Characterizing Migratory Signaling Pathways Of Transplantable Retinal Progenitor Cells And Photoreceptor Precursor Cells Toward Restoration Of Degenerative Retina \u27 A Systems Biology Approach

A common feature of several heterogeneous diseases that result in retinal degeneration (RD) is photoreceptor loss, leading to an irreversible decline in visual function [15-17]. There are no cell replacement treatments available for RD diseases such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP). Although many RD cases are of a genetic origin, a promising strategy to treat diseased phenotypes is by replacing lost photoreceptor cells, for synaptic integration and restoration of visual function. To advance photoreceptor-replacement strategies as a practical therapy, in light of highly restricted integration rates reported across studies, this body of research focused on defining the molecular mechanisms facilitating migration of transplantable photoreceptor precursors in the retinal microenvironment. To accomplish this work we utilized bioinformatics, bioengineering and molecular biologic techniques for a systems level approach. Guided by classic neuronal migration models, we hypothesized that transplanted photoreceptor precursors navigate to specific retinal lamina in part due to cell surface receptor expression and in response to spatially gradated directional ligand cues provided by the host retinal microenvironment. Given the neural origin of the mammalian retinal system, we also predicted that these chemotactic receptor-ligand pairs trigger intracellular signaling events in migrating photoreceptors analogous to canonical migration pathways exhibited by neuronal precursors. For a comprehensive account of these motility-deterministic biochemical interactions, we first performed in silico bioinformatics modeling of PPC transplantation into light-damaged retina by matching microarray datasets between PPC receptors and ligands in the light-damaged retinal microenvironment. We then refined the gene expression network data to focus on motility deterministic interactions at the interface of the PPC cell-surface receptors and extracellular ligands of the damaged retina. Our in silico network modeling generated a library of ligand-receptor pairs associated with cellular movement specific for this retinal transplantation paradigm and the intracellular signaling pathways induced by candidate chemotactic ligands. Working from predicted interactions of in silico paired PPC receptors and retinal ligands, we then performed cell migration analysis to evaluate whether exposure to stromal derived factor-1α (SDF-1α) would guide the motility of PPCs and RPCs in vitro. We also assessed the chemotactic effects of epidermal growth factor (EGF) on RPCs. Cell surface expression of C-X-C chemokine receptor type 4 (CXCR4) receptors on PPCs and RPCs, and EGF receptor expression on RPCs were verified via immunocytochemical staining and validated by Western blot analysis. Boyden chamber analysis was used as an initial high-throughput screen to verify the motogenic effects of the ligands on PPCs and RPCs. We determined that RPC motility was optimally stimulated in these chambers by EGF concentrations in the range of 20-400ng/ml, with decreased stimulation at higher concentrations, suggesting concentration-dependence of EGF-induced motility. Both RPCs and PPCs also demonstrated a concentration-dependent chemotactic response to an optimal SDF-1α concentration of 100ng/ml. Using bioinformatics downstream signaling pathway analysis of the EGF and SDF-1α ligands in a retina-specific gene network, we predicted a chemotactic function for EGF involving the MAPK and JAK-STAT intracellular signaling pathways. Based on targeted inhibition studies, we show that ligand binding, phosphorylation of EGFR and activation of the intracellular STAT3 and PI3Kinase signaling pathways are necessary to drive RPC motility. The JAK-STAT pathway was also implicated in transducing similar motogenic effects on PPCs with SDF-1α induction. To test our hypothesis of the gradated nature of ECM ligand effects on both ontogenetic retinal cell types, we employed engineered microfluidic devices to generate quantifiable steady-state gradients of EGF and SDF-1α coupled with live-cell tracking, and analyzed the dynamics of individual RPC and PPC motility. Microfluidic analysis, including center of mass and maximum accumulated distance, revealed that EGF induced motility is chemokinetic in EGFR expressing RPCs with optimal activity observed in response to low concentration gradients. On the other hand, PPCs and RPCs exhibited significant chemotaxis towards the source of SDF-1α with longer accumulated Euclidean distances and Center of Mass (COM) compared to controls. We also ascertained that receptor mediated signaling was requisite for ligand-induced motility by using the CXCR4 inhibitor, AMD 3100, to antagonize the SDF-1α receptor. CXCR4 receptor inhibition resulted in decreases of PPC and RPC movement in uniform and steady state gradients for a number of migration indices measured. To advance translational application of the characterized chemotactic signaling potential of transplantable photoreceptor precursors, we performed computational drug analysis of our newly identified motility-deterministic networks, to develop a library of FDA approved drugs and small molecules predicted to potentially influence the expression of target motility signaling mechanisms in photoreceptor progenitor cells. Using the Expression2Kinases software and LINCS drug computational algorithm, we were able to identify pharmacological drug targets that modulate the biochemical activity of transcriptional regulatory genes which govern the expression of candidate receptor protein targets, and provide preliminary results validating the up-regulatory effect of candidate drug aminophenazone on SDF-1α receptor CXCR4 expression. Results from this study demonstrate the applicability of our systems level in silico modeling of matched transplantable cell surface-receptors and transplantation site ligands to predict molecular signaling guiding migration. Verification of in silico predictions, using molecular and microfluidic analysis provide important data for defining cell response properties to specific ligands present during transplantation into the retinal microenvironment. The drug computational analysis provides a translational perspective to our in silico modeling paradigms extending its applicability. Future studies will validate the functionality of resolved ligand-receptor pairs from our in silico library and characterize down-stream signaling guiding motility and homing. This systems level paradigm can effectively be applied to defining the molecular basis of transplantable cell migration in vivo toward improved efficiency for repair of retina and other neural tissue types

Chemotactic Migration of Clustered Central Nervous System Progenitor Cells

Clustering of central nervous system (CNS) cells is often utilized for cell growth and characterization, as well as investigated for tissue regeneration and disease progression. Collective CNS cell migration, however, has been largely unstudied. Cell cluster formation and migration play a critical part of modeling in vivo conditions and in development of therapies. Three distinct CNS cell types, medulloblastoma (MB), medulloblastoma-derived glial progenitor cells (MGPC), and retinal progenitor cells (RPC), were investigated for cluster formation, upregulation of CXCR4, the receptor for Stromal-Derived Growth Factor (SDF-1), and Connexin 43 expression, a gap junction hemichannel. A microfluidic platform was used to examine the the migration of clusters and single cells in response to controlled concentration gradients of SDF-1. All cell types illustrated self-clustering, as well as upregulated CXCR4 surface expression and increased Connexin 43 expression upon ligand stimulation. Further, RPC clusters exhibited collective, chemotactic migration along SDF-1 concentration gradients, while MB clusters illustrated inconsistent collective migration, and MGPCs clusters did not exhibit adhesion-based migration

Effects of Extreme Climate Events on Tea (Camellia sinensis) Functional Quality Validate Indigenous Farmer Knowledge and Sensory Preferences in Tropical China

Climate change is impacting agro-ecosystems, crops, and farmer livelihoods in communities worldwide. While it is well understood that more frequent and intense climate events in many areas are resulting in a decline in crop yields, the impact on crop quality is less acknowledged, yet it is critical for food systems that benefit both farmers and consumers through high-quality products. This study examines tea (Camellia sinensis; Theaceae), the world’s most widely consumed beverage after water, as a study system to measure effects of seasonal precipitation variability on crop functional quality and associated farmer knowledge, preferences, and livelihoods. Sampling was conducted in a major tea producing area of China during an extreme drought through the onset of the East Asian Monsoon in order to capture effects of extreme climate events that are likely to become more frequent with climate change. Compared to the spring drought, tea growth during the monsoon period was up to 50% higher. Concurrently, concentrations of catechin and methylxanthine secondary metabolites, major compounds that determine tea functional quality, were up to 50% lower during the monsoon while total phenolic concentrations and antioxidant activity increased. The inverse relationship between tea growth and concentrations of individual secondary metabolites suggests a dilution effect of precipitation on tea quality. The decrease in concentrations of tea secondary metabolites was accompanied by reduced farmer preference on the basis of sensory characteristics as well as a decline of up to 50% in household income from tea sales. Farmer surveys indicate a high degree of agreement regarding climate patterns and the effects of precipitation on tea yields and quality. Extrapolating findings from this seasonal study to long-term climate scenario projections suggests that farmers and consumers face variable implications with forecasted precipitation scenarios and calls for research on management practices to facilitate climate adaptation for sustainable crop production

Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina

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

Characterization of Induced Pluripotent Stem Cell Microvesicle Genesis, Morphology and Pluripotent Content

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

Effects of water availability and pest pressures on tea (Camellia sinensis) growth and functional quality

Extreme shifts in water availability linked to global climate change are impacting crops worldwide. The present study examines the direct and interactive effects of water availability and pest pressures on tea (Camellia sinensis; Theaceae) growth and functional quality. Manipulative greenhouse experiments were used to measure the effects of variable water availability and pest pressures simulated by jasmonic acid (JA) on tea leaf growth and secondary metabolites that determine tea quality. Water treatments were simulated to replicate ideal tea growing conditions and extreme precipitation events in tropical southwestern China, a major centre of tea production. Results show that higher water availability and JA significantly increased the growth of new leaves while their interactive effect was not significant. The effect of water availability and JA on tea quality varied with individual secondary metabolites. Higher water availability significantly increased total methylxanthine concentrations of tea leaves but there was no significant effect of JA treatments or the interaction of water and JA. Water availability, JA treatments or their interactive effects had no effect on the concentrations of epigallocatechin 3-gallate. In contrast, increased water availability resulted in significantly lower concentrations of epicatechin 3-gallate but the effect of JA and the interactive effects of water and JA were not significant. Lastly, higher water availability resulted in significantly higher total phenolic concentrations but there was no significant impact of JA and their interaction. These findings point to the fascinating dynamics of climate change effects on tea plants with offsetting interactions between precipitation and pest pressures within agro-ecosystems, and the need for future climate studies to examine interactive biotic and abiotic effects

Characterization of Induced Pluripotent Stem Cell Microvesicle Genesis, Morphology and Pluripotent Content

Microvesicles (MVs) are lipid bilayer-covered cell fragments that range in diameter from 30 nm-1 uM 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.S

Effects of Precipitation Variability on Tea Prices.

<p>Farmers at the study site have experienced an average decrease of 51% in on-farm tea prices received during the Monsoon tea harvests compared to the dry spring tea harvests.</p

Effects of Precipitation Variability on Tea Growth.

<p>Increased precipitation from the spring drought to the monsoon tea harvest significantly increased tea leaf weight and length. Sampling periods not connected by the same letters are significantly different. Values are means ± one standard error.</p

Historical Precipitation Trends During the Dry Spring and Monsoon Tea Harvests.

<p>Comparison of average rain rate for dry spring (top) and monsoon (bottom) tea harvests during the following periods: a) 1979–1984, b) 1985–1989, c) 1990–1994, d) 1995–1999, e) 2000–2004, f) 2005–2010. The study site is located at the dot in the middle of each map.</p