Date of Degree

9-2017

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor(s)

Stephen Redenti

Committee Members

Maria Canto Soler

Moira Sauane

Mark Emmerson

Hyungsik Lim

Subject Categories

Biology

Keywords

mouse retina progenitor cells; mouse induced pluripotent stem cell; human induced pluripotent stem cell; extracellular vesicles; exosomes; microparticles; retina cups;

Abstract

Extracellular vesicles (EVs) have emerged as a novel cell-to-cell communication mechanism in recent years. EVs are membrane-covered cell fragments released into the extracellular environment by all cell types in the normal physiological and pathological conditions. These membranous extracellular organelles include exosomes (30-100 nm) and microparticles (100-1000 nm), which are believed to play a pivotal role in intercellular communication. EVs represent the way for intercellular transfer of proteins and RNAs. By transfer of genetic materials, EVs are involved in reprogramming and tissue repair. We predict that during retinal development, retinal progenitor cells release EVs containing temporally expressed mRNA, proteins and miRNA, which may influence functioning and fate of recipient cells. As the first step to validate this hypothesis, we isolated EVs from multi-passages of mouse retinal progenitor cells (RPCs). We describe the concentration, ultrastructure and contents of EVs released from mouse retinal progenitor cells (mRPCs). The data reveals that mRPC derived EVs contain mRNA, miRNA and proteins associated with multipotency and retinal development. EVs released from mRPCs contain mRNA for the transcription factors Nestin, Pax6, Hes1, Ki-67, and Sox2, each of which is involved in retinogenesis. Proteomic analysis revealed a wide number of transcription factors, growth factors and retinal morphogen proteins contained in mRPC EVs. Imaging revealed, EV binding to and uptake by mRPCs. Finally, mRPC-derived EVs were shown to transfer this material to non-GFP RPCs.

In summary, the data supports a novel paradigm of EV genetic material encapsulation and transfer within RPC populations. RPC EV transfer may influence recipient RPC transcriptional and post-transcriptional regulation, representing a novel mechanism of differentiation and fate determination during retinal development.

In addition to RPC-derived EVs, we characterized the EVs from mouse induced pluripotent stem cell (iPSCs) content and fusion to retinal progenitor cells (RPCs) in vitro was analyzed. Electron microscopic analysis of iPSCs revealed cytoplasmic origin of EVs and release via lipid bilayer budding. The mRNA content of iPSC EVs was characterized and revealed the presence of the transcription factors Oct-3/4, Nanog, Klf4, and C-Myc. Isolated iPSC EVs were shown to fuse with RPCs in vitro at multiple points on the plasma membrane. These findings reveal that the mRNA and protein cargo in iPSC EVs have established roles in maintenance of pluripotency. Building on this work, iPSC derived EVs may be shown to be involved in maintaining cellular pluripotency and may have application in regenerative strategies for neural tissue.

To begin to characterize EVs present during human retinal development, we also analyzed the morphology and content of EVs released from hiPSC derived 3D retinas cups at three developmental time points, D42, D63 and D93, which are three important developmental stage for retinal lamination in vitro. Analysis of small RNAs contained in EVs using next generation sequencing revealed the presence of a range of non-coding RNAs such as micro, piwi- and transfer-RNA species with predicted regulatory functions. This work provides the first evidence of small RNAs contained within EVs released from a model of human retinal development.

Taken together, our work shows EVs from mouse retinal progenitor cells and hiPSC derived 3D retinas cups contain important genetic materials, which may involve in retinal development.

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