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Thyroid hormone (T3) plays a vital role in brain development and its dysregulation can impact behavior, nervous system function, and cognitive development. Large case-cohort studies have associated abnormal maternal T3 during early pregnancy to epilepsy, autism, and attention deficit hyperactivity disorder (ADHD) in children. Recent experimental findings have also shown T3's influence on the fate of neural precursor cells and raise the question of its convergence with embryonic neural progenitors. Our objective was to investigate how T3 treatment affects neuronal development and functionality at the cellular level. In vitro experiments using neural precursor cells (NPCs) measured cell growth and numbers after exposure to varying T3 concentrations. Time points included week 0 (W0) representing NPCs treated with 100 nM T3 for 5 days, and differentiated cortical neurons assessed at weeks 3 (W3), 6 (W6), and 8 (W8). Techniques such as single-cell calcium imaging and whole-cell patch clamp were utilized to evaluate neuronal activity and function. IHC staining detected mature neuron markers, and RNA sequencing enabled molecular profiling. W6 and W8 neurons exhibited higher action potential frequencies, with W6 showing increased peak amplitudes and shortened inter-spike intervals by 50%, indicating enhanced activity. Transcriptomic analysis revealed that W6 T3-treated neurons formed a distinct cluster, suggesting accelerated maturation. Comparison with the whole transcriptome further unveiled a correlation between W6 neurons treated with T3 and neuronal regulatory elements associated with autism and ADHD. These findings provide insights into T3's impact on neuronal development and potential mechanisms of T3 dysregulation and neurodevelopmental disorders.

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α-Synuclein (αSyn) is a small, disordered protein that becomes aggregated in Lewy body diseases, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Human induced pluripotent stem cells (hiPSCs) potentially provide a tractable disease model to monitor early molecular changes associated with PD/DLB. We and others have previously derived hiPSC lines from patients with duplication and triplication of the SNCA gene, encoding for αSyn. It is now recognised that to perform meaningful disease modelling with these hiPSC lines, it is critical to generate isogenic control cell lines that lack the disease causing mutations. In order to complement the existing and emerging hiPSC models for PD/DLB, we have generated an allelic series of αSyn over-expressing hESC lines on the same isogenic background. An unresolved question is whether pluripotent stem cell lines, with elevated levels of αSyn, can undergo efficient differentiation into dopaminergic and cortical neurons to model PD and DLB, respectively. We took advantage of our isogenic collection of hESC lines to determine if increased expression of αSyn affects neural induction and neuronal differentiation. Clonal hESC lines with significantly different levels of αSyn expression proliferated normally and maintained expression of pluripotent markers, such as OCT4. All cell lines efficiently produced PAX6+ neuroectoderm and there was no correlation between αSyn expression and neural induction efficiency. Finally, global transcriptomic analysis of cortical differentiation of hESC lines with low or high levels of αSyn expression demonstrated robust and similar induction of cortical neuronal expression profiles. Gene expression differences observed were unrelated to neural induction and neuronal differentiation. We conclude that elevated expression of αSyn in human pluripotent stem cells does not adversely affect their neuronal differentiation potential and that collections of isogenic cell lines with differing levels of αSyn expression are valid and suitable models to investigate synucleinopathies.

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BACKGROUND: Autism is a heterogeneous collection of disorders with a complex molecular underpinning. Evidence from postmortem brain studies have indicated that early prenatal development may be altered in autism. Induced pluripotent stem cells (iPSCs) generated from individuals with autism with macrocephaly also indicate prenatal development as a critical period for this condition. But little is known about early altered cellular events during prenatal stages in autism. METHODS: iPSCs were generated from 9 unrelated individuals with autism without macrocephaly and with heterogeneous genetic backgrounds, and 6 typically developing control individuals. iPSCs were differentiated toward either cortical or midbrain fates. Gene expression and high throughput cellular phenotyping was used to characterize iPSCs at different stages of differentiation. RESULTS: A subset of autism-iPSC cortical neurons were RNA-sequenced to reveal autism-specific signatures similar to postmortem brain studies, indicating a potential common biological mechanism. Autism-iPSCs differentiated toward a cortical fate displayed impairments in the ability to self-form into neural rosettes. In addition, autism-iPSCs demonstrated significant differences in rate of cell type assignment of cortical precursors and dorsal and ventral forebrain precursors. These cellular phenotypes occurred in the absence of alterations in cell proliferation during cortical differentiation, differing from previous studies. Acquisition of cell fate during midbrain differentiation was not different between control- and autism-iPSCs. CONCLUSIONS: Taken together, our data indicate that autism-iPSCs diverge from control-iPSCs at a cellular level during early stage of neurodevelopment. This suggests that unique developmental differences associated with autism may be established at early prenatal stages.

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Directed differentiation of human pluripotent stem cells varies in specificity and efficiency. Stochastic, genetic, intracellular, and environmental factors affect maintenance of pluripotency and differentiation into early embryonic lineages. However, factors affecting variation in in vitro differentiation to defined cell types are not well understood. To address this, we focused on a well-established differentiation process to cerebral cortex neural progenitor cells and their neuronal progeny from human pluripotent stem cells. Analysis of 162 differentiation outcomes of 61 stem cell lines derived from 37 individuals showed that most variation occurs along gene expression axes reflecting dorsoventral and rostrocaudal spatial expression during in vivo brain development. Line-independent and line-dependent variations occur, with the latter driven largely by differences in endogenous Wnt signaling activity. Tuning Wnt signaling during a specific phase early in the differentiation process reduces variability, demonstrating that cell-line/genome-specific differentiation outcome biases can be corrected by controlling extracellular signaling.

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Three-dimensional (3D) brain organoids derived from human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), have become a powerful system to study early development events and to model human disease. Cerebral organoids are generally produced in static culture or in a culture vessel with active mixing, and the two most widely used systems for mixing are a large spinning flask and a miniaturized multi-well spinning bioreactor (also known as Spin Omega (SpinΩ)). The SpinΩ provides a system that is amenable to drug testing, has increased throughput and reproducibility, and utilizes less culture media. However, technical limitations of this system include poor stability of select components and an elevated risk of contamination due to the inability to sterilize the device preassembled. Here, we report a new design of the miniaturized bioreactor system, which we term Spinfinity (Spin∞) that overcomes these concerns to permit long-term experiments. This updated device is amenable to months-long (over 200 days) experiments without concern of unexpected malfunctions.