Singh Kalra, Priti Pritipal; (2023) Characterisation of Porcine Anorectal Transition Zone Multipotential Stem Cells and their healing potential in an In Vivo Fistula Model. Doctoral thesis (Ph.D), UCL (University College London).

Abstract

Patients with Crohn’s disease often suffer from perianal fistulas, an incurable and debilitating condition where a tunnel forms between the rectal mucosa and anal skin, passes through muscle and fat, and opens outside the body. Crohn’s perianal fistulas develop at the junction of the rectal mucosa and anal skin; a tissue transition zone (TZ) of two developmental lineages: endoderm and ectoderm. Epithelial transition zones are located throughout the body: cornea-conjunctiva, esophagogastric, gastro-duodenal, endo-ectocervix, ileocecal, and the anorectal transition zone (ATZ). In addition to perianal fistulas, TZs are also prone to developing cancer. We developed an intestinal crypt-derived porcine organoids model in preparation to characterise stem cells in the intestinal mucosa and anal skin of Crohn’s disease patients. Organoids are 3- dimensional in-vitro clusters of cells that recapitulate the tissue of origin’s in vivo architecture and physiology. Hence, they show great potential in disease modelling. Acquiring patient’s GI biopsy are rare and requires them to be under general anaesthesia (GA) for biopsy of the anal region. Porcine organoids model share anatomical and physiological similarities to humans and serve as a healthy benchmark for Crohn’s disease patients. This thesis describes the multipotential stem cell characteristics of freshly isolated ATZ-derived crypts compared to rectal, colon, and small intestine (SI) and their differentiation capability Exposure of the fistula to gut contents elicits a state of chronic inflammation which requires periodic clearing of tissue debris. Cell therapy with allogeneic adipose-derived mesenchymal stem cells (AdMSC) delays the build-up of inflammatory tissue to a limited extent. In vivo capacity of allogeneic anorectal transition zone (ATZ) stem cells was assessed in a hydrogel scaffold for its capacity to differentiate and heal fistulas in a healthy porcine model. Histological analysis showed that ATZ cells in a scaffold remodelled the fistula tract with fibrotic tissue containing immature blood vessels, collagen, fibroblasts, smooth muscle cells, and immature adipose tissue. In preparation to elucidate the genetic mutations in Crohn’s fistulising disease for drug screening, methodology was developed and validated to identify mutations at the individual crypt organoid level for familial adenomatous polyposis (FAP), an inherited genetic disorder and a precursor condition to colorectal cancer (CRC). FAP patients are born with a mutation in one allele of the tumour suppressor gene, APC. This thesis describes the FAP organoid model and genomic sequencing to identify the status of the APC gene on the wild type allele and mutational heterogeneity of organoids. We have established a long-term biobank of FAP organoids to be used as a disease modelling platform and molecular profiling of key genes in CRC using next generation sequencing.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as JSONCSVXML

Archive Staff Only

View Item