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Supervisors names
Conor McCann
Paolo De Coppi
Background
Hirschsprung disease (HSCR), a life-threatening intestinal disorder affecting 1 in 5000 live births, is caused by the absence of enteric neurons in the distal bowel, which ultimately results in intestinal obstruction. The current treatment is surgical resection of the affected bowel. Whilst lifesaving, this surgical treatment can result in life-long gastrointestinal problems including constipation, faecal incontinence, and enterocolitis, which significantly impact on quality of life. Recent pre-clinical evidence in animal models suggests that human pluripotent stem cell (hPSC)-derived enteric nervous system (ENS) progenitor transplantation, aimed at replacing lost neurons, is potentially viable. These data provide a strong basis for further translational investigation of this novel cell therapy. However, a major limitation, remains a lack of knowledge surrounding the integration of hPSC-derived ENS progenitors in human tissue and the effects of immunosuppression on the efficacy of any ENS progenitor therapeutics.
Aims/Objectives and Methods:
This overarching aim of this project is to utilise a proven in vitro ENS progenitor generation protocol5 to strengthen the pre-clinical basis for a regenerative medicine approach to treat HSCR tissue.
Specifically, this project will:
1) Evaluate the ability of hPSC-derived ENS progenitors to integrate within human HSCR patient-derived gut samples in a novel ex vivo culture system.
To test the ability of our hPSC-derived ENS progenitors to integrate within human HSCR tissue, the student will transplant candidate hPSC-derived ENS progenitor populations into isolated segments of human HSCR diseased aganglionic tissue ex vivo to examine their functionality in a human disease context. The integration and functional output of transplanted cells, within recipient colonic tissues, will be assessed using immunohistochemistry, molecular biology techniques and organ bath contractility/calcium imaging.
2) Examine the effects of current clinical immunosuppression protocols on hPSC-derived ENS progenitors. To examine the response of hPSC-derived ENS progenitors to immunosuppressive agents the student will compare the ability of hPSCs to efficiently generate ENS progenitors (quantitatively defined by expression of key ENS progenitor markers: p75, RET/TRKC) in the presence or absence of clinically relevant immunosuppressants using cell culture methods. The student will further examine the ability ENS progenitors to form key ENS derivatives (e.g., enteric neurons, glia and neuronal subtypes) both in vitro and within ex vivo transplanted HSCR tissue in the presence of immunosuppressive agents using a combination of state-of-the-art imaging techniques, fluorescence activated cell sorting (FACS) and transcriptional analyses.
3) Generate “universal” hPSC-derived ENS progenitors to overcome potential immune response barriers.
To generate hypoimmune “universal” hPSC-derived ENS progenitors the student will use gene editing approaches (i.e., CRISPR/Cas9) to target MHC-I and II complexes within a target hPSCs. Following targeting, hypoimmune donor cells will be used to generate hPSC-derived ENS progenitors using our established protocol. The student will subsequently assess the capacity of hypoimmune hPSC to generate ENS progenitors, and their derivatives, compared to non-targeted hPSC using a combination of immunohistochemistry, FACS and RNAseq approaches in both cell and ex vivo organotypic culture using patient derived HSCR tissue.
Timeline:
- Objective 1 (Months 0-12)
- Objective 2 (Months 3-21)
- Objective 3 (Months 6-30)
- Thesis write up/submission (Months 30-36)
References:
(1) Obermayr et al. (2013) Nat Rev Gastroenterol Hepatol 10:43-57. (2) Catto-Smith et al. (2007) J. Gastroent. Hepatol. 22:2273-2282. (3) Conway et al. (2007) J. Ped. Surg. 42:14291432. (4) Burns AJ et al. (2016) Dev Biol 417:229-251. (5) Frith et al. (2020) Stem Cell Reports. 3:557-565.
Contact
Conor McCann (conor.mccann@ucl.ac.uk)