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Rett syndrome arises from loss-of-function mutations in the X-linked chromatin regulator MECP2, yet the earliest molecular derailments in development are poorly defined. Using isogenic human embryonic stem cell (hESC) models carrying three patient-derived MECP2 mutations, we followed the transcriptome from pluripotency through neuroectoderm, neural stem/progenitor stages. Developmental stage dominated transcriptional variance, but mutants shared a secondary program enriched for synaptic-membrane and extracellular matrix genes. Single-cell/bulk profiling at the embryonic stem cell (ESC) stage revealed partial naïve-like drift, marked by the up-regulation of the naïve-enriched factor ZFP42/REX1 and related markers in MECP2-mutant lines. Among convergently dysregulated genes, the cortical determinant EMX1 showed an abnormal developmental trajectory, early repression followed by overshoot, and was consistently altered across independent Rett PSC models. Single-nucleus RNA-seq of cerebral organoids uncovered allele-specific yet convergent disturbances in cortical lineage allocation. These data chart a continuous developmental trajectory for MECP2-mutant cells and nominate naïve-like drift and mis-timed EMX1 expression as tractable entry points for dissecting Rett pathogenesis. Graphical abstract Highlights•MECP2 mutations induce an early naïve-like transcriptional drift in hESCs•EMX1 shows a conserved abnormal developmental trajectory across Rett models•Shared transcriptional programs emerge during neural induction in MECP2 mutants•Rett cerebral organoids display mutation-specific shifts in lineage allocation In this article, Flamier and colleagues show that MECP2 mutations perturb human neurodevelopment from the pluripotent stage onward. Using isogenic hESC and organoid models, they identify an early naïve-like transcriptional drift, abnormal EMX1 timing, and convergent defects in cortical lineage allocation, revealing continuous developmental vulnerability in Rett syndrome.

Highlights•Preformation of aggregates tuned by cell density enable cultivation of hiPSCs in scale-down shear environments.•Scale-down systems utilizing preformation protocols achieve comparable fold expansion with commercial systems.•Expression of pluripotency markers and functional differentiation capacity is maintained following passage in scale-down culture.•Successful application of hiPSC protocols at < 20 mL scales enable rapid and cost-effective research into cell phenotype under dynamic conditions. Human induced pluripotent stem cell (hiPSC) derived therapeutics require clinically relevant quantities of high-quality cell populations for applications in regenerative medicine. The lack of efficacy exhibited across clinical trials suggests deeper understanding of the networks governing phenotype is needed. Further, costs limit study throughput in characterizing the artificial niche relative to outcomes. We present herein an optimized strategy to enable high-throughput hiPSC expansion at <20 mL research scale. We assessed viability of single cell inoculation and aggregate preformation to facilitate proliferation. We modeled aggregate characteristics against agitation rate. Our results demonstrate tunable control with fold expansion comparable to commercial systems. Marker quantification and teratoma assay confirm functional pluripotency. This approach constitutes a scalable protocol to accelerate hiPSC research, and a significant step in advancing the rate of progress in elucidating links to derivative functionality. This work will enable statistically rigorous studies targeting hiPSC and downstream phenotype for clinical manufacturing. Graphical abstractImplementation of adapted protocols enable scale-down systems as a tool for high-throughput iPSC biomanufacturing research, in platforms conducive to scale-up for clinical manufacturing.Image, graphical abstract

Maturation of human pluripotent stem (hPS) cell-derived cardiomyocytes is critical for their use as a model system. Here we mimic human heart maturation pathways in the setting of hPS cell-derived cardiac organoids (hCOs). Specifically, transient activation of 5′ AMP-activated protein kinase and estrogen-related receptor enhanced cardiomyocyte maturation, inducing expression of mature sarcomeric and oxidative phosphorylation proteins, and increasing metabolic capacity. hCOs generated using the directed maturation protocol (DM-hCOs) recapitulate cardiac drug responses and, when derived from calsequestrin 2 ( CASQ2 ) and ryanodine receptor 2 ( RYR2 ) mutant hPS cells exhibit a pro-arrhythmia phenotype. These DM-hCOs also comprise multiple cell types, which we characterize and benchmark to the human heart. Modeling of cardiomyopathy caused by a desmoplakin ( DSP ) mutation resulted in fibrosis and cardiac dysfunction and led to identifying the bromodomain and extra-terminal inhibitor INCB054329 as a drug mitigating the desmoplakin-related functional defect. These findings establish DM-hCOs as a versatile platform for applications in cardiac biology, disease and drug screening. Subject terms: Tissue engineering, Differentiation, Cardiomyopathies