient phenotype to test new therapeutic strategies. [20][21][22][23][24][25][26] Then, using confocal microscopy and flow cytometry, we demonstrated the capability of liposomes to transport and deliver recombinant cytochrome b 558 to the membrane of X 0 -linked CGD (X 0 -CGD) iPSC-derived macrophages. Finally, the therapeutic potential of the NOX2/p22 phox liposomes was evidenced by the restoration of the NADPH oxidase activity of these ROS-deficient cells using the nitroblue tetrazolium (NBT) chloride test.…”

“…In normal macrophages, cytochrome b 558 is first located in the plasma membrane and then in the phagosomal membrane after phagocytosis. 64 In this study, we focused on macrophages because of their easy production from iPSCs 23 but first of all because of their long lifetime in the body (several months in some tissues), making them an advantageous target for protein therapy. The in vitro macrophages used for the experiments were nondividing cells, like their counterparts in the blood circulation.…”