Signal pathways that regulate retinal differentiation from embryonic stem cells
Open Access
- Author:
- Lu, Amy Qi
- Graduate Program:
- Biomedical Sciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 06, 2017
- Committee Members:
- Colin James Barnstable, Dissertation Advisor/Co-Advisor
Colin James Barnstable, Committee Chair/Co-Chair
Alistair J Barber, Committee Member
Sarah Bronson, Committee Member
James Robert Connor, Outside Member
Sinisa Dovat, Outside Member - Keywords:
- retina development
photoreceptor precursor
photoreceptor development
activin signaling
embryonic stem cell - Abstract:
- Retinal differentiation from embryonic stem cells (ESCs) can be used to examine the intricate interactions between extrinsic and intrinsic mechanisms that regulate mammalian retina development. We utilized this differentiation process to study the roles of exogenous factors acting through multiple signaling pathways with the overall goal of understanding how photoreceptors develop from multipotential anterior neural tissue and to improve the generation of cells suitable for photoreceptor replacement. ESCs can reproducibly generate several cell types, including retinal progenitor cells (RPCs). However, the subsequent derivation of photoreceptor precursors is variable and relatively inefficient. We compared the effects of exogenous signaling factors and physical culture conditions on the derivation of retinal cells from ESCs and propose a relatively simple protocol that is optimized for guiding ESCs through stages that correspond to the sequence of in vivo retinal development and also optimized for studying the sequence of steps spanning the time between eye field formation and photoreceptor precursor generation in the embryonic retina. We demonstrated the usefulness of this ESC-retinal induction protocol in screening for factors that improve photoreceptor precursor yield by evaluating responses to Activin signaling. Application of Activin A during a time that corresponds to early embryonic retinogenesis increased generation of CRX+ photoreceptor precursors and decreased PAX6+ RPCs. Following treatment, Activin signaling through SMAD2/3 in RPCs regulated expression of transcription factors involved in cell type determination to promote photoreceptor lineage specification. Furthermore, we tested the ability of ESCs to model developmental stages before and after the transition of RPCs into photoreceptor precursors. ESC-retinal induction appeared to mimic in vivo effects of altering factors implicated in development at these stages. Specifically, Ikaros might govern the determination of retinal cell types by acting as a switch between early versus late RPC subpopulations, which have different propensities for generating lineage-specific daughter cells. Preliminary studies also suggested that terminal differentiation of ESC-derived photoreceptor precursors is affected by alterations in STAT3 and PDK1 signaling. Our findings demonstrate that ESC differentiation follows the temporal sequence of in vivo retinal development and this progression can be manipulated with exogenous factors. By optimizing the activities of signal pathways during this process, post-mitotic photoreceptors precursors can be efficiently generated. Further investigation into post-transplant outcomes of ESC-derived photoreceptor precursors will allow us to evaluate the effectiveness of these precursors in rescuing visual function and lead to optimization of the transplant protocol and post-transplant environmental requirements. Thus, ESC-retinal differentiation can be used both as a way to screen to factors that improve target cell yield and as source of starting material for optimizing transplant outcomes after cell replacement. This work can contribute to the production of photoreceptors for therapy.