Using human pluripotent stem cell-based models to understand a congenital deglycosylation disorder-induced abnormality in cerebral development

Background: Human pluripotent stem cells (hPSCs) provide significant promise for regenerative medicine and in addressing questions relevant to the disease physiopathology. Therefore, the generation of hPSCs using somatic cells obtained by noninvasive approaches would be ideal for securing patient samples and further implement the notion of personalized insight into disease and treatment. Neurodysfunction caused by a congenital disorder of deglycosylation, NGLY1 deficiency, was recently identified. How NGLY1 deficiency disturbs normal cerebral development and causes neurological abnormalities in the pediatric population is unknown. Purpose: Our desire is to establish a hPSC-based disease model of NGLY1 deficiency and study the pathogenic neurogenesis associated with NGLY1 loss and its influences on early development of the human brain. Method: Urinary epithelial cells from urine specimens of normal individuals and patient skin biopsy-derived fibroblasts were collected and reprogrammed to generate transgene-free human induced pluripotent stem cells (hiPSCs). Through gene editing, NGLY1-deficient hPSCs were obtained from the normal hPSCs which included human embryonic stem cells (hESCs) and hiPSCs. The NGLY1-deficient hPSCs and their normal counterparts were differentiated into neural cells using 2D- and 3D-differentiation protocols optimized by our group. Systems biology and 3D-imaging techniques were employed to characterize the cellular and molecular features of NGLY1-deficient neurodevelopment. Results: Urine-derived hiPSCs were pluripotent and capable of forming cerebral organoids that contain multiple types of neural cells in a self-organized and layered 3D structure closely mimicking the early human forebrain development. Like normal hPSCs, NGLY1-deficient hPSCs also form cerebral organoids. Unique gene expression patterns in NGLY1-deficient samples at different stages of development were identified by transcriptomic analysis. Structural abnormalities were also observed in NGLY1-deficient organoids compared with normal organoids. Conclusions: We have streamlined the production of cerebral organoids using hiPSCs reprogrammed from non-invasively collected cells via urine specimens and NGLY1-deficient hPSCs. Additionally, we demonstrate our ability to recapitulate NGLY1 deficiency down to the molecular level, and to continually uncover insights into a pathologically enigmatic disease. In doing so, we clearly exemplify a beginning to end approach that can be used to study other neurological diseases, as well as, assay their potential therapies in an all human-based and personalizable system.