Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction

Abstract

Probing a wide range of cellular phenotypes in neurodevelopmental disorders using patient-derived neural progenitor cells (NPCs) can be facilitated by 3D assays, as 2D systems cannot entirely recapitulate the arrangement of cells in the brain. Here, we developed a previously unidentified 3D migration and differentiation assay in layered hydrogels to examine how these processes are affected in neurodevelopmental disorders, such as Rett syndrome. Our soft 3D system mimics the brain environment and accelerates maturation of neurons from human induced pluripotent stem cell (iPSC)-derived NPCs, yielding electrophysiologically active neurons within just 3 wk. Using this platform, we revealed a genotype-specific effect of methyl-CpG-binding protein-2 (MeCP2) dysfunction on iPSC-derived neuronal migration and maturation (reduced neurite outgrowth and fewer synapses) in 3D layered hydrogels. Thus, this 3D system expands the range of neural phenotypes that can be studied in vitro to include those influenced by physical and mechanical stimuli or requiring specific arrangements of multiple cell types.3D hydrogels | neuronal migration and maturation | 3D RTT modeling N euronal migration and maturation is a key step in brain development. Defects in this process have been implicated in many disorders, including autism (1) and schizophrenia (2). Thoroughly understanding how neural progenitor cell (NPC) migration is affected in neurodevelopmental disorders requires a means of dissecting the process using cells with genetic alterations matching those in patients. Existing in vitro assays of migration generally involve measurement of cell movement across a scratch or gap or through a membrane toward a chemoattractant in 2D culture systems. Although widely used, such assays may not accurately reveal in vivo differences, as neuronal migration is tightly regulated by physical and chemical cues in the extracellular matrix (ECM) that NPCs encounter as they migrate.In vitro 3D culture systems offer a solution to these limitations (3-7). Compared with 2D culture, a 3D arrangement allows neuronal cells to interact with many more cells (4); this similarity to the in vivo setting has been shown to lengthen viability, enhance survival, and allow formation of longer neurites and more dense networks in primary neurons in uniform matrices or aggregate culture (8, 9). Indeed, 3D culture systems have been used to study nerve regeneration, neuronal and glial development (10-12), and amyloid-β and tau pathology (13). Thus, measuring neuronal migration through a soft 3D matrix would continue this trend toward using 3D systems to study neuronal development and pathology.We sought to develop a 3D assay to examine potential migration and neuronal maturation defects in Rett syndrome (RTT), a genetic neurodevelopmental disorder that affects 1 in 10,000 children in the United States and is caused by mutations in the X-linked methyl-CpG-binding protein-2 (MECP2) gene (14). Studies using induced pluripotent stem cells (iPSCs) from RTT patients in traditiona...

Dysfunctional mTORC1 Signaling: A Convergent Mechanism between Syndromic and Nonsyndromic Forms of Autism Spectrum Disorder?

“…Notably, studies in RTT mouse models have suggested that cell autonomous and non-cell autonomous mechanisms drive neuronal morphology and function [229,230,231]. In addition, recent in vitro models of RTT using MECP2 -deficient neurons derived from human pluripotent stem cells have recapitulated many neurological features of RTT [232,233,234,235,236], and have also shown neuronal migration defects [236] (Table 1). Interestingly, in contrast to the majority of the ASD-associated mTORopathies, neurons from Mecp2 −/− and Mecp2 +/− mice [43], as well as from MECP2 -deficient human pluripotent stem cells [233], show decreased mTORC1 signaling activity, transcription and protein synthesis rate.…”

Fusion of Regionally Specified hPSC-Derived Organoids Models Human Brain Development and Interneuron Migration

“…Moreover, cell transplantation or explant cultures have been used to model interneuron migration in vivo or in vitro (Bellion et al, 2005; Maroof et al, 2013; Nicholas et al, 2013), but these studies have largely relied upon xenografts of human cells into immunodeficient mice. To recapitulate 3-D neuronal migration in vitro , biomaterial-based methods were recently established (Zhang et al, 2016). However, such systems still lack a physiological cellular environment, necessitating 3-D culture systems to model interneuron migration in the context of human developing cortex, which have been recently reported (Bagley et al, 2017; Birey et al, 2017).…”

Mechanical Stimulation of Cells Through Scaffold Design for Tissue Engineering

“…Some scientists have been focused on imitating paterns, forms, textures and speciic characteristics such as mechanical resistance and chemical structure. For example, Zhang et al [33] constructed a three-dimensional system to create tissue architecture. The scafold systems were synthesised with an elastic modulus similar to brain tissue.…”