Three-dimensional single-cell imaging for the analysis of RNA and protein expression in intact tumour biopsies.

Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

p53 controls genomic stability and temporal differentiation of human neural stem cells and affects neural organization in human brain organoids.

In this study, we take advantage of human induced pluripotent stem (iPS) cell-derived neural stem cells and brain organoids to study the role of p53 during human brain development. We knocked down (KD) p53 in human neuroepithelial stem (NES) cells derived from iPS cells. Upon p53KD, NES cells rapidly show centrosome amplification and genomic instability. Furthermore, a reduced proliferation rate, downregulation of genes involved in oxidative phosphorylation (OXPHOS), and an upregulation of glycolytic capacity was apparent upon loss of p53. In addition, p53KD neural stem cells display an increased pace of differentiating into neurons and exhibit a phenotype corresponding to more mature neurons compared to control neurons. Using brain organoids, we modeled more specifically cortical neurogenesis. Here we found that p53 loss resulted in brain organoids with disorganized stem cell layer and reduced cortical progenitor cells and neurons. Similar to NES cells, neural progenitors isolated from brain organoids also show a downregulation in several OXPHOS genes. Taken together, this demonstrates an important role for p53 in controlling genomic stability of neural stem cells and regulation of neuronal differentiation, as well as maintaining structural organization and proper metabolic gene profile of neural progenitors in human brain organoids.

Broadly Active Antiviral Compounds Disturb Zika Virus Progeny Release Rescuing Virus-Induced Toxicity in Brain Organoids.

RNA viruses have gained plenty of attention during recent outbreaks of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zika virus (ZIKV), and Ebola virus. ZIKV is a vector borne Flavivirus that is spread by mosquitoes and it mainly infects neuronal progenitor cells. One hallmark of congenital ZIKV disease is a reduced brain size in fetuses, leading to severe neurological defects. The World Health Organization (WHO) is urging the development of new antiviral treatments against ZIKV, as there are no efficient countermeasures against ZIKV disease. Previously, we presented a new class of host-targeting antivirals active against a number of pathogenic RNA viruses, such as SARS-CoV-2. Here, we show the transfer of the image-based phenotypic antiviral assay to ZIKV-infected brain cells, followed by mechanism-of-action studies and a proof-of-concept study in a three-dimensional (3D) organoid model. The novel antiviral compounds showed a therapeutic window against ZIKV in several cell models and rescued ZIKV-induced neurotoxicity in brain organoids. The compound's mechanism-of-action was pinpointed to late steps in the virus life cycle, impairing the formation of new virus particles. Collectively, in this study, we expand the antiviral activity of new small molecule inhibitors to a new virus class of Flaviviruses, but also uncover compounds' mechanism of action, which are important for the further development of antivirals.

Single cell analysis of autism patient with bi-allelic NRXN1-alpha deletion reveals skewed fate choice in neural progenitors and impaired neuronal functionality.

We generated human iPS derived neural stem cells and differentiated cells from healthy control individuals and an individual with autism spectrum disorder carrying bi-allelic NRXN1-alpha deletion. We investigated the expression of NRXN1-alpha during neural induction and neural differentiation and observed a pivotal role for NRXN1-alpha during early neural induction and neuronal differentiation. Single cell RNA-seq pinpointed neural stem cells carrying NRXN1-alpha deletion shifting towards radial glia-like cell identity and revealed higher proportion of differentiated astroglia. Furthermore, neuronal cells carrying NRXN1-alpha deletion were identified as immature by single cell RNA-seq analysis, displayed significant depression in calcium signaling activity and presented impaired maturation action potential profile in neurons investigated with electrophysiology. Our observations propose NRXN1-alpha plays an important role for the efficient establishment of neural stem cells, in neuronal differentiation and in maturation of functional excitatory neuronal cells.