Final Report Summary - ORALSTEM (Role of CD133 in controlling oral epithelial stem cell fate determination)
The project “Role of CD133 in controlling oral epithelial stem cell fate determination” aimed to investigate the synergistic roles of Notch and CD133 in regulating oral epithelial stem cell fate. The original project objectives contain two connected research directions: (1) Researching into the functional interaction between Notch and CD133 signaling in oral epithelial cell survival and differentiation, and (2) Investigating the role of Notch-CD133 connection in oral cancer. The project involved in different in vivo and in vitro approaches to study the functional interaction, common targets between Notch and CD133 signaling, in oral epithelial stem cell survival as well as the biological significance of the two pathways in oral cancer and potential clinical implications.
Using continuously growing mouse incisor tooth as a model, Dr. Bing Hu’s group first analyzed the expression pattern of CD133 epitopes and Notch pathway members in the different populations of epithelial cells, including stem cells, transit amplifying cells and differentiated cells. Second, the group compared CD133 knockout and epithelial specific Notch knockout mouse models concerning epithelial cell population components, cellular structure integrity and cell fate by employing different technical approaches including immunohistochemistry, confocal microscopy followed by three-dimensional reconstruction and quantification, real time RT-PCR, as well as a newly developed fluorescent cell cycle indicator system. To investigate the additional crosstalk between CD133 and the other pathways, the group has used PCR array, chromatin immunoprecipitation, as well as Co-immunoprecipitation. Epigenetic assays of CpG islands methylation was used to investigate the gene activation/silencing mechanisms. For the new pathways that has been identified involving in the molecular networks with CD133, the group has received strong supports from the collaborators for providing different transgenic mouse models, as well as biochemical tools to introduce point mutation into the gene. For the induction of oral cancer in the mouse models, the group has applied 4-Nitroquinoline 1-oxide induced oral model onto the CD133 knockout and control mice.
Our key findings can be reflected by the following two manuscripts that currently under review and revision that can be summarized as below:
1. We have identified that CD133, one widely used stem cell marker, has distinct expression epitope specific patterns in the tooth epithelium. Unexpectedly, the CD133 c-terminal is located in the cytoplasm and sometimes in nuclei in the undifferentiated tooth epithelial cells and the extracellular loop of CD133 only expresses in the transit amplifying cells, suggesting potentially this molecule has different roles in controlling epithelial cell fate based on its epitopes. Phenotypically I have first identified that CD133 knockout mice have significant tooth enamel development defects, like the human genetic disease: amelogenesis imperfecta, which highly affects enamel development. The tooth phenotypes are also similar to the mice with specific loss of RBPjkappa, the key Notch pathway transcription factor, in the tooth epithelium. Currently my group is performing further study to illustrate the functions of the CD133 epitopes and their molecular partners, as well as downstream targets.
2. We have found that CD133 can coordinate ciliary dynamics in stem cells by recruiting ciliary membrane components, histone deacetylase and transcription factors. Nuclear translocation of CD133, together with the other molecules, is particularly evident in transit amplifying cells, the immediate derivatives of SCs. In the absence of CD133, impaired ciliary dynamics results in disrupted stem cell function, including quiescence, maintenance and activation, leading to abolishment of Sonic Hedgehog stimulation impacts on SCs, and a failure in terminal cell differentiation.
Both works suggest CD133 is a key regulator required to ensure that SCs receive and respond appropriately to extracellular signals that has important implications in development, regeneration and diseases. In addition, we have also found that the CD133 knock out mice are less prone to develop oral cancers.
We expect our major researches on the projects will be eventually published in 2018 on two high impact international peer-reviewed journals. In parallel, Dr. Hu's group has identified that autophagy is a key cellular event that can preserve the dental stem cells. The relevant results have been published as 2 papers. In addition, with the Marie Curie CIG grant as a platform, with support from Plymouth University Dr. Bing Hu has been appointed as associate professor/reader, a permanent position. In four years, Dr. Hu has been able to quickly expand my independent group into a team consisting of 15 researchers, including 6 PhD students and 1 postdoctoral researcher and 3 associated lecturers, 2 visiting scientists and 2 volunteer research students. 3 additional postdoctoral researchers received training at the group as well. Proudly, Dr. Hu’s students have won several key research prizes including twice for the Unilever Poster Prize, a highly considered dental research prize and the prestige the UK Science Council RSci CPD Awards. Those significant progresses have helped Dr. Hu to secure two additional major research grants from the EU and Biotechnology and Biological Sciences Research Council (UK) and several other smaller grants that in total worth more than 1.5 million pounds. In addition, Dr. Hu has 3 published papers and 2 patents filed, and 3 papers currently under revision. In total, Dr. Hu has established collaborations with more than 20 international and national universities and 5 industry partners. In a summary, the EU Marie Curie CIG fellowship has enabled Dr. Hu to establish the research and progress career towards a leader in international oral biology and cancer researches.
Using continuously growing mouse incisor tooth as a model, Dr. Bing Hu’s group first analyzed the expression pattern of CD133 epitopes and Notch pathway members in the different populations of epithelial cells, including stem cells, transit amplifying cells and differentiated cells. Second, the group compared CD133 knockout and epithelial specific Notch knockout mouse models concerning epithelial cell population components, cellular structure integrity and cell fate by employing different technical approaches including immunohistochemistry, confocal microscopy followed by three-dimensional reconstruction and quantification, real time RT-PCR, as well as a newly developed fluorescent cell cycle indicator system. To investigate the additional crosstalk between CD133 and the other pathways, the group has used PCR array, chromatin immunoprecipitation, as well as Co-immunoprecipitation. Epigenetic assays of CpG islands methylation was used to investigate the gene activation/silencing mechanisms. For the new pathways that has been identified involving in the molecular networks with CD133, the group has received strong supports from the collaborators for providing different transgenic mouse models, as well as biochemical tools to introduce point mutation into the gene. For the induction of oral cancer in the mouse models, the group has applied 4-Nitroquinoline 1-oxide induced oral model onto the CD133 knockout and control mice.
Our key findings can be reflected by the following two manuscripts that currently under review and revision that can be summarized as below:
1. We have identified that CD133, one widely used stem cell marker, has distinct expression epitope specific patterns in the tooth epithelium. Unexpectedly, the CD133 c-terminal is located in the cytoplasm and sometimes in nuclei in the undifferentiated tooth epithelial cells and the extracellular loop of CD133 only expresses in the transit amplifying cells, suggesting potentially this molecule has different roles in controlling epithelial cell fate based on its epitopes. Phenotypically I have first identified that CD133 knockout mice have significant tooth enamel development defects, like the human genetic disease: amelogenesis imperfecta, which highly affects enamel development. The tooth phenotypes are also similar to the mice with specific loss of RBPjkappa, the key Notch pathway transcription factor, in the tooth epithelium. Currently my group is performing further study to illustrate the functions of the CD133 epitopes and their molecular partners, as well as downstream targets.
2. We have found that CD133 can coordinate ciliary dynamics in stem cells by recruiting ciliary membrane components, histone deacetylase and transcription factors. Nuclear translocation of CD133, together with the other molecules, is particularly evident in transit amplifying cells, the immediate derivatives of SCs. In the absence of CD133, impaired ciliary dynamics results in disrupted stem cell function, including quiescence, maintenance and activation, leading to abolishment of Sonic Hedgehog stimulation impacts on SCs, and a failure in terminal cell differentiation.
Both works suggest CD133 is a key regulator required to ensure that SCs receive and respond appropriately to extracellular signals that has important implications in development, regeneration and diseases. In addition, we have also found that the CD133 knock out mice are less prone to develop oral cancers.
We expect our major researches on the projects will be eventually published in 2018 on two high impact international peer-reviewed journals. In parallel, Dr. Hu's group has identified that autophagy is a key cellular event that can preserve the dental stem cells. The relevant results have been published as 2 papers. In addition, with the Marie Curie CIG grant as a platform, with support from Plymouth University Dr. Bing Hu has been appointed as associate professor/reader, a permanent position. In four years, Dr. Hu has been able to quickly expand my independent group into a team consisting of 15 researchers, including 6 PhD students and 1 postdoctoral researcher and 3 associated lecturers, 2 visiting scientists and 2 volunteer research students. 3 additional postdoctoral researchers received training at the group as well. Proudly, Dr. Hu’s students have won several key research prizes including twice for the Unilever Poster Prize, a highly considered dental research prize and the prestige the UK Science Council RSci CPD Awards. Those significant progresses have helped Dr. Hu to secure two additional major research grants from the EU and Biotechnology and Biological Sciences Research Council (UK) and several other smaller grants that in total worth more than 1.5 million pounds. In addition, Dr. Hu has 3 published papers and 2 patents filed, and 3 papers currently under revision. In total, Dr. Hu has established collaborations with more than 20 international and national universities and 5 industry partners. In a summary, the EU Marie Curie CIG fellowship has enabled Dr. Hu to establish the research and progress career towards a leader in international oral biology and cancer researches.