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    Multiple endocrine neoplasia type 2B: modelling the disease in human cells and avian embryos
    Abu-Bonsrah, Kwaku Dad ( 2017)
    Paediatric cancer initiation is difficult to study because the early stages are often prenatal. Patient cells at the time of detection already have complex genetic (including epigenetic) changes, and the cancer cell population is heterogeneous. One way to model cancer initiation at the organism level is to engineer the specific initiating mutation into the appropriate cell lineage of an experimental animal embryo. For the human cell context, an ideal cell model would start with the normal human cell of origin and create candidate initiating mutations. Multiple Endocrine Neoplasia type 2B (MEN2B) is an autosomal dominant complex oncologic disease of the neural crest (NC) cell lineage, a so-called neurocristopathy. It presents with i) multiple mucosal ganglioneuromas including hyperplasia of the enteric (gut) nervous system leading to gastrointestinal disorders, ii) pheochromocytoma, with sympathoadrenal (SA) hyperplasia and catecholamine disturbance, and iii) medullary thyroid carcinoma with C-cell hyperplasia, elevated calcitonin and calcium metabolism disturbance. In addition, patients have marfanoid facial features. MEN2B results from de novo germline gain-of-function mutations in the gene RET, most often M918T. RET codes for the signalling receptor for the growth factor ligand GDNF, hence in MEN2B cells, RET signalling is divorced from GDNF availability. MEN2B is rare but it is often misdiagnosed especially early in life. This is due to the nature and diversity of the lineages affected; SA and enteric NC-lineage cells and thyroid C-cells, the latter being of foregut endodermal entero-endocrine lineage. We were the first to successfully use CRISPR/Cas9 to mutate the developing chicken embryo in vivo, showing phenotypic abnormality. This included creating a single point mutation by homology directed repair in vivo in NC cells at the avian MEN2B homologous site (M910T). For the human cell context, we combined the CRISPR/Cas9 technology and knowledge of embryo development and cell differentiation to create MEN2B M918T cells using the human embryonic stem cell (hESC) lines H9, HES3 and MEL2. We modified a hESC differentiation protocol to produce enteric NC-like cells, showing in vitro that these cells upregulated key NC and enteric genes. Functionally we also showed higher proliferation and greater axon production in the MEN2B mutant cells: this is consistent with the ganglioneuroma phenotype. In addition, we developed a new differentiation protocol to produce human SA progenitors and medullary chromaffin-like cells, as marked by expression of key catecholamine genes TH and PNMT, and expression of adrenaline and noradrenaline by HPLC. These cells are affected in MEN2B patient pheochromocytoma. We then developed a novel differentiation protocol for thyroid C-cell-like cells from hESCs via Definitive Endodermal Cells. These cells produce Calcitonin and we validated their functionality by ELISA assay and compared the MEN2B clones with the control hESCs. These cells are affected in MEN2B patients resulting in medullary thyroid carcinoma.