Medical Biology - Theses

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Type: Masters Research thesis × Author: Cong, Yuyang Jr ×

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    Establishing an in vitro screening platform for haematopoietic progenitor cell development
    Cong, Yuyang Jr ( 2017)
    Before the emergence of haematopoietic stem cells (HSC), haematopoiesis in the embryo depends on the activity of haematopoietic progenitor cells (HPC). During embryogenesis, HPCs of broadening lineage develop in the yolk sac. Understanding the underlying regulatory genes that facilitate, or instruct HPC development will help to improve methods for in vitro HPC generation. To identify genes involved in yolk sac HPC development, an in vitro screening platform was established to identify genes required for HPC development. Briefly the target genes will be knocked out from E8.5 and E9.5 yolk sac HPCs using the CRISPR/Cas9 system. Candidate genes that are differentially expressed between early and late yolk sac HPCs were for the first round screening. These genes were identified using single cell RNA sequencing dataset within the Taoudi Laboratory. During the knockout period, HPCs will be cultured on a stromal cell monolayer to preserve the HPC state (facilitating survival and impeding differentiation). Then the manipulated HPCs will be harvested for functional tests using the in vitro colony forming assays. To optimize this screening platform, I performed stepwise modifications by studies on wild-type yolk sac HPCs. For the screening to be high throughput in the future, I developed an automated image analysis macro instruction (macro). This macro is capable of counting haematopoietic colonies and collecting their morphology features at a higher speed compared with manual assessment and at a higher accuracy compared with existing automated algorithms recognizing round objects, which promises its potential application to image analysis in other fields. Due to time constrains, the evaluation of the sensitivity and reproducibility of the screening platform was incomplete. Given more time, this would be performed by knocking out control genes that play important roles in HPC survival or development. Loss-of-function screening of differentially expressed genes between early and late HPCs using this in vitro screening platform will help to identify regulatory genes involved in HPC development, and provide hints for improvements of in vitro HPC generation using reprogramming strategies.