Development and application of human cortical organoids: A 1q21.1 deletion study

Cerebral organoids are quickly becoming an essential research model in the study of neurodevelopmental disorders. With the aim of generating resources for investigating such neurodevelopmental disorders, new methodology was devised for immunocytochemistry (ICC) quantification of cerebral organoids. A high throughput ICC analysis pipeline was developed, with post-hoc corrections for apoptotic cell death and creation of a universal constant for normalising morphological features and dead cell count data. In addition to these analysis techniques, revised human induced pluripotent stem cell (hIPSC)-derived cortical organoid (hCO) protocols were trialled with the intention of improving on current guided cerebral organoid models. Each protocol’s hCOs were scrutinised for dorsal forebrain characteristics such as cortical layering and organised neural progenitors, of which varied significantly between protocols. Extended ROCK inhibition during early hCO differentiation proved counterproductive to developing dorsal forebrain identity. With improved analysis methodologies and a characterised hCO protocol, hCOs were generated from patients of the rare, pathogenic copy number variant (CNV), 1q21.1 deletion (1qDel). 1qDel is associated with developmental delay, intellectual disability, schizophrenia and microcephaly. From the onset of hCO differentiation, 1qDel hCOs were microcephalus until neuronal maturation. As the neuroepithelium developed, 1qDel hCOs also exhibited early neurogenesis and disruption of the cell cycle, hypothesised to be a result of repressed NOTCH and Wnt signalling due to 1qDel. Neuronal maturation alleviated these phenotypes, but expansion of ventral forebrain progenitors at Day 30 was suspected to detrimentally affect cortical layering. By two months of age, 1qDel hCOs were predominantly indistinguishable from controls, with the exception of an increase in GABA-ergic presynaptic markers suggesting an excitatory/inhibitory imbalance in neuronal activity. This is the first in vitro example of the frontal lobe-specific microcephaly found in 1qDel patients, as well as providing future insights into CNV-associated cortical dysfunction

Using induced pluripotent stem cells to investigate human neuronal phenotypes in 1q21.1 deletion and duplication syndrome

Copy Number Variation (CNV) at the 1q21.1 locus is associated with a range of neurodevelopmental and psychiatric disorders in humans, including abnormalities in head size and motor deficits. Yet, the functional consequences of these CNVs (both deletion and duplication) on neuronal development remain unknown. To determine the impact of CNV at the 1q21.1 locus on neuronal development, we generated induced pluripotent stem cells from individuals harbouring 1q21.1 deletion or duplication and differentiated them into functional cortical neurons. We show that neurons with 1q21.1 deletion or duplication display reciprocal phenotype with respect to proliferation, differentiation potential, neuronal maturation, synaptic density and functional activity. Deletion of the 1q21.1 locus was also associated with an increased expression of lower cortical layer markers. This difference was conserved in the mouse model of 1q21.1 deletion, which displayed altered corticogenesis. Importantly, we show that neurons with 1q21.1 deletion and duplication are associated with differential expression of calcium channels and demonstrate that physiological deficits in neurons with 1q21.1 deletion or duplication can be pharmacologically modulated by targeting Ca2+ channel activity. These findings provide biological insight into the neuropathological mechanism underlying 1q21.1 associated brain disorder and indicate a potential target for therapeutic interventions

Somatic mutation rates scale with lifespan across mammals.

The rates and patterns of somatic mutation in normal tissues are largely unknown outside of humans1-7. Comparative analyses can shed light on the diversity of mutagenesis across species, and on long-standing hypotheses about the evolution of somatic mutation rates and their role in cancer and ageing. Here we performed whole-genome sequencing of 208 intestinal crypts from 56 individuals to study the landscape of somatic mutation across 16 mammalian species. We found that somatic mutagenesis was dominated by seemingly endogenous mutational processes in all species, including 5-methylcytosine deamination and oxidative damage. With some differences, mutational signatures in other species resembled those described in humans8, although the relative contribution of each signature varied across species. Notably, the somatic mutation rate per year varied greatly across species and exhibited a strong inverse relationship with species lifespan, with no other life-history trait studied showing a comparable association. Despite widely different life histories among the species we examined-including variation of around 30-fold in lifespan and around 40,000-fold in body mass-the somatic mutation burden at the end of lifespan varied only by a factor of around 3. These data unveil common mutational processes across mammals, and suggest that somatic mutation rates are evolutionarily constrained and may be a contributing factor in ageing