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postgraduate thesis: Establishment and direct differentiation of induced pluripotent stem cells from a Hirschsprung's patient

TitleEstablishment and direct differentiation of induced pluripotent stem cells from a Hirschsprung's patient
Authors
Issue Date2014
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Yung, S. J. [容心怡]. (2014). Establishment and direct differentiation of induced pluripotent stem cells from a Hirschsprung's patient. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5435660
AbstractHirschsprung’s (HSCR) disease is a congenital disorder in which some enteric ganglion cells are absent in the colon due to incomplete colonization of neural crest cells (NCCs) in the hindgut, causing chronic constipation. A significant number of HSCR patients also clinically present with other NC- associated disorders, such as ventricular and atrial septal defects (VSD/ASD). A hypomorphic allele or SNP of a major gene, RET, causes or imparts susceptibility to HSCR. In particular, SNP (rs2435357) residing in the intron 1 of RET gene was found to be highly associated with HSCR and lead to reduced RET expression. However, the molecular basis of syndromic HSCR with VSD/ASD is largely unclear. In our project, with the use of the induced pluripotent stem cell (iPSC) technology, we aim to establish a patient-specific model unravel the etiology of HSCR and the associated disorders. To this end, 3 iPSC clones from a syndromic HSCR patient with VSD/ASD, carrying the RET risk allele in rs2435357 were generated. We attempted to use different protocols to directly differentiate iPSCs into NCCs with unique HOX expression patterns, corresponding to anterior cranial/vagal or posterior vagal/trunk NCCs. Consistently, the patient iPSCs displayed similar capacities in generating NCCs at all axial levels, marked by HNK-1 and 〖p75〗^NTR. Nevertheless, the patient NCCs and their derivatives exhibited severe migration and/ or differentiation defects in making neurons and smooth muscle cells. In particular, HNK-1+〖p75〗^NTR+ HOX+ (vagal/trunk) NCCs derived from patient-iPSCs were less migratory compared to the control NCCs, while no obvious migration defect was observed in their cranial counterpart, indicating that the migration defect was only restricted to the more posterior NCCs. In addition, these patient NCCs were less capable in generating neurons and readily biased toward generating glial cells. Intriguingly, the neural differentiation defects were restricted to NC lineage. The capacity of patient iPSCs to make various types of CNS progenitors and neurons was comparable to that of the control iPSCs, nicely recapitulating the patient’s phenotype where only enteric neurons, but not CNS progenitors were affected. Subsequent expression analysis revealed that patient NCCs express lower level of RET which is known to be regulating enteric NCC migration and differentiation. Whole transcriptome RNA sequencing analysis also revealed an enhanced expression of genes associated with gliogenesis and a reduced expression in genes associated with neurogenesis and migration. Moreover, the expression of a new candidate gene ALDH3B1 was shown to be significantly reduced in the HSCR-iPSC-derived NCCs that might contribute to the disease pathogenesis. In summary, these data suggests that reduced RET expression in HSCR patient NCCs may at least partly account for the disease phenotypes.
DegreeDoctor of Philosophy
SubjectStem cells
Hirschsprung's disease - Etiology
Dept/ProgramSurgery
Persistent Identifierhttp://hdl.handle.net/10722/209471

 

DC FieldValueLanguage
dc.contributor.authorYung, Sum-yee, Jasmine-
dc.contributor.author容心怡-
dc.date.accessioned2015-04-23T23:10:47Z-
dc.date.available2015-04-23T23:10:47Z-
dc.date.issued2014-
dc.identifier.citationYung, S. J. [容心怡]. (2014). Establishment and direct differentiation of induced pluripotent stem cells from a Hirschsprung's patient. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5435660-
dc.identifier.urihttp://hdl.handle.net/10722/209471-
dc.description.abstractHirschsprung’s (HSCR) disease is a congenital disorder in which some enteric ganglion cells are absent in the colon due to incomplete colonization of neural crest cells (NCCs) in the hindgut, causing chronic constipation. A significant number of HSCR patients also clinically present with other NC- associated disorders, such as ventricular and atrial septal defects (VSD/ASD). A hypomorphic allele or SNP of a major gene, RET, causes or imparts susceptibility to HSCR. In particular, SNP (rs2435357) residing in the intron 1 of RET gene was found to be highly associated with HSCR and lead to reduced RET expression. However, the molecular basis of syndromic HSCR with VSD/ASD is largely unclear. In our project, with the use of the induced pluripotent stem cell (iPSC) technology, we aim to establish a patient-specific model unravel the etiology of HSCR and the associated disorders. To this end, 3 iPSC clones from a syndromic HSCR patient with VSD/ASD, carrying the RET risk allele in rs2435357 were generated. We attempted to use different protocols to directly differentiate iPSCs into NCCs with unique HOX expression patterns, corresponding to anterior cranial/vagal or posterior vagal/trunk NCCs. Consistently, the patient iPSCs displayed similar capacities in generating NCCs at all axial levels, marked by HNK-1 and 〖p75〗^NTR. Nevertheless, the patient NCCs and their derivatives exhibited severe migration and/ or differentiation defects in making neurons and smooth muscle cells. In particular, HNK-1+〖p75〗^NTR+ HOX+ (vagal/trunk) NCCs derived from patient-iPSCs were less migratory compared to the control NCCs, while no obvious migration defect was observed in their cranial counterpart, indicating that the migration defect was only restricted to the more posterior NCCs. In addition, these patient NCCs were less capable in generating neurons and readily biased toward generating glial cells. Intriguingly, the neural differentiation defects were restricted to NC lineage. The capacity of patient iPSCs to make various types of CNS progenitors and neurons was comparable to that of the control iPSCs, nicely recapitulating the patient’s phenotype where only enteric neurons, but not CNS progenitors were affected. Subsequent expression analysis revealed that patient NCCs express lower level of RET which is known to be regulating enteric NCC migration and differentiation. Whole transcriptome RNA sequencing analysis also revealed an enhanced expression of genes associated with gliogenesis and a reduced expression in genes associated with neurogenesis and migration. Moreover, the expression of a new candidate gene ALDH3B1 was shown to be significantly reduced in the HSCR-iPSC-derived NCCs that might contribute to the disease pathogenesis. In summary, these data suggests that reduced RET expression in HSCR patient NCCs may at least partly account for the disease phenotypes.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.lcshStem cells-
dc.subject.lcshHirschsprung's disease - Etiology-
dc.titleEstablishment and direct differentiation of induced pluripotent stem cells from a Hirschsprung's patient-
dc.typePG_Thesis-
dc.identifier.hkulb5435660-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineSurgery-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b5435660-

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