A range of cell types, including embryonic stem cells, neurons and astrocytes have been shown to release extracellular vesicles (EVs) containing molecular cargo. Across cell types, EVs facilitate transfer of mRNA, microRNA and proteins between cells. Here we describe the release kinetics and content of EVs from mouse retinal progenitor cells (mRPCs). Interestingly, mRPC derived EVs contain mRNA, miRNA and proteins associated with multipotency and retinal development. Transcripts enclosed in mRPC EVs, include the transcription factors Pax6, Hes1, and Sox2, a mitotic chromosome stabilizer Ki67, and the neural intermediate filaments Nestin and GFAP. Proteomic analysis of EV content revealed retinogenic growth factors and morphogen proteins. mRPC EVs were shown to transfer GFP mRNA between cell populations. Finally, analysis of EV mediated functional cargo delivery, using the Cre-loxP recombination system, revealed transfer and uptake of Cre+ EVs, which were then internalized by target mRPCs activating responder loxP GFP expression. In summary, the data supports a 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.

The mechanisms underlying retinal development have not been completely elucidated. Extracellular vesicles (EVs) are novel essential mediators of cell-to-cell communication with emerging roles in developmental processes. Nevertheless, the identification of EVs in human retinal tissue, characterization of their cargo, and analysis of their potential role in retina development has not been accomplished. Three-dimensional retinal tissue derived from human induced pluripotent stem cells (hiPSC) provide an ideal developmental system to achieve this goal. Here we report that hiPSC-derived retinal organoids release exosomes and microvesicles with small noncoding RNA cargo. EV miRNA cargo-predicted targetome correlates with Gene Ontology (GO) pathways involved in mechanisms of retinogenesis relevant to specific developmental stages corresponding to hallmarks of native human retina development. Furthermore, uptake of EVs by human retinal progenitor cells leads to changes in gene expression correlated with EV miRNA cargo predicted gene targets, and mechanisms involved in retinal development, ganglion cell and photoreceptor differentiation and function.

Interleukin 24 (IL-24) is a tumor-suppressing protein, which inhibits angiogenesis and induces cancer cell-specific apoptosis. We have shown that IL-24 regulates apoptosis through phosphorylated eukaryotic initiation factor 2 alpha (eIF2α) during endoplasmic reticulum (ER) stress in cancer. Although multiple stresses converge on eIF2α phosphorylation, the cellular outcome is not always the same. In particular, ER stress-induced apoptosis is primarily regulated through the extent of eIF2α phosphorylation and activating transcription factor 4 (ATF4) action. Our studies show for the first time that cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) activation is required for IL-24-induced cell death in a variety of breast cancer cell lines and this event increases ATF4 activity. We demonstrate an undocumented role for PKA in regulating IL-24-induced cell death, whereby PKA stimulates phosphorylation of p38 mitogen-activated protein kinase and upregulates extrinsic apoptotic factors of the Fas/FasL signaling pathway and death receptor 4 expression. We also demonstrate that phosphorylation and nuclear import of tumor suppressor TP53 occurs downstream of IL-24-mediated PKA activation. These discoveries provide the first mechanistic insights into the function of PKA as a key regulator of the extrinsic pathway, ER stress, and TP53 activation triggered by IL-24.

Microvesicles (MVs) are lipid bilayer-covered cell fragments that range in diameter from 30 nm–1uM and are released from all cell types. An increasing number of studies reveal that MVs contain microRNA, mRNA and protein that can be detected in the extracellular space. In this study, we characterized induced pluripotent stem cell (iPSC) MV genesis, content and fusion to retinal progenitor cells (RPCs) in vitro. Nanoparticle tracking revealed that iPSCs released approximately 2200 MVs cell/hour in the first 12 hrs with an average diameter of 122 nm. Electron and light microscopic analysis of iPSCs showed MV release via lipid bilayer budding. The mRNA content of iPSC MVs was characterized and revealed the presence of the transcription factors Oct-3/4, Nanog, Klf4 and C-Myc. The protein content of iPSCs MVs, detected by immunogold electron microscopy, revealed the presence of the Oct-3/4 and Nanog. Isolated iPSC MVs were shown to fuse with RPCs in vitro at multiple points along the plasma membrane. These findings demonstrate that the mRNA and protein cargo in iPSC MVs have established roles in maintenance of pluripotency. Building on this work, iPSC derived MVs may be shown to be involved in maintaining cellular pluripotency and may have application in regenerative strategies for neural tissue.

To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α – CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment.

Central nervous system (CNS) cells cultured in vitro as neuroclusters are useful models of tissue regeneration and disease progression. However, the role of cluster formation and collective migration of these neuroclusters to external stimuli has been largely unstudied in vitro. Here, 3 distinct CNS cell types, medulloblastoma (MB), medulloblastoma-derived glial progenitor cells (MGPC), and retinal progenitor cells (RPC), were examined with respect to cluster formation and migration in response to Stromal-Derived Growth Factor (SDF-1). A microfluidic platform was used to distinguish collective migration of neuroclusters from that of individual cells in response to controlled concentration profiles of SDF-1. Cell lines were also compared with respect to expression of CXCR4, the receptor for SDF-1, and the gap junction protein Connexin 43 (Cx43). All cell types spontaneously formed clusters and expressed both CXCR4 and Cx43. RPC clusters exhibited collective chemotactic migration (i.e. movement as clusters) along SDF-1 concentration gradients. MGPCs clusters did not exhibit adhesion-based migration, and migration of MB clusters was inconsistent. This study demonstrates how controlled microenvironments can be used to examine the formation and collective migration of CNS-derived neuroclusters in varied cell populations.