Human Retinal Development in an in situ Whole Eye Culture System

Abstract

Phenotypic characterization of human retinogenesis may be facilitated by use of the tissue culture system paradigm. Traditionally, most culture protocols involve isolation of retinal tissue and/or cells, imposing various degrees of trauma, which in many cases leads to abnormal development. In this paper, we present a novel culture technique using whole embryonic eyes to investigate whether the retina in situ can develop normally in vitro. All procedures were carried out in accordance with the Declaration of Helsinki. Human embryos were obtained from elective abortions with the informed consent of the women seeking abortion. A total of 19 eyes were enucleated. The ages of the embryonic retinas were 6–7.5 weeks. Eyecups from 2 eyes were fixed immediately, to be used as controls. The remaining 17 eyes were placed on culture plates and divided into 3 groups kept for 7 (n = 4), 14 (n = 7) and 28 (n = 6) days in vitro (DIV). After fixation, the specimens were processed for hematoxylin and eosin staining, immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Antibodies against recoverin (rods and cones), protein kinase C (PKC; rod bipolar cells) and vimentin (Müller cells) were used. TUNEL was used to detect apoptotic cells. In hematoxylin- and eosin-stained sections, the control retinas displayed a neuroblast cell layer (NBL) and an inner marginal zone. Specimens kept 7–14 DIV had a similar appearance, while 28-day specimens consisted of an NBL with almost no marginal zone. Thirteen of the 17 cultured retinas displayed completely normal lamination without rosettes or double folds. Pyknotic cells were found at the inner margin of the retinas, and the proportion of these cells increased with time in vitro. TUNEL staining revealed a few scattered cells in 7-DIV specimens, and the amount of stained cells in the inner part of the retinas progressively increased in 14- and 28-DIV specimens. Vimentin labeling showed cells arranged in a vertical pattern in all retinas. Labeling with recoverin revealed photoreceptors in 4 of the retinas kept for 14 DIV, and in all retinas kept for 28 DIV. After 28 DIV, 2 of the eyes labeled with PKC contained rod bipolar cells with minimal axons. Here we showed that human embryonic retinas can be kept in culture in situ within the eye for at least 4 weeks. Abnormal lamination is not as frequent as in isolated full-thickness retinas, indicating that physical and biochemical contact with surrounding tissues is vital for proper development. Several types of the retina-specific neuronal and glial cells were seen to differentiate according to the in vivo schedule. The results are important for future studies of retinal development, and the technique can also be used for testing the effects of various drugs on the immature retina.

Altered cortical and subcortical local coherence in PTSD: evidence from resting-state fMRI

“…The thalamus and putamen had been implicated in a number of discrete, distributed corticalstriatal-thalamic circuits involved in specific cognitive functions (25). We suppose the alterations in regionally subcortical connectivity may be attributable to the high level of arousal and result in symptoms such as dissociation and flashback in PTSD (26), and the changes in subcortical regions during resting state may provide more insight into the neuronal circuitry underlying the re-experience and reawakening of traumatic events (27).…”

Cell replacement and visual restoration by retinal sheet transplants

“…Gosh and collaborators have developed procedures for culturing fetal retinal sheets (Ghosh et al, 2009) and whole eye cups (Engelsberg and Ghosh, 2011). Tissue culture induces selective loss of inner retinal cells.…”

A novel whole tooth‐in‐jaw‐bone culture of rat molars: Morphological, immunohistochemical, and laser capture microdissection analysis

“…Organ tissue cultures may best mimic natural replication circumstances and thus provide a third approach that bridges the gap between standard cell culture and animal models (Griffith et al,2006; Yamada and Cukierman,2007). Organ tissue culture systems in the medical field has been established for studies of the retina (Engelsberg and Ghosh,2011; Moritoh et al,2010; Thangaraj et al,2011), central nervous system (Zhang et al,2011), nasal epithelium (Bochkov et al,2011), liver (Baptista et al,2011), gastrointestinal tissue (Randall et al,2011), fallopian tubes or uterine tubes (Levanon et al,2010), intervertebral disc (Jim et al,2011; Korecki et al,2007), and bone (Papadimitropoulos et al,2011). Under conventional organ culture condition, however, it is usually difficult to preserve tissue viability for >1 week.…”