Response of human limbal epithelial cells to wounding on 3D RAFT tissue equivalents: effect of airlifting and human limbal fibroblasts.

Limbal epithelial stem cell deficiency can cause blindness but may be treated by human limbal epithelial cell (hLE) transplantation, normally on human amniotic membrane. Clinical outcomes using amnion can be unreliable and so we have developed an alternative tissue equivalent (TE), RAFT (Real Architecture for 3D Tissue), which supports hLE expansion, and stratification when airlifted. Human limbal fibroblasts (hLF) may be incorporated into RAFT TEs, where they support overlying hLE and improve phenotype. However, the impact of neither airlifting nor hLF on hLE function has been investigated. hLE on RAFT TEs (±hLF and airlifting) were wounded using heptanol and re-epithelialisation (fluorescein diacetate staining), and percentage putative stem cell marker p63α and proliferative marker Ki67 expression (wholemount immunohistochemistry), measured. Airlifted, hLF- RAFT TEs were unable to close the wound and p63α expression was 7 ± 0.2% after wounding. Conversely, non-airlifted, hLF- RAFT TEs closed the wound within 9 days and p63α expression was higher at 22 ± 5% (p < 0.01). hLE on both hLF- and hLF+ RAFT TEs (non-airlifted) closed the wound and p63α expression was 26 ± 8% and 36 ± 3% respectively (ns). Ki67 expression by hLE increased from 1.3 ± 0.5% before wounding to 7.89 ± 2.53% post-wounding for hLF- RAFT TEs (p < 0.01), and 0.8 ± 0.08% to 17.68 ± 10.88% for hLF+ RAFT TEs (p < 0.05), suggesting that re-epithelialisation was a result of proliferation. These data suggest that neither airlifting nor hLF are necessarily required to maintain a functional epithelium on RAFT TEs, thus simplifying and shortening the production process. This is important when working towards clinical application of regenerative medicine products.

Heptanol application to the mouse round window: a model for studying cochlear lateral wall regeneration.

OBJECTIVE: Identify cells supporting cochlear lateral wall regeneration. STUDY DESIGN: Prospective controlled trial. SETTING: Laboratory. Human presbyacusis occurs, in part, secondary to age-related degeneration of cochlear lateral wall structures such as the stria vascularis and spiral ligament fibrocytes. This degeneration is likely linked to the diminished regenerative capacity of lateral wall cells with age. While lateral wall regeneration is known to occur after an acute insult, this process remains poorly understood and the cells capable of self-replication unidentified. We hypothesized that spiral ligament fibrocytes constitute these proliferative cells. SUBJECTS AND METHODS: To test the hypothesis, an acute ototoxic insult was created in 65 normal-hearing, young adult mice via cochlear exposure to heptanol. Sacrifice occurred at 1 to 60 days posttreatment. Auditory brainstem responses, 5-ethynyl-2'-deoxyuridine assay, and immunostaining were used to assess regeneration. RESULTS: Posttreatment hearing thresholds were elevated in nearly all treated mice. Selective fibrocyte apoptosis and strial injury were observed at the time of peak hearing loss around 1 to 7 days posttreatment. Cellular proliferation was detected in the region of type II fibrocytes during this time. Hearing thresholds plateaued at 7 days posttreatment followed by a significant recovery of both hearing and morphologic appearance. Permanent outer hair cell degeneration was observed. CONCLUSIONS: Heptanol application to the round window of young adult mice is a rapid, selective, and reliable technique for investigating proliferation in the cochlear lateral wall. The data indirectly showed that spiral ligament fibrocytes may be the proliferative cells of the cochlear lateral wall. Further studies of this process are needed.

Microphysiological systems and low-cost microfluidic platform with analytics.

A multiorgan, functional, human in vitro assay system or 'Body-on-a-Chip' would be of tremendous benefit to the drug discovery and toxicology industries, as well as providing a more biologically accurate model for the study of disease as well as applied and basic biological research. Here, we describe the advances our team has made towards this goal, as well as the most pertinent issues facing further development of these systems. Description is given of individual organ models with appropriate cellular functionality, and our efforts to produce human iterations of each using primary and stem cell sources for eventual incorporation into this system. Advancement of the 'Body-on-a-Chip' field is predicated on the availability of abundant sources of human cells, capable of full differentiation and maturation to adult phenotypes, for which researchers are largely dependent on stem cells. Although this level of maturation is not yet achievable in all cell types, the work of our group highlights the high level of functionality that can be achieved using current technology, for a wide variety of cell types. As availability of functional human cell types for in vitro culture increases, the potential to produce a multiorgan in vitro system capable of accurately reproducing acute and chronic human responses to chemical and pathological challenge in real time will also increase.

Ventricular arrhythmogenesis following slowed conduction in heptanol-treated, Langendorff-perfused mouse hearts.

Arrhythmogenic effects of slowed action potential conduction produced by the gap junction and sodium-channel inhibitor heptanol (0.1-2 mM) were explored in Langendorff-perfused mouse hearts. Monophasic action potential recordings showed that 2 mM heptanol induced ventricular tachycardia in the absence of triggered activity arising from early or after-depolarizations during regular 8 Hz pacing and programmed electrical stimulation (PES). It also increased activation latencies and ventricular effective refractory periods (VERPs), but did not alter action potential duration (APD), thereby reducing local critical intervals for re-excitation given by APD(90) - VERP. Bipolar electrogram recordings showed that 2 mM heptanol increased electrogram duration (EGD) and ratios of EGDs obtained at the longest to those obtained at the shortest S1S2 intervals studied during PES, suggesting increased dispersion of conduction velocities. These findings show, for the first time in the mouse heart, that slowed conduction induces reversible arrhythmogenic effects despite repolarization abnormalities expected to reduce arrhythmogenicity.

Corneal surface reconstruction using adult mesenchymal stem cells in experimental limbal stem cell deficiency in rabbits.

PURPOSE: To investigate the ability of mesenchymal stem cells (MSC) to transdifferentiate to corneal epithelial cells in experimental limbal stem cell deficiency in rabbits. METHODS: Total limbal stem cell deficiency was produced in 21 right eyes of 21 New Zealand rabbits; 6 eyes served as controls (group 1, G1). After removal of the conjunctival overgrowth, five eyes received amniotic membrane transplantation (AMT; G2). In four eyes, autologous limbal stem cell transplantation from the healthy eye was performed with AMT (G3). In another six eyes, enriched autologous MSC were injected under the amniotic membrane (AM) (G4). Within 280 days, corneoscleral discs were analysed for goblet cells, cytokeratin (CK) 3/12, connexin 43, ß(1) -integrin, CK 19, α-enolase, p63 and ATP-binding cassette transporter subtype G-2 (ABCG-2) distribution patterns. RESULTS: Cultivated MSC were positive for CK 3/12 and α-enolase, but negative for ABCG-2, p63 and connexin 43. On rabbit corneas, CK 3/12 was expressed in all corneal regions in all groups, but with significantly different intensities. Among all other parameters, expression levels of ABCG-2, ß(1) -integrin and connexin 43 were significantly different between the transplanted groups and the control group. After a mean follow-up time of 172 (47-280) days, goblet cells were rarely present in the central cornea (G1-4). CONCLUSION: CK 3/12 is not highly specific for differentiated corneal epithelium. Further, goblet cells are not a reliable marker for conjunctivalization in rabbits. Expression of ABCG-2, ß(1)-integrin and connexin 43 after mesenchymal stem cell transplantation may indicate their ability to maintain their stem cell character or to transdifferentiate to epithelial progenitor cells.

Arabinogalactan as active compound in the management of corneal wounds: in vitro toxicity and in vivo investigations on rabbits.

PURPOSE: Aims of the present investigation were to prove that natural polysaccharide arabinogalactan (AG) is well tolerated after ocular administration and exerts a high restoring effect on corneal epithelium abrasions. MATERIALS AND METHODS: AG interactions with corneal cells, as well as its effect on their proliferation, were evaluated employing rabbit corneal epithelial cell cultures. The effects due to the presence of benzalkonium chloride (BAK) were also studied on cell cultures, ex vivo on rabbit isolated corneas, evaluating the hydration level, and on the healing rate of experimental corneal wounds in rabbits. Furthermore, the healing process of corneal lesions treated with an experimental 5.0% AG solution was studied and compared with those obtained applying solutions of hyaluronic acid and tamarind seed polysaccharide, both chosen as a reference by virtue of their well-known adjuvant properties on corneal trophism; the study was carried out by light and transmission electron microscopy. RESULTS: BAK showed toxic effects on corneal epithelium in all experiments. AG proved to stimulate the growth of the corneal epithelial cells by interacting at the level of the cell plasma membrane. The microscopy observations of the epithelial surface of AG-treated damaged corneas revealed a well-restored and histologically organized ultrastructure characterized by fully formed microvilli and glycocalyx; the healing process resulted faster with respect to spontaneously recovered untreated corneas. CONCLUSION: Our results suggest that AG can interact with corneal epithelial cells without any toxic side effect; moreover, it proved to stimulate cell proliferation, thus promoting tissue re-epithelialization and reorganization just 48 hr post-wounding.

Effect of retinol palmitate eye drops on experimental keratoconjunctival epithelial damage induced by n-heptanol in rabbit.

PURPOSE: To investigate ability of vitamin A (VA, retinol palmitate) to promote wound healing after injury in rabbit eye. METHODS: After keratoconjunctival injury by 20 microL of n-heptanol, VA eye drops at 500, 1000, or 1500 IU/mL were started 6 times a day for 11 days. Fluorescein and rose bengal staining and histological analysis were performed. RESULTS: Fluorescein staining was significantly reduced by 1000 and 1500 IU/mL of VA and rose bengal staining by all concentrations. Histological examination revealed acceleration of wound healing after 7 days (1500 IU/mL). CONCLUSIONS: VA may be useful in treatment of keratoconjunctival injury.

Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits.

PURPOSE: To determine factors affecting the outcome of corneal surface reconstruction in rabbits with total limbal stem cell deficiency (LSCD), by using autologous limbal epithelial stem cells (LSC) ex vivo, expanded on rabbit amniotic membrane (AM). METHODS: Left eyes of 52 rabbits were rendered totally limbal stem cell deficient by n-heptanol debridement of the entire corneal epithelium followed by surgical removal of 360 degrees of limbal rim. After cytologic verification of LSCD, the fibrovascular pannus of each cornea was removed. Group I (n = 10) received a rabbit AM transplant, whereas groups II, III, and IV (n = 42) underwent transplantation of LSCs cultured on rabbit AM (LSC-AM graft) derived from a small limbal biopsy specimen from the right eye. Clinical outcome was graded as a success if a smooth, avascular corneal surface was restored, a partial success if more than two quadrants of corneal surface were smooth, or a failure if the corneal surface was revascularized and irregular. RESULTS: A long-term follow-up of more than 1 year was achieved. Compared with the 100% failure rate in group I, inclusion of expanded LSCs resulted in variable success rates in groups II, III, and IV (all P < 0.001). Kaplan-Meier survival analysis showed that different suturing techniques, subconjunctival injection of long-acting steroid, and tarsorrhaphy used in groups II (n = 17) and III (n = 13) did not significantly alter the outcome (P = 0.89). However, the use of a larger graft and human AM as a temporary patch with the explant retained for 1 week in group IV (n = 12) significantly improved the success rate to 83% (P = 0.002). Among eyes showing clinical failure, there was a significant correlation between the logarithm of the first day when an epithelial defect was noted and the time of graft failure (r(2) = 0.60, P < 0.001). Furthermore, the presence of severe lid deformity was borderline significant when correlated with failure cases in all four groups (P = 0.069). CONCLUSIONS: Ex vivo expansion of LSCs can be achieved by using rabbit AM culture. Such expanded LSCs can successfully reconstruct corneal surfaces affected by total LSCD. This animal model is useful to investigate culturing variables affecting epithelial stemness so that surgical reconstruction of corneas with total LSCD can be successfully performed. Furthermore, this model can be used to test the feasibility of gene therapies targeting LSCD in the future.