Tittle: Modelling Addiction in stem cell derived cerebral organoids
An overview of the project:
Cerebral organoids represent a novel model system in which neuronal differentiation, fate specification and organisation can be investigated in vitro and have been proposed as relevant models to study the influence of prenatal drug exposure on human foetal brain. Maternal addictive substance use has been estimated at around 5%, with epidemiological studies suggesting as high as 750,000 cocaine exposed pregnancies every year. While the consequences of maternal drug abuse on the mother are well described, the effects on the unborn child are more difficult to quantify yet include premature birth, low birth weight and associated skeletal growth deficits. Moreover, foetal cocaine exposure leading to post natal persistent cognitive, information processing and learning deficits in children highlight the requirement for more research on the molecular and cellular effects of cocaine exposure in the developing human brain. Here we will explore the use of layered multiomic approaches , principally scRNA-seq and scATAC-seq to study the effects of cocaine exposure on cell fate using cerebral organoids. Human iPSCs will be differentiated to form brain organoids over a period of 36 days, after which time organoids will be maintained with or without cocaine for 48 hours and subsequently characterised by immunohistochemistry and real time PCR. Single cell libraries for RNA-seq and ATAC-seq have been generated using the 10X Genomics platform and sequence on Illumina Nextseq 500. (5000 cells to a depth of 30,000 reads per cel). Initial processing has been performed using Cell Ranger version 3.1.0. Clustering and data exploration will be performed in R 3.6.3 using Seurat version 3.1.5 (Stuart and Butler et al., 2018). Subsequent analyses will focus on integration of chromatin accessibility and gene expression data in response to acute exposure to cocaine with comparison to existing foetal and adult brain data and validated by advanced molecular and microscopy approaches.

Main points must be included in the Introduction:

First, the idea of addiction. We use three models to investigate addiction, in a realativily simple model NT2 differentiation and more complex model of cerebral organoids and the single cell cerebral organoid experiment. So these are the three models that we have been using to investigate the effects of cocaine on differentiation.
Then, the struggle that we had actually in modelling this. We can not use human test subject for this, animal models are a lot of limitations as animal brains not all are similar to human brain especially in the kind of affects that addiction has on animal models. So we need a new solution and that new solution hopefully would be the cerebral organoids that we are working on.
Then, talk about the origin of cerebral organoid and how we using it in this project.
Then, bioinformatics. Individual labelling of cells and the whole genome sequencing of each Individual cell and be able to merge all of that data in to a way that we can investigate sprete gene expression.

Addiction
Cocaine addiction
Brain development + organoid methods
Bioinformatics – scRNAseq
Project aims – models
Main points must be included in the Results/Discussion:

NT2 Westerns
NT2 ICC images
Organoids images
Organoid WB
Organoid immunofluorescence images
Bioinformatics