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

摘要

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 gp91^phox, p47^phox, and p22^phox 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.