Masatoshi Haruta

Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity

We previously identified a stromal cell-derived inducing activity (SDIA), which induces differentiation of neural cells, including midbrain tyrosine hydroxylase-positive (TH+) dopaminergic neurons, from mouse embryonic stem cells. We report here that SDIA induces efficient neural differentiation also in primate embryonic stem cells. Induced neurons contain TH+ neurons at a frequency of 35% and produce a significant amount of dopamine. Interestingly, differentiation of TH+ neurons from undifferentiated embryonic cells occurs much faster in vitro (10 days) than it does in the embryo (≈5 weeks). In addition, 8% of the colonies contain large patches of Pax6+-pigmented epithelium of the retina. The SDIA method provides an unlimited source of primate cells for the study of pathogenesis, drug development, and transplantation in degenerative diseases such as Parkinsons disease and retinitis pigmentosa.

Induction of photoreceptor-specific phenotypes in adult mammalian iris tissue

We show that iris tissue in the adult rat eye, which is embryonically related to the neural retina, can generate cells expressing differentiated neuronal antigens. In addition, the Crx gene transfer induced the specific antigens for rod photoreceptors in the iris-derived cells, which was not seen in the adult hippocampus-derived neural stem cells. Our findings demonstrate a remarkable plasticity of adult iris tissue with potential clinical applications, as autologous iris tissue can be feasibly obtained with peripheral iridectomy.

Neuronal differentiation of adult rat hippocampus-derived neural stem cells transplanted into embryonic rat explanted retinas with retinoic acid pretreatment.

The purpose of this study was to evaluate the effects of the retinal environment and retinoic acid (RA) pretreatment on the differentiation of transplanted adult rat hippocampus-derived neural stem cells (AHSCs). AHSCs were transplanted into embryonic (E18) or neonatal (P6) rat retinal explants and the mixture was cultured for 2 weeks. Other AHSCs were stimulated by 0.5 microM all-trans RA for 6 days before transplantation. Immunofluorescent double staining showed that a larger number of AHSCs became beta-tubulin III-positive neurons in the E18 than in P6 retinas. In addition, many AHSCs became MAP2ab-positive and MAP5-positive neurons following RA pretreatment and transplantation. Only a few AHSCs became HPC-1-, calbindin-, PKC- or rhodopsin-positive cells under these conditions. We conclude that the microenvironment supplied by embryonic retinas is conductive to neuronal differentiation in general. RA stimulation before transplantation was also effective in stimulating differentiation.

Depleting Rac1 in mouse rod photoreceptors protects them from photo-oxidative stress without affecting their structure or function

In nonphagocytic cells, Rac1 is a component of NADPH oxidase that produces reactive oxygen species [Ushio-Fukai M (2006) Sci STKE 2006:re8]. Rac1 is expressed abundantly in mammalian retinal photoreceptors, where it is activated in response to light stimuli [Balasubramanian N, Slepak VZ (2003) Curr Biol 13:1306–1310]. We used Cre-LoxP conditional gene targeting to knock down Rac1 expression in mouse rod photoreceptors and found protection against light-induced photoreceptor death compared with WT litter-mates. We also found a similar protective effect on rods using apocynin, which inhibits NADPH oxidase activity. These results implicate both neuronal Rac1 and NADPH oxidase in cell death in this model of CNS degeneration. Studies in which dominant-mutants of Rac1 were expressed in transgenic Drosophila species demonstrated that Rac1 is a key regulator of photoreceptor morphogenesis and polarity [Chang HY, Ready DF (2000) Science 290:1978–1980]. However, we found that diminished Rac1 expression in mouse rods had no effect on retinal structure or function examined by light microscopy, electron microscopy, rhodopsin measurement, electroretinogram activity, and visual acuity, indicating rod outer segment morphogenesis proceeded normally in Rac1 conditional knockout mice. The lack of structural or functional effect of Rac1 depletion on photoreceptors, but protection under conditions of stress, indicate that the Rac1 pathway warrants exploration as a target for therapy in retinal neurodegenerative diseases.

Different characteristics of rat retinal progenitor cells from different culture periods

Embryonic retina is one of the possible cell sources that will repair degenerated retina such as retinitis pigmentosa. Retinal progenitor cells isolated from embryonic rats could be cultured and expanded in serum free medium with both epidermal growth factor and basic fibroblast growth factor. We analyzed the properties of two different retinal progenitor cells in terms of culture periods. Retinal progenitor cells from embryonic retina could be expanded keeping immature cell properties and had the ability to migrate into degenerated adult retina from subretinal space after transplantation. They differentiated into neurons and glias, even into photoreceptor cells both in vitro and in vivo. However, they appeared to lose their tissue specificity after a long-term culture.

Upregulated expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in differentiated neural stem cells

Adult rat hippocampus-derived neural stem cells are incorporated into neural tissues, and differentiate to neuronal and glial cells. However, the cell surface protein molecules are, to date, undefined. RT-PCR, immunoblotting and immunocytochemistry showed the increased expression of N-syndecan, a transmembrane heparan sulfate proteoglycan, in the neural stem cells after the differentiation induced by retinoic acid. Our data indicate that N-syndecan may be involved in the differentiation of neural stem cells.

Vitrectomy for optic disc pit-associated maculopathy with or without preoperative posterior vitreous detachment

Background The purpose of this study was to evaluate the efficacy of pars plana vitrectomy for the treatment of optic disc pit-associated maculopathy with or without preoperative posterior vitreous detachment. Methods We reviewed the clinical records of four consecutive patients who underwent pars plana vitrectomy in one eye for the treatment of optic disc pit-associated maculopathy, with an emphasis on the preoperative condition of the posterior hyaloid membrane. Results Two of four eyes were confirmed to have an attached posterior hyaloid membrane, which was subsequently removed during surgery. Following vitrectomy, these two eyes experienced an improvement in visual acuity with complete retinal attachment of the macula. However, the other two eyes, which already had a posterior vitreous detachment at the time of surgery, showed a decrease in visual acuity with persistent maculopathy postoperatively. Conclusion Pars plana vitrectomy for optic disc pit-associated maculopathy was beneficial for improving visual acuity in two eyes without preoperative posterior vitreous detachment but not in two eyes with preoperative posterior vitreous detachment. Our study suggests that preoperative assessment of a posterior hyaloid membrane is clinically important in predicting the surgical outcome of optic disc pit-associated maculopathy.

Sympathetic ophthalmia after 23-gauge transconjunctival sutureless vitrectomy

Purpose We report a case of a sympathetic ophthalmia that occurred after 23-gauge transconjunctival sutureless vitrectomy for a retinal detachment. Case report A 41-year-old Japanese woman underwent combined phacoemulsification with intraocular lens implantation and 23-gauge transconjunctival sutureless vitrectomy for a rhegmatogenous retinal detachment in the right eye. Endolaser photocoagulation and silicone oil tamponade were used to manage inferior retinal holes. Four weeks after the surgery, she returned with a 5-day history of reduced vision and metamorphopsia in her left eye. Slit-lamp examination showed a shallow anterior chamber in the right eye and moderate anterior uveitis bilaterally. Silicone oil bubbles and pigment dispersion were observed in the subconjunctival space adjacent to the right eye’s superonasal sclerotomy site. Fundus examination showed multifocal serous retinal detachments in both eyes. A diagnosis of sympathetic ophthalmia was made and the patient was treated with intensive topical and systemic steroids. The subretinal fluid cleared in both eyes following treatment. Twelve months after the onset of inflammation, the patient’s condition was stable on a combination of oral cyclosporine and topical steroids. Sunset glow retinal changes remain, but there has been no evidence of recurrent inflammation. Conclusion Sympathetic ophthalmia can develop after 23-gauge transconjunctival sutureless vitrectomy despite its smaller sclerotomy size. We recommend that special care should be taken to inspect for adequate closure of sclerotomy sites at the end of this operation.

Derivation and Characterization of Lentoid Bodies and Retinal Pigment Epithelial Cells From Monkey Embryonic Stem Cells In Vitro

For cell replacement therapies of retinal diseases, the most-needed cells are retinal pigment epithelial cells and photoreceptor cells; lens cells are needed for replacement of the cataract. Stromal cell-derived inducing activity induces the differentiation of mouse embryonic stem (ES) cells into neural cells, including midbrain tyrosine hydroxylase-positive dopaminergic neurons, and ocular cells; the method also works with primate ES cells. We describe the methods to induce retinal pigment epithelial and lens cells from monkey ES cells using stromal cell-derived inducing activity and show the characteristics of those cells in vitro and in vivo.

Iridociliary melanocytoma with suspected pulmonary metastasis.

Melanocytoma is a benign tumor that is usually located over the optic nerve head, but may occur rarely in the iris, ciliary body, or choroid. 1 Because this deeply pigmented tumor may grow progressively, 2 invade the surrounding ocular tissues, 3 or cause secondary glaucoma, 4 it is sometimes difficult to clinically differentiate melanocytoma from malignant melanoma prior to histopathologic diagnosis. This report concerns a case of iridociliary melanocytoma with pulmonary mass lesion that initially simulated malignant melanoma with pulmonary metastasis.