Using human genetically engineered stem cell in vitro models to reveal new insights into Crohn's disease pathogenesis

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Deng, Boping (2022). Using human genetically engineered stem cell in vitro models to reveal new insights into Crohn's disease pathogenesis. University of Birmingham. Ph.D.

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Abstract

Crohn's disease (CD) is becoming a global challenge to public health. Unfortunately, the pathogenesis of CD is still unclear, and mouse models are not optimal in studying this complex human disease. CRISPR-Cas9-based genome editing was combined with in vitro differentiation of human induced pluripotent stem cells (iPSCs) to generate models for exploring CD pathogenesis. Two CD risk-associated genes NOD2 (a pattern-recognition receptor) and LRRK2 (a large protein of undefined function that has been implicated in a wide range of biological processes) were knocked out in iPSCs from a healthy donor, and the cells were differentiated into isogenic human macrophages and intestinal organoids in vitro. RNA sequencing was used to explore the biological changes caused by the knockouts. Findings from the research indicated that the loss of NOD2 led to a large defect in the response to interferon-gamma (IFN-γ) of the macrophages, whilst loss of LRRK2 did not, though the gene was found to be significantly up regulated in macrophages treated by IFN-γ. Meanwhile, the effects from LRRK2 deficiency were identified to focus on mitochondria in macrophages. Moreover, this work also revealed a correlation between NOD2 (the major susceptible gene for Crohn's disease) and LRRK2 (the major susceptible gene for Parkinson's disease (PD)) expression in macrophages treated by IFN-γ, which suggested a shared pathogenesis between CD and PD. Additionally, a pipeline for preparing single cell RNA-seq samples from CD patients' resections was established to compare those in vitro models with the in vivo situation of the disease. Monocytes/macrophages cell populations were identified in single cell RNA-seq data, which showed the subtle differences between resections from the same patient but with agreed with the IFN-γ effect seen in in vitro macrophages. Data from CD patient's resections also showed an altered IFN-γ response and additional effects on mitochondrial gene expression were associated with the severity of inflammation in macrophages. This finding from patients' samples supports the clinical relevance of the in vitro macrophage models.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):