Saiko Uchida

Neurotrophic function of conditioned medium from human amniotic epithelial cells.

Human amniotic epithelial cells (HAEC) may have pluripotent function because they are formed from the epiblast cells at the 8th day of fertilization. Previously, we reported that HAEC have the capacity to synthesize and release acetylcholine and catecholamine associated with the binding sites of catecholamine receptors. We show the neurotrophic function of a conditioned medium from HAEC using cultured cortical neurons of E18 rats. Extensive analyses with various techniques demonstrated that HAEC and immortalized HAEC synthesize and release brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT‐3) and nerve growth factor (NGF). Other neurotrophic factors were not detected in a cultured medium of HAEC by enzyme immunoassay. Various neurotrophic factors or growth factors did not show neurotrophic effects on E18 rat neuron except for EGF. Because EGF was not detected in the conditioned medium of HAEC, these data indicate an unidentified neurotrophic factor presently that is synthesized and released from HAEC. The amniotic membrane may have a significant role in supplying neurotrophic factors to the amniotic fluid as well as neurotransmitters, suggesting an important function to the early stages of neural development in the embryo. J. Neurosci. Res. 62:585–590, 2000.

Human amnion mesenchyme cells express phenotypes of neuroglial progenitor cells

Previous studies from our laboratory showed that human amnion epithelial cells (AECs) have multiple functions, such as synthesis and release of catecholamines, acetylcholine, neurotrophic factors, activin, and noggin. In this study, we investigated the identity of neural progenitor cells in human amnion mesenchyme cells (AMCs), which lie immediately adjacent to the AECs. Cryostat sections revealed that vimentin expression was detected in the AMCs and CK19 in AECs. Vimentin‐positive cells made up 97.5% of total cells tested in cultured AMCs. Interestingly, 3.6% of total AMCs expressed the phenotype CK19+/vimentin+, indicating coexpression of epithelial and mesenchyme cell markers. In culturing with bromodeoxyuridine (BrdU) for 24 hr, 66–82% of cells were found to be BrdU positive, suggesting that they have proliferating potency. By using RT‐PCR, AMCs express mRNA of nestin and Musashi1. With a neural cell differentiating protocol, cell bodies extended long bipolar or complex multipolar processes. Nestin (87.7% of total cells tested) and Musashi1 (93.1%) were expressed in undifferentiated cells, and their positively stained cells increased in number slightly after induction. Undifferentiated cells were stained by anti‐Tuj1 and NF‐M, and their positively stained cells increased significantly in number after induction, to 72.8% and 46.0%, respectively. Meanwhile, glial fibrillary acidic protein‐positive cells increased from 25.4% to 43.2% after induction. These studies demonstrate that AMCs have phenotypes of neuroglial progenitor cells and can be differentiated into neuroglial phenotypes by optimal differentiation protocol. Eventually, AMC‐derived stem cells may be a favorable cell vehicle in regenerative medicine.

Corneal stromal and endothelial cell precursors

Purpose: To isolate precursors of human corneal stromal and endothelial cells in vitro and to compare the distribution of rabbit corneal endothelial cell precursors in the central and peripheral regions of the cornea. Methods: Sphere-forming cells were isolated from human corneal stromal and endothelial cells by a reaggregation-free neurosphere assay. To promote differentiation of the cells, the isolated sphere colonies were plated in wells with a medium containing fetal bovine serum. Expression of various proteins was examined in the sphere colonies and their progeny by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Corneal endothelium and Descemet membrane of rabbits were demarcated into peripheral (6.0-10.0 mm in diameter) and central (6.0 mm in diameter) regions. Precursor cells were isolated from these 2 regions by sphere-forming assay, and the number of sphere colonies and size of spheres were compared in the central and peripheral regions. Results: Human corneal stromal and endothelial cells formed sphere-shaped colonies that expressed mesenchymal and neural markers. The rate of sphere formation in the peripheral region of rabbit corneal endothelium was significantly higher than that in the central cornea. Conclusion: Adult human corneal stroma contains precursor cells that have a strong propensity to differentiate into mesenchymal fibroblasts, but these cells can also differentiate into neuronal lineage. Adult human corneal endothelium contains precursors with a propensity to differentiate into corneal endothelial cells, but these cells can also produce neuronal and mesenchymal cell proteins. The peripheral endothelium of rabbit cornea contains more precursors than the central region.

Factors secreted by human amniotic epithelial cells promote the survival of rat retinal ganglion cells.

We evaluated whether factors secreted by human amniotic epithelial cells (HAECs) have the neuroprotective effect on rat retinal ganglion cells (RGCs) purified by immunopanning. After culture in B27 complete medium containing B27 supplement, brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and forskolin, the medium was changed to: (1). medium containing N2 supplement and forskolin (N2 basal medium); (2). medium conditioned by HAECs containing N2 supplement and forskolin (HAEC-CM); and (3). N2 basal medium containing several neurotrophic factors. HAEC-CM promoted the RGC survival compared to N2 basal medium. The effect of HAEC-CM was significantly higher than that of BDNF, neurotrophin-3 and CNTF. These results suggest that HAECs may produce unknown neuroprotective factors, suggesting its potential for the treatment of RGC degenerative diseases.

Long-term follow-up of initial 5-fluorouracil trabeculectomy in primary open-angle glaucoma in Japanese patients.

PurposeTo evaluate the long-term efficacy and safety of initial trabeculectomy with subconjunctival 5-fluorouracil in Japanese patients with primary open-angle glaucoma. Subjects and MethodsClinical records of 117 eyes from 117 patients with records of up to 14 years were retrospectively analyzed using the appropriate statistical methods. ResultsThe mean follow-up period was 6.2 ± 3.6 years. Criteria for successful intraocular pressure control and success probability by Kaplan-Meier method at 12 years were intraocular pressure < 21 mm Hg without medication plus a 30% or more reduction in intraocular pressure, 50.4 ± 5.2% (SE) 38.2 ± 5.3%; and intraocular pressure < 16 mm Hg without medication plus a 30% or more reduction in intraocular pressure, 45.9 ± 5.1% (36.5 ± 5.2%). Cox multiple regression analysis showed that a younger age was associated with a higher success probability (P <0.01). Eyes with lower postoperative intraocular pressure tended to show more positive value of the MD slope (P = 0.0669), whereas 7% of successfully treated eyes showed a negative postoperative MD slope (P < 0.1). The probabilities for developing bleb leaks and bleb-related infections in eyes with a functioning bleb were 28.9 ± 12.5% and 13.0 ± 10.4% at 12 years. ConclusionTrabeculectomy with 5-fluorouracil as an initial surgery in Japanese patients with primary open-angle glaucoma was effective for long-term control of glaucoma. However, the probability of late-onset bleb-related complications was not low in eyes with a functioning bleb, stressing the importance of constant care regarding bleb status.

Synthesis and release of activin and noggin by cultured human amniotic epithelial cells.

Recent studies suggest that extra‐embryonic tissues may be essential sources of early organizing signals for the mouse embryo. In vitro studies of human amniotic epithelial cells (HAEC) have shown that the amnion can produce various biologically active substances. In this study, the synthesis and release of activin A and noggin, and the activin signaling pathway, was investigated in HAEC. Conditioned medium from cultured HAEC contained activin A which was functionally active in Xenopus laevis animal cap assays. Immunohistochemistry, western blotting and reverse transcription–polymerase chain reaction confirmed that HAEC also synthesize and release noggin. Noggin transcripts were induced by the addition of recombinant activin A, and activin A was inhibited by activin antibody except in the presence of cycloheximide (CHX). These data demonstrate that noggin mRNA expression is induced directly by activin A without new protein synthesis, indicating that noggin is a primary response gene. The results suggest that there is an activin signaling pathway in HAEC, and that the human amnion might therefore be involved in neural formation during early development.

Retinal nerve fibre layer damage after indocyanine green assisted vitrectomy

Recently, indocyanine green (ICG) has been used to stain and visualise the internal limiting membrane (ILM) during vitrectomy.1 Some case series showed that visual field defects on the nasal side can occur after the surgery through unknown cause.2,3 Here, we report a case in which nasal visual field defects occurred after ICG assisted ILM peeling for epiretinal membrane (ERM). Detailed examination revealed that the superior and inferior retinal nerve fibre is severely damaged in this case. A 60 year old woman who received ICG assisted ILM peeling for ERM in her right eye was referred to our hospital. The preoperative best corrected visual acuity (BCVA) was 20/60 in the right eye. According to the referring ophthalmologist, 25 mg of ICG (Diagnogreen; Daiichi Pharmaceuticals) was dissolved in 10 ml of distilled water, which was further diluted by a viscoelastic material (Healon; Pharmacia) to give 0.16% ICG solution. To stain ILM, ICG was injected into an air filled eye and the dye …

Erratum: Human amnion mesenchyme cells express phenotypes of neuroglial progenitor cells (Journal of Neuroscience Research (2004) 78 (208-214))

Norio Sakuragawa,* Kenichi Kakinuma, Aiko Kikuchi, Hideyuki Okano, Saiko Uchida, Isao Kamo, Mamoru Kobayashi, and Yasunobu Yokoyama Department of Regenerative Medicine, Toho University School of Medicine, Sagamihara, Kanagawa, Japan Center for Molecular Genetics and Cytogenetics, SRL, Inc., Hino Tokyo, Japan Department of Ultrastructure Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan Department of Physiology, Keio University School of Medicine, Tokyo, Japan Department of Ophthalmology, Tokyo University School of Medicine, Tokyo, Japan