Modeling human neurogenesis in vitro using pluripotent stem cells: the evolution from 2D to 3D
Document TypePhD thesis
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© 2018 Dr. Cristiana Mattei
Progress in stem cell technologies is based on fundamental research on exploring culture conditions that improve and optimize in vitro generation of human cell types and tissues. To date, there has been significant progress in developing protocols for in vitro neural differentiation of human pluripotent stem cells (hPSCs). Despite these advances, there are still large gaps in our understanding in how to create the ideal microenvironment in vitro to support ‘normal’ cellular processes occurring in vivo. Addressing these gaps would significantly advance the stem cell-based approach for in vitro modeling and drug screening studies as well as for regenerative medicine applications. In the last five years, a significant advancement in stem cell biology has been the shift to developing three dimensional (3D) hPSC cell culture systems. Establishing a faithful in vitro model of human neurogenesis is to date one of the main challenges of the field. To this end, in this thesis we explored the utilization of innovative 3D culture systems for deriving human neural-like tissue in vitro. Our main approach was the use of a rotary cell culture system (RCCS) to generate hPSC-derived neural organoids, which mimics microgravity conditions. Our results show that although neural organoids could be generated and maintained in microgravity conditions, there were changes in expression of rostral-caudal neural patterning genes and cortical markers compared to organoids generated in standard conditions. In particular, we showed that RCCS-derived organoids are capable of supporting otic-like specification and recapitulate some characteristics of inner ear development, including generation of hair cells displaying vestibular-like morphological and physiological phenotypes that resemble developing human fetal inner ear hair cells. Another aim of this thesis was to interrogate the application of a conductive 3D scaffold, the graphene foam, to support in vitro culture of hPSCs-derived neurons. Our data demonstrated biocompatibility of the graphene foam for human neurons and suggested it may be a suitable scaffold for developing 3D in vitro platforms for studies examining neural connectivity and circuit formation. Finally, we explored the application of hPSC culture systems for in vitro disease modeling with regard to Autism Spectrum Disorders (ASD). Establishing a valuable in vitro model of ASD would be of a great benefit for advancing our understanding of this complex condition and designing appropriate treatments. Our study provided preliminary but promising characterization data of three ASD induced pluripotent stem cell (PSC) line-derived cortical-like tissue. In conclusion, we propose alternative 3D in vitro systems for modeling several aspects of human neurogenesis. Our data provide new insights for using hPSC to study human neurodevelopment and related neurodevelopmental diseases.
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