Human iPSCs-based CNS systems to model Autosomal Dominant Leukodystrophy

Autosomal Dominant Leukodystrophy (ADLD) is a rare, progressive, and ultimately fatal neurological disorder caused by overexpression of the nuclear envelope protein Lamin B1 (LMNB1), typically resulting from duplications of the LMNB1 coding region or deletions in upstream noncoding regulatory elements. Although LMNB1 is ubiquitously expressed, neurons and glial cells are particularly sensitive to dosage imbalances, leading to widespread white matter abnormalities and progressive neurological decline. Despite advances in elucidating the genetic underpinnings of ADLD, the cellular and molecular mechanisms linking LMNB1 dysregulation to disease pathology remain poorly understood, in part due to the absence of suitable human model systems. To address this critical gap, we generated human induced pluripotent stem cells (hiPSCs) from ADLD patients harboring either a LMNB1 duplication or an upstream regulatory deletion, as well as from an asymptomatic carrier with a LMNB1 duplication. Reprogramming was achieved using a non-integrating Sendai virus-based method, ensuring the generation of genetically stable, integration-free pluripotent lines. The resulting hiPSC lines were extensively validated for pluripotency markers and differentiation capacity. Using these patient-derived hiPSCs, we established both two-dimensional (2D) and three-dimensional (3D) in vitro models of neurodevelopment by directing differentiation into neuronal and glial lineages under defined conditions. This approach enabled the generation of adherent monolayer cultures and brain organoids that recapitulate key features of early brain architecture and cell-type interactions. Structural analyses revealed nuclear abnormalities characteristic of LMNB1 overexpression in both neurons and astrocytes across monolayer cultures and brain organoids. These findings provide direct evidence linking LMNB1 dysregulation to altered nuclear architecture in human neural cells. Functional studies further demonstrated impairments in network activity: multi-electrode array (MEA) recordings and calcium imaging identified deficits in spontaneous firing, synchronization, and network maturation in patient-derived neurons, neuron-astrocyte co-cultures, and cerebellar organoids compared to controls. Collectively, these hiPSC-based 2D and 3D models constitute powerful platforms to investigate the cellular consequences of LMNB1 overexpression in human neural cells. They offer unique opportunities to dissect neuron-glia interactions underlying neurodegeneration in ADLD and to explore molecular mechanisms and therapeutic strategies for this devastating disorder.