A pancreatic stem cell could provide the tissue necessary for widespread β-cell transplantation therapy for diabetes. It is disputed whether pancreatic stem cells or β-cell replication are responsible for maintenance and regeneration of endocrine cells. Evidence presented here shows that pancreatic stem cells express insulin and produce multiple endocrine, exocrine and neural cells in vitro and in vivo. The human pancreas also contains stem cells that produce functional β-cells capable of reducing blood sugar levels in a diabetic mouse. Initial studies of pancreatic stem cells grown clonally in vitro indicated that they produced large numbers of neurons, suggesting they may be derived from the neural crest. Evidence shows that there are at least two distinct developmental origins for stem cells in the pancreas; one from the pancreatic lineage that produces endocrine and exocrine cells and one from the neural crest lineage that produces neurons and Schwann cells. Furthermore, pancreatic stem cells require the developmental transcription factor, Pax6, for endocrine cell formation suggesting they are using expected differentiation pathways. There is an interesting evolutionary connection between pancreatic β-cells and neurons which was applied to the derivation of pancreatic stem cells from human embryonic stem cells by using a clonal neural stem cell assay. These pancreatic stem cells express pancreatic and neural markers, self-renew and differentiate into insulin-expressing cells. The overexpression of SOX17 in these cells increases stem cell formation and self-renewal but inhibits differentiation. Overall I will show that there is a genuine stem cell in the adult mammalian pancreas capable of producing functional β-cells, that this stem cell is derived from the pancreatic developmental lineage but the pancreas also contains stem cells from the neural crest lineage, and that the neural stem cell assays that have identified these adult stem cells can be applied to the derivation of a pancreatic stem cell from hESCs.

A pancreatic stem cell could provide the tissue necessary for widespread β-cell transplantation therapy for diabetes. It is disputed whether pancreatic stem cells or β-cell replication are responsible for maintenance and regeneration of endocrine cells. Evidence presented here shows that pancreatic stem cells express insulin and produce multiple endocrine, exocrine and neural cells in vitro and in vivo. The human pancreas also contains stem cells that produce functional β-cells capable of reducing blood sugar levels in a diabetic mouse. Initial studies of pancreatic stem cells grown clonally in vitro indicated that they produced large numbers of neurons, suggesting they may be derived from the neural crest. Evidence shows that there are at least two distinct developmental origins for stem cells in the pancreas; one from the pancreatic lineage that produces endocrine and exocrine cells and one from the neural crest lineage that produces neurons and Schwann cells. Furthermore, pancreatic stem cells require the developmental transcription factor, Pax6, for endocrine cell formation suggesting they are using expected differentiation pathways. There is an interesting evolutionary connection between pancreatic β-cells and neurons which was applied to the derivation of pancreatic stem cells from human embryonic stem cells by using a clonal neural stem cell assay. These pancreatic stem cells express pancreatic and neural markers, self-renew and differentiate into insulin-expressing cells. The overexpression of SOX17 in these cells increases stem cell formation and self-renewal but inhibits differentiation. Overall I will show that there is a genuine stem cell in the adult mammalian pancreas capable of producing functional β-cells, that this stem cell is derived from the pancreatic developmental lineage but the pancreas also contains stem cells from the neural crest lineage, and that the neural stem cell assays that have identified these adult stem cells can be applied to the derivation of a pancreatic stem cell from hESCs.

Neural crest cells (NCC) arise from the neural tube (NT) and migrate through given regions of embryos, where they generate most of the peripheral nervous system (PNS), facial skeleton and pigment cells. Defective NCC development gives rises to malformations in multiple NCC-derived structures, collectively known as neurocristopathies. NCC from the NT vagal and trunk levels express Hoxb5 plus a number of other Hox proteins. Hoxb5 is a member of Hox transcription factors family that binds to specific target nucleotide sequences in the genome via their DNA-binding domain, where they regulate gene expressions. Vagal NCC migrate to the intestine and generate the enteric nervous system (ENS). To test the Hoxb5 function in vagal NCC, we made use a transgenic mouse line (enb5) and showed that perturbation of Hoxb5 signaling in NCC resulted in down-regulation of Ret and defective ENS, indicating that normal Hoxb5 function was required for the development of vagal NCC. Current project aims to investigate the function of Hoxb5 in trunk NCC development. Transgenic mouse enb5 can be induced by Cre recombinase to express a hybrid protein namely engrailed-Hoxb5 (enb5), in which the transactivation domain of the mouse Hoxb5 is replaced with a repressor domain of the Drosophila engrailed (en) protein. With the intact DNA-binding domain, enb5 binds to target genes of Hoxb5, repressing the expression of target genes instead of induction. Therefore, enb5 produces a dominant negative effect on the developmental pathways that normally require Hoxb5. In this study, enb5 mice were crossed to Wnt1-Cre mice to express enb5 in NCC that arose from the entire length of NT. Wnt1-Cre/enb5 mutants displayed apoptosis of NCC, skin hypopigmentation and PNS defects (hypoplastic dorsal root ganglion and defective ENS). Expression of Sox9, Foxd3 and Ret was down-regulated in Wnt1-Cre/enb5 embryos. Conditional deletion of Sox9 and Foxd3 by Wnt1-Cre, or conventional deletion of Ret in mice produced NCC phenoptypes similar to those of Wnt1-Cre/enb5. Taken all these prompted me to further investigate if Hoxb5 functioned in the same pathway as Sox9 and Foxd3 for NCC development using multiple experimental approaches. In ovo electroporation of enb5 in chick embryos induced apoptosis of NT, and co-electroporation of Hoxb5, Ret, Sox9 or Foxd3 rescued enb5-induced cell death. By bioinformatics analysis, Hoxb5 binding sites were identified in SOX9 and FOXD3 promoter sequences. Binding of Hoxb5 protein onto these binding sites of SOX9 and FOXD3 promoters was revealed by electro-mobility shift assay and further confirmed by chromatin immuno-precipitation assay. In addition, enb5 was also shown to bind to the same regions of SOX9 and FOXD3 promoters as Hoxb5. Using dual luciferase reporter assay, Hoxb5 was shown to induce transcription from SOX9 and FOXD3 promoters, and enb5 blocked the induction. Taken all these indicate that (i) Hoxb5 binds and induces transcriptions from SOX9 and FOXD3 promoters, (ii) enb5 blocks the induction. In summary, Hoxb5 regulates NCC development by controlling the expression of Sox9, Foxd3 and Ret, and perturbation of Hoxb5 signaling results in NCC death and neurocristopathies. / published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

abstract / Surgery / Master / Master of Philosophy

abstract / Biochemistry / Master / Master of Philosophy

To determine whether exposure to simulated microgravity (SMG) affects cranial neural crest (CNC)-derived tissues, zebrafish embryos were exposed to SMG starting at one of three developmental stages corresponding to CNC migration. Juvenile and adult fish were analyzed after exposure to SMG using statistics and geometric morphometrics for changes in melanophore surface area and number, and changes in skull morphology. Analyses reveal an initial increase in the surface area of melanophores present on the dorsal view of the juvenile skull and a decrease in melanophore number over the period of a week. Additionally, buckling is observed in CNC-derived frontal bones in juvenile fish after exposure. The effects on the melanophores are transient and the effects on CNC-derived bones are short-term. Surprisingly, severe long-term effects occurred in mesoderm-derived bones, such as the parasphenoid. In summary, exposure to SMG affects both CNC- and mesoderm-derived tissues in the juvenile and adult zebrafish head.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 96-106) Also available in print.

Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 118-137).