A protocol for isolation and culturing of mouse primary postmitotic photoreceptors and isolation of extracellular vesicles.

Here, we present a protocol for isolating and culturing mouse photoreceptors in a minimal, chemically defined medium free from serum. We describe steps for retina dissection, enzymatic dissociation, photoreceptor enrichment, cell culture, extracellular vesicles (EVs) enrichment, and EV ultrastructural analysis. This protocol, which has been verified for cultured cells derived from multiple murine strains, allows for the study of several aspects of photoreceptor biology, including EV isolation and nanotube formation. For complete details on the use and execution of this protocol, please refer to Kalargyrou et al. (2021).1.

ChipSeg: An Automatic Tool to Segment Bacterial and Mammalian Cells Cultured in Microfluidic Devices.

Extracting quantitative measurements from time-lapse images is necessary in external feedback control applications, where segmentation results are used to inform control algorithms. We describe ChipSeg, a computational tool that segments bacterial and mammalian cells cultured in microfluidic devices and imaged by time-lapse microscopy, which can be used also in the context of external feedback control. The method is based on thresholding and uses the same core functions for both cell types. It allows us to segment individual cells in high cell density microfluidic devices, to quantify fluorescent protein expression over a time-lapse experiment, and to track individual mammalian cells. ChipSeg enables robust segmentation in external feedback control experiments and can be easily customized for other experimental settings and research aims.

A Microfluidic/Microscopy-Based Platform for on-Chip Controlled Gene Expression in Mammalian Cells.

Applications of control engineering to mammalian cell biology have been recently implemented for precise regulation of gene expression. In this chapter, we report the main experimental and computational methodologies to implement automatic feedback control of gene expression in mammalian cells using a microfluidics/microscopy platform.

A tunable dual-input system for on-demand dynamic gene expression regulation.

Cellular systems have evolved numerous mechanisms to adapt to environmental stimuli, underpinned by dynamic patterns of gene expression. In addition to gene transcription regulation, modulation of protein levels, dynamics and localization are essential checkpoints governing cell functions. The introduction of inducible promoters has allowed gene expression control using orthogonal molecules, facilitating its rapid and reversible manipulation to study gene function. However, differing protein stabilities hinder the generation of protein temporal profiles seen in vivo. Here, we improve the Tet-On system integrating conditional destabilising elements at the post-translational level and permitting simultaneous control of gene expression and protein stability. We show, in mammalian cells, that adding protein stability control allows faster response times, fully tunable and enhanced dynamic range, and improved in silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to modulate levels of signalling pathway components in mouse Embryonic Stem Cells.

IGF-1 Signaling Plays an Important Role in the Formation of Three-Dimensional Laminated Neural Retina and Other Ocular Structures From Human Embryonic Stem Cells.

We and others have previously demonstrated that retinal cells can be derived from human embryonic stem cells (hESCs) and induced pluripotent stem cells under defined culture conditions. While both cell types can give rise to retinal derivatives in the absence of inductive cues, this requires extended culture periods and gives lower overall yield. Further understanding of this innate differentiation ability, the identification of key factors that drive the differentiation process, and the development of clinically compatible culture conditions to reproducibly generate functional neural retina is an important goal for clinical cell based therapies. We now report that insulin-like growth factor 1 (IGF-1) can orchestrate the formation of three-dimensional ocular-like structures from hESCs which, in addition to retinal pigmented epithelium and neural retina, also contain primitive lens and corneal-like structures. Inhibition of IGF-1 receptor signaling significantly reduces the formation of optic vesicle and optic cups, while exogenous IGF-1 treatment enhances the formation of correctly laminated retinal tissue composed of multiple retinal phenotypes that is reminiscent of the developing vertebrate retina. Most importantly, hESC-derived photoreceptors exhibit advanced maturation features such as the presence of primitive rod- and cone-like photoreceptor inner and outer segments and phototransduction-related functional responses as early as 6.5 weeks of differentiation, making these derivatives promising candidates for cell replacement studies and in vitro disease modeling.

Pain and neurological sequelae of cluster munitions on children and adolescents in South Lebanon.

This paper aims at evaluating the neurological repercussions arising from injuries sustained due to cluster munitions in children up to 18 years in South Lebanon following the 2006 conflict. Data on neurological and pain symptoms suffered during and after treatment because of sub-munitions in South Lebanon from August 2006 till late 2011 were prospectively recorded. Patients were divided into subcategories; children aged 12 and under and adolescents aged between 13 and 18. During the study period, there were 407 casualties, 122 (30%) of which were aged 18 years or younger. There were 116 (95%) males and six (5%) females. Average age was 14 years. 10 (8.2%), all males, died as a result of their injuries. 42 (34.4%) were children and 80 (65.6%) were adolescents. 112 had surgical treatments for their injuries. 83 out of 112 patients (74%) with non-lethal injuries had amputations, 67% children and 78% adolescents. Among those who had amputations, 31 (37.4%) suffered from phantom limb pain and 71% suffered from stump/residual limb pain. 88% of patients were diagnosed with post-traumatic stress disorder (44% children and 77% adolescents) and 41% were diagnosed with post-concussion syndrome. Four patients (3.6%) suffered from traumatic brain injuries, both penetrating and closed. Pain syndromes were found in all patients who had amputation. The injury related comorbidities together with many post-concussion syndrome cases, and fewer traumatic brain injuries lead into a high level of physical, psychosocial and economic burdens on the community.