The complexity of the human brain has made it difficult to study many brain disorders in model organisms, and highlights the need for an in vitro model of human brain development. We have developed a human pluripotent stem cell-derived 3D organoid culture system, termed cerebral organoid, which develops various discrete though interdependent brain regions. These include cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNAi and patient-specific iPS cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could explain the disease phenotype. Our data demonstrate that 3D organoids can recapitulate development and disease of even this most complex human tissue.
Cellular differentiation involves profound remodeling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPSC formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as a novel regulator of somatic cell identity during transcription factor-induced cell fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.
To determine the dynamics of allelic-specific expression during mouse development, we analyzed RNA-seq data from 23 F1 tissues from different developmental stages, including 19 female tissues allowing X chromosome inactivation (XCI) escapers to also be detected. We demonstrate that allelic expression arising from genetic or epigenetic differences is highly tissue-specific. We find that tissue-specific strain-biased gene expression may be regulated by tissue-specific enhancers or by post-transcriptional differences in stability between the alleles. We also find that escape from X-inactivation is tissue-specific, with leg muscle showing an unexpectedly high rate of XCI escapers. By surveying a range of tissues during development, and performing extensive validation, we are able to provide a high confidence list of mouse imprinted genes including 18 novel genes. This shows that cluster size varies dynamically during development and can be substantially larger than previously thought, with the Igf2r cluster extending over 10 Mb in placenta.DOI: http://dx.doi.org/10.7554/eLife.25125.001
Glycosylation, the covalent attachment of carbohydrate structures onto proteins, is the most abundant post-translational modification. Over 50% of human proteins are glycosylated, which alters their activities in diverse fundamental biological processes. Despite the importance of glycosylation in biology, the identification and functional validation of complex glycoproteins has remained largely unexplored. Here we develop a novel quantitative approach to identify intact glycopeptides from comparative proteomic data sets, allowing us not only to infer complex glycan structures but also to directly map them to sites within the associated proteins at the proteome scale. We apply this method to human and mouse embryonic stem cells to illuminate the stem cell glycoproteome. This analysis nearly doubles the number of experimentally confirmed glycoproteins, identifies previously unknown glycosylation sites and multiple glycosylated stemness factors, and uncovers evolutionarily conserved as well as species-specific glycoproteins in embryonic stem cells. The specificity of our method is confirmed using sister stem cells carrying repairable mutations in enzymes required for fucosylation, Fut9 and Slc35c1. Ablation of fucosylation confers resistance to the bioweapon ricin, and we discover proteins that carry a fucosylation-dependent sugar code for ricin toxicity. Mutations disrupting a subset of these proteins render cells ricin resistant, revealing new players that orchestrate ricin toxicity. Our comparative glycoproteomics platform, SugarQb, enables genome-wide insights into protein glycosylation and glycan modifications in complex biological systems.
A 20-year-old man with a 6-year history of infrequent nocturnal focal motor seizures with secondary generalization attended an epilepsy clinic. During the seizures, the patient was stiff and rigid, exhibiting left-side convulsive movements. After each episode he had a short-lived left hemiparesis with associated intense vasomotor disturbance. There was no tongue biting or incontinence, but in the morning the patient would complain of being stiff and sore. These seizures had occurred once every 2 to 4 months. He had never had any daytime episodes, although there was a history of occasional daytime jerking of the limbs. Otherwise the patient had been fit and well. Clinical examination was normal, and it was believed that these seizures originated from the right hemisphere. Brain EEG and CT were unremarkable at the time.Three months later, the patient's condition had deteriorated dramatically. His affect seemed inappropriate, and he had marked ataxia, as well as cerebellar signs in the left arm. He had bilateral grasp reflexes and he exhibited generalized myoclonic movements. He scored 25 of 30 on testing with the Mini-Mental State Examination. The clinical features suggest a possible diagnosis of new variant Creutzfeldt-Jakob disease (nvCJD). The patient was admitted for evaluation.Routine hematologic and biochemical indices were normal. No acanthocytes were seen in the peripheral blood film. Thyroid function and vitamin E levels were normal. CSF was essentially normal. Autoantibodies and treponemal antibodies were negative. Measles antibodies were not elevated. Biochemical testing excluded Wilson's disease. Brain MRI showed a high-density change in the posterior thalamic regions (a feature noted previously in nvCJD). 1 Psychometric testing demonstrated a verbal IQ of 79, a performance IQ of 61, and a full-scale IQ of 71, compared with a predicted premorbid full-scale IQ of 95.
Sumatriptan is a highly effective treatment for migraine in adults but its efficacy in children has not been determined. Fourteen children with migraine (6.4 to 9.8 years of age; seven girls, six with aura) participated in a randomized double-blind placebo-controlled crossover study to evaluate the efficacy of sumatriptan nasal spray. After sumatriptan, 12 of 14 (versus 6 of 14 after placebo) reported a decrease in pain intensity (p = 0.031); complete headache relief was obtained in 9 of 14 after sumatriptan versus 2 of 14 after placebo (p = 0.016). Migraine-associated symptoms were also significantly reduced by sumatriptan.
In this series of patients with SMA I through III who underwent sural nerve biopsy, there was significant sensory nerve pathology in severely affected patients with SMA type I, whereas there were no sensory nerve alterations clinically or morphologically in patients with milder SMA type II or III.
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