Propagation of fetal human RPE cells: Preservation of original culture morphology after serial passage

Song, Mi-Kyoung; Lui, Ge Ming

doi:10.1002/jcp.1041430127

Cited by 86 publications

(73 citation statements)

References 18 publications

(14 reference statements)

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“…Cells so obtained were then transferred to 35 mm ECM (extracellular matrix)-coated culture plates containing Dulbecco's modified Eagle's medium supplemented with 15 % fetal calf serum. 1 ng/ml basic fibroblast growth factor (bFGF), and antibiotics (Song & Lui, 1990). Cells were kept at 37 'C with 10% C02-90 % air and fed every other day.…”

Section: Methodsmentioning

confidence: 99%

Whole‐cell K+ currents in fresh and cultured cells of the human and monkey retinal pigment epithelium.

Wen

Lui

Steinberg

1993

The Journal of Physiology

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SUMMARY1. Whole-cell potassium currents of freshly isolated human (adult and fetal) and monkey (adult) retinal pigment epithelial (RPE) cells, as well as cultured human and monkey RPE cells were studied using the patch-clamp technique.2. In freshly isolated adult cells of both species, two currents were observed in the voltage range from -150 to + 50 mV: an outwardly rectifying current and an inwardly rectifying current. These currents were also found in cultured cells of both species.3. The outwardly rectifying current in freshly isolated adult human and monkey cells and some cultured cells was evoked by depolarizing voltage pulses more positive that -30 mV. The current activated with a sigmoidal time course after a brief delay, and was virtually non-inactivating. The conductance associated with the current was half-maximal at -16 4 mV for fresh human cells and -13 5 mV for fresh monkey cells, but was shifted 16 0 and 17 7 mV in the positive direction in cultured human and monkey cells, respectively. The reversal potential of the current in both human and monkey cells matched the potassium equilibrium potential (EK) over a wide range of external potassium concentrations. This current was blocked by 20 mM tetraethylammonium.4. A membrane current that exhibited inward rectification was observed with hyperpolarizing voltage pulses. The zero-current potential of this current was close to EK. This current was blocked by 2 mm Ba25 and 2 mm Cs'. In cultured human and monkey cells, but not in fresh cells, this current exhibited an inactivation when voltage pulses were more negative than -120 mV. External Na+ was responsible for the inactivation, as the inactivation was removed in a Na+-free solution.5

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Section: Methodsmentioning

confidence: 99%

Whole‐cell K+ currents in fresh and cultured cells of the human and monkey retinal pigment epithelium.

Wen

Lui

Steinberg

1993

The Journal of Physiology

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“…RPE sheets were dissected carefully from the choroid at the end of the incubation and gently trimmed to form microaggregates. RPE microaggregates were then plated on BCE-ECM [28] or ECM ligand-coated cultured dishes/wells. Cells were cultured in DMEM containing 15 percent fetal bovine serum, 2 mM L-glutamine, 2.5 g/mL amphotericin B, 0.05 mg/mL gentamicin, and 1 ng/mL basic fibroblastic growth factor at 37 °C in 10 percent carbon dioxide.…”

Section: Isolation Of Human Rpe Microaggregatesmentioning

confidence: 99%

Migration and proliferation of retinal pigment epithelium on extracellular matrix ligands

Wang¹,

Patten²,

Sugino³

et al. 2006

JRRD

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Abstract-We compared aged adult and fetal retinal pigment epithelium (RPE) migration and proliferation in cell culture. Aged adult RPE resurfaced on dishes coated with human or mouse laminin, human or rat collagen I, human or mouse collagen IV, or human fibronectin. Resurfacing was best on bovine corneal endothelial cell extracellular matrix. Resurfacing was worst on mouse laminin. Fetal RPE resurfacing was robust on all surfaces studied except human and mouse laminin. In general, fetal RPE resurfacing was much greater than that seen with aged RPE on comparable surfaces. The degree of cell proliferation (labeled by Ki-67) was much greater in fetal than adult RPE cells.

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“…Primary cultures of normal human RPE cells from a 35 year old male donor (apoE phenotype E3/E3) were prepared as described (34,35). Cells from passages 5-10 were used for the experiments described below.…”

Section: Cell Culturementioning

confidence: 99%

Regulated expression of apolipoprotein E by human retinal pigment epithelial cells

Ishida

Bailey

Duncan

et al. 2004

Journal of Lipid Research

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In early age-related macular degeneration (AMD), lipid-containing deposits (drusen) accumulate in Bruch's membrane underlying the retinal pigment epithelium (RPE).Recent studies indicate that apolipoprotein E (apoE) may play a role in lipid trafficking in AMD. Compared with the apoE3 allele, the apoE4 and apoE2 alleles are associated with decreased and increased risk for AMD, respectively; drusen contain high levels of apoE, and apoE null mice develop lipid deposits in Bruch's membrane similar to those observed in AMD. Primary cultures of human RPE cells expressing the apoE3 allele were grown on Transwell ® culture plates. Western blotting, ELISA assay, and mass spectrometry confirmed that apoE3 was secreted into the apical and basal chambers and that secretion was upregulated by thyroid hormone, 9-cis -retinoic acid, and 22( R )-hydroxycholesterol. In addition, basally secreted apoE associated with exogenously added HDL. These results indicate that apoE secretion can be regulated by specific hormones and that apoE associates with HDL. The findings are consistent with a role for apoE in lipid trafficking through Bruch's membrane and may be relevant to AMD. Age-related macular degeneration (AMD) is the leading cause of severe visual loss in the developed world (1, 2). In the early stages of the disease, before visual loss occurs from choroidal neovascularization, there is progressive accumulation of lipids in Bruch's membrane (3-6). Bruch's membrane lies at the critical juncture between the outer retina and its blood supply, the choriocapillaris. Lipid deposition causes reduced hydraulic conductivity and macromolecular permeability in Bruch's membrane and is thought to impair retinal metabolism (7-9). Interestingly, lipid accumulation in Bruch's membrane similar to that in AMD has been observed in apolipoprotein E (apoE) null mice (10, 11). Because of the additional association between apoE alleles and other age-related degenerations, such as Alzheimer's disease and atherosclerosis, there has been recent investigation into a potential role for apoE in AMD.Several studies of apoE polymorphism in AMD have been conducted (12)(13)(14). In contrast to Alzheimer's disease, the apoE4 allele has been associated with a reduced prevalence of AMD. The apoE2 allele is slightly increased in patients with AMD. Further supporting a role in AMD pathogenesis, apoE has been detected in drusen, the Bruch's membrane deposits that are the hallmark of AMD (13,15). Immunohistochemical studies of postmortem eyes have demonstrated apoE in the basal aspect of the retinal pigment epithelium (RPE) (15). Cultured RPE cells synthesize high levels of apoE mRNA, comparable to the levels found in brain (15).Although the role of apoE in AMD is not established, this apolipoprotein has several functions that may affect the course of this disease. ApoE has anti-angiogenic (16), anti-inflammatory (17), and anti-oxidative (18) effects. These are all considered atheroprotective attributes of apoE, but they may also be important in protecting a...

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Propagation of fetal human RPE cells: Preservation of original culture morphology after serial passage

Cited by 86 publications

References 18 publications

Whole‐cell K+ currents in fresh and cultured cells of the human and monkey retinal pigment epithelium.

Whole‐cell K+ currents in fresh and cultured cells of the human and monkey retinal pigment epithelium.

Migration and proliferation of retinal pigment epithelium on extracellular matrix ligands

Regulated expression of apolipoprotein E by human retinal pigment epithelial cells

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