Survival and Functionality of hESC-Derived Retinal Pigment Epithelium Cells Cultured as a Monolayer on Polymer Substrates Transplanted in RCS Rats.

Purpose: To determine the safety, survival, and functionality of human embryonic stem cell-derived RPE (hESC-RPE) cells seeded on a polymeric substrate (rCPCB-RPE1 implant) and implanted into the subretinal (SR) space of Royal College of Surgeons (RCS) rats.

Methods: Monolayers of hESC-RPE cells cultured on parylene membrane were transplanted into the SR space of 4-week-old RCS rats. Group 1 (n = 46) received vitronectin-coated parylene membrane without cells (rMSPM+VN), group 2 (n = 59) received rCPCB-RPE1 implants, and group 3 (n = 13) served as the control group.

In vivo detection of hESC-RPE cells via confocal near-infrared fundus reflectance.

Background And Objective: To investigate whether the confocal near-infrared reflectance (NIR) imaging modality could detect the in vivo presence of retinal pigment epithelium cells derived from embryonic human stem cells (hESC-RPE) implanted into the subretinal space of the Royal College of Surgeons (RCS) rat.

Materials And Methods: Monthly NIR images were obtained from RCS rats implanted with either hESC-RPE seeded on a parylene membrane (n = 14) or parylene membrane without cells (n = 14). Two independent, masked investigators graded the images.

Subretinal implantation of retinal pigment epithelial cells derived from human embryonic stem cells: improved survival when implanted as a monolayer.

Purpose: To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM).

Methods: Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36).

A novel approach for subretinal implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium monolayer.

Objective: To evaluate the feasibility of a new technique for the implantation of ultrathin substrates containing stem cell-derived retinal pigment epithelium (RPE) cells into the subretinal space of retina-degenerate Royal College of Surgeon (RCS) rats.

Methods: A platform device was used for the implantation of 4-µm-thick parylene substrates containing a monolayer of human embryonic stem cell-derived RPE (hESC-RPE). Normal Copenhagen rats (n = 6) and RCS rats (n = 5) were used for the study.

Autoimmune dacryoadenitis and sialadenitis induced in rabbits by intravenous injection of autologous lymphocytes activated ex vivo against lacrimal antigens.

Purpose: Autologous peripheral blood lymphocytes, activated in a mixed cell reaction when cocultured with purified rabbit lacrimal epithelial cells, are known to induce a severe autoimmune dacryoadenitis when injected directly into the donor animal's remaining inferior lacrimal gland (LG) or subcutaneously at a site remote from the LG. The purpose of the present study was to determine the ability of intravenously (IV) injected autologous stimulated lymphocytes to home to the LG and salivary gland (SG) and induce disease.

Methods: One inferior LG was surgically excised from each rabbit.

Changes of chloride channels in the lacrimal glands of a rabbit model of Sjögren syndrome.

Purpose: To test the hypothesis that expressions of Na-K-2Cl cotransporter-1 (NKCC1), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride channel 2 γ subunit (ClC2γ) in the lacrimal glands (LGs) of rabbits with induced autoimmune dacryoadenitis (IAD) are changed.

Methods: LGs were obtained from adult female rabbits with IAD and age-matched female control rabbits. LGs were processed for laser capture microdissection, real-time reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence.

Changes of aquaporins in the lacrimal glands of a rabbit model of Sjögren's syndrome.

Aims: To test the hypothesis that the expression of aquaporins (AQPs) 4 and 5 is altered in the lacrimal glands (LG) of rabbits with induced autoimmune dacryoadenitis (IAD).

Materials And Methods: LGs were obtained from adult female rabbits with IAD, and age-matched female control rabbits. LGs were processed for laser capture microdissection (LCM), real time RT-PCR, Western blot, and immunofluorescence for the detection and quantification of protein and mRNAs of AQP4 and AQP5 in whole LGs, and purified acinar cells and duct cells from specific duct segments.

Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

Purpose: To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease.

Methods: Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS).

Adeno-associated virus-mediated IL-10 gene transfer suppresses lacrimal gland immunopathology in a rabbit model of autoimmune dacryoadenitis.

Purpose: To evaluate the effect of adeno-associated virus (AAV) vector-mediated viral (v)IL-10 gene expression on lacrimal gland (LG) immunopathology and ocular surface disease in a rabbit model of induced autoimmune dacryoadenitis (ID).

Methods: Autologous peripheral blood lymphocytes, activated in a mixed-cell reaction when cocultured with purified rabbit lacrimal epithelial cells, induce a Sjögren's-like autoimmune dacryoadenitis when injected directly back into the donor animal's inferior LG. Four weeks after disease induction, AAV vector expressing the vIL-10 gene under control of a tetracycline-inducible promoter was injected into the inferior LG of the treatment group (ID/Rx), and doxycycline was fed orally to induce transgene expression.

Long-term topical cyclosporine treatment improves tear production and reduces keratoconjunctivitis in rabbits with induced autoimmune dacryoadenitis.

Purpose: To use a rabbit model of induced autoimmune dacryoadenitis to evaluate the efficacy of topical ophthalmic cyclosporine A (CsA).

Methods: Autoimmune dacryoadenitis was induced by injecting autologous peripheral blood lymphocytes, which had been activated in a mixed cell reaction with acinar cells isolated from one inferior lacrimal gland (LG), back into the donor animal's remaining inferior LG. Schirmer's test, tear breakup time, and rose Bengal staining were assessed.

Microporous poly(L-lactic acid) membranes fabricated by polyethylene glycol solvent-cast/particulate leaching technique.

With the eventual goal of developing a tissue-engineered tear secretory system, we found that primary lacrimal gland acinar cells grown on solid poly(L-lactic acid) (PLLA) supports expressed the best histiotypic morphology. However, to be able to perform vectorial transport functions, epithelia must be supported by a permeable substratum. In the present study, we describe the use of a solvent-cast/particulate leaching technique to fabricate microporous PLLA membranes (mpPLLAm) from PLLA/polyethylene glycol blends.

Transduced viral IL-10 is exocytosed from lacrimal acinar secretory vesicles in a myosin-dependent manner in response to carbachol.

The purpose of this study was to determine the intracellular trafficking and release pathways for the therapeutic protein, viral IL-10 (vIL-10), from transduced acinar epithelial cells from rabbit lacrimal gland. Primary cultured rabbit lacrimal gland acinar cells (LGACs) were transduced with adenovirus serotype 5 containing viral interleukin-10 (AdvIL-10). The distribution of vIL-10 was assessed by confocal fluorescence microscopy.

Ocular surface reconstruction: recent advances and future outlook.

Purpose Of Review: Ocular surface disorder underlies a diverse group of prevalent diseases in the United States, caused by biological aging, autoimmune conditions, trauma, or iatrogenic factors. Left untreated, these conditions can progress to vision loss or destruction of the globe itself. This review discusses the most recent and relevant clinical and experimental advances in the treatment options for ocular surface disorders.

Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds.

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na(+),K(+)-ATPase and the tight junction-associated protein occludin.

Identification of Notch-1 expression in the limbal basal epithelium.

Purpose: To determine whether Notch-1, a ligand-activated transmembrane receptor known to maintain cells in an undifferentiated state, primarily progenitor cells in other systems, could be used as a stem cell marker in human limbal epithelium.

Methods: Human corneoscleral tissues obtained from the Doheny Eye & Tissue Transplant Bank were prepared for cross section and whole mount analysis. Tissue for whole mount was incubated in dispase; the epithelial sheet was removed and fixed in 4% paraformaldehyde.

Current status of gene delivery and gene therapy in lacrimal gland using viral vectors.

Gene delivery is one of the biggest challenges in the field of gene therapy. It involves the efficient transfer of transgenes into somatic cells for therapeutic purposes. A few major drawbacks in gene delivery include inefficient gene transfer and lack of sustained transgene expression.

Tissue engineering: current and future approaches to ocular surface reconstruction.

Although cells have been cultured outside the body for many years, research has only recently begun to develop complex three-dimensional tissue constructs that will, ideally, mature into fully functional tissues and organs. Tissue engineering is an emerging field in the area of biotechnology that combines the principles and methods of life sciences with those of engineering for the purpose of regenerating, repairing, or replacing diseased tissues. In this review, we describe the recent advances and current development of tissue engineering approaches as related to the ocular surface system, which comprises the three main integrated tissue units: conjunctiva, cornea and lacrimal glands.

Tissue-engineered tear secretory system: functional lacrimal gland acinar cells cultured on matrix protein-coated substrata.

Dry eye is a general term that refers to a myriad of ophthalmic disorders resulting in the inadequate wetting of the corneal surface by the tear film. Dry eyes are typically treated by the application of artificial tears. However, patients with lacrimal insufficiencies such as Stevens-Johnson syndrome, chemical and thermal injuries, or ocular cicatricial pemphigoid have very limited options because of the short duration and action of lubricating agents.

Identification of estrogen receptor beta expression in Chinese hamster ovary (CHO) cells and comparison of estrogen-responsive gene transcription in cells adapted to serum-free media.

Most cultured cell lines require addition of serum to the medium to maintain their proliferative capacity. For studies examining the cellular effects of estrogens serum is charcoal-stripped to remove steroids. Nonetheless, addition of the selective estrogen receptor modulator (SERM) 4-hydroxytamoxifen (4-OHT) inhibits the basal transcriptional activity of estrogen receptors alpha or beta (ERalpha or ERbeta) in transfected cells.