Human Cerebral Organoids Reveal Early Spatiotemporal Dynamics and Pharmacological Responses of UBE3A

Abstract

Summary Angelman syndrome is a complex neurodevelopmental disorder characterized by delayed development, intellectual disability, speech impairment, and ataxia. It results from the loss of UBE3A protein, an E3 ubiquitin ligase, in neurons of the brain. Despite the dynamic spatiotemporal expression of UBE3A observed in rodents and the potential clinical importance of when and where it is expressed, its expression pattern in humans remains unknown. This reflects a common challenge of studying human neurodevelopment: prenatal periods are hard to access experimentally. In this work, human cerebral organoids reveal a change from weak to strong UBE3A in neuronal nuclei within 3 weeks of culture. Angelman syndrome human induced pluripotent stem cell-derived organoids also exhibit early silencing of paternal UBE3A , with topoisomerase inhibitors partially rescuing UBE3A levels and calcium transient phenotypes. This work establishes human cerebral organoids as an important model for studying UBE3A and motivates their broader use in understanding complex neurodevelopmental disorders.

Evaluation of UBE3A antibodies in mice and human cerebral organoids

“…The H9 UBE3Am−/p− hESC cell line was generated and validated by Sirois and colleagues 9 . Our group also confirmed the deletion in these cells in an earlier study 15 . All cell lines were maintained in 6-well tissue culture dishes (Fisher Scientific) coated with 0.5 mg/cm 2 Vitronectin (VTN-N) (Thermo Fisher) in E8 medium (Thermo Fisher) and passaged using standard protocols.…”

“…In addition, both Bethyl Laboratories antibodies showed reduced signal similar to their background levels in the neuronal regions (MAP2+/SOX2−) of both AS model hCOs (H9 UBE3A m−/p+ and hiPSC ASdel ) indicating imprinting and silencing of the paternal allele, as was previously observed 15 (Figs. 3 B and S3 B).…”

“…In addition to hPSCs, we and others have previously used human stem cell-derived neurons 17 , 18 and hCOs 15 , 19 as promising experimental models for AS research. In particular, in a recent study our group showed that hCOs can recapitulate the important disease relevant molecular changes surrounding UBE3A including paternal imprinting and nuclear localization in neurons 15 . Here, we conducted IF experiments to evaluate not only antibody specificities, but also the ability of the selected antibodies to capture these salient molecular features of UBE3A using 12 week hCOs generated from seven different hPSC lines including three AS models (Figs.…”

“…Although a few commercial UBE3A antibodies have been rigorously validated in the published literature for mouse brain tissue 12 and human stem cell derived neurons 9 , others are less well documented. Furthermore, except for the mouse monoclonal antibody from Sigma-Aldrich (SAB1404508) 9 and rabbit polyclonal antibody from Bethyl Laboratories (A300-351A) 15 , the specificities of most antibodies in human samples remain unknown, and even when validated, were tested only in limited cell types. With the growing use of human pluripotent stem cell-derived neuronal cultures and three-dimensional cerebral organoids as models to study human UBE3A biology, it will be important to assess the specificities of UBE3A antibodies in human stem cell-derived systems as well as mouse models.…”

Effects of early geometric confinement on the transcriptomic profile of human cerebral organoids

“…hCOs were washed 3 times in cold PBS. Total RNA was extracted as previously described [41]. Briefly, Matrigel was dissolved by incubating the hCOs in chilled Cell Recovery Solution (Corning, cat.…”