Invited Mini Review : Regeneration of the retina: toward stem cell therapy for degenerative retinal diseases

( So Hee Jeon ),( Il Hoan Oh ) 생화학분자생물학회(구 한국생화학분자생물학회) 2015 BMB Reports Vol.48 No.4

Degenerative retinal diseases affect millions of people worldwide, which can lead to the loss of vision. However, therapeutic approaches that can reverse this process are limited. Recent efforts have allowed the possibility of the stem cell-based regeneration of retinal cells and repair of injured retinal tissues. Although the direct differentiation of pluripotent stem cells into terminally differentiated photoreceptor cells comprises one approach, a series of studies revealed the intrinsic regenerative potential of the retina using endogenous retinal stem cells. Muller glial cells, ciliary pigment epithelial cells, and retinal pigment epithelial cells are candidates for such retinal stem cells that can differentiate into multiple types of retinal cells and be integrated into injured or developing retina. In this review, we explore our current understanding of the cellular identity of these candidate retinal stem cells and their therapeutic potential for cell therapy against degenerative retinal diseases. [BMB Reports 2015; 48(4): 193-199]

Protective Effects of Epigallocatechin Gallate after UV Irradiation in Cultured Human Retinal Pigment Epithelial Cells

( Seong Won Yang ),( Byung Rae Lee ),( Jae Woong Koh ) 대한안과학회 2007 Korean Journal of Ophthalmology Vol.21 No.4

Purpose: To evaluate the protective effects of Epigallocatechin gallate (EGCG) against UV irradiation in cultured human retinal pigment epithelial (RPE) cells. Methods: UV irradiation was produced by a UV lamp for 30 seconds with an irradiance of 3.3 mW/cm2. After 5 minutes and 1 hour, we administered different concentrations of EGCG (0, 5, 10, 15, 25, 50, 100 uM). The cell count was determined under a microscope using a counting chamber and the cell activity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Results: The cell count of cultured human RPE cells after UV irradiation was markedly increased in the EGCG administration group, compared with the non-administrated group. The cell activity of the cultured human RPE cells after UV irradiation was markedly increased in the EGCG administration group and was increased in a dose-dependent way as determined by the MTT assay. Conclusions: The administration of EGCG increased the cell count and the cell activity after UV irradiation in cultured human retinal pigment epithelial cells; this suggests that EGCG provided protection against UV damage in cultured human retinal pigmented epithelial cells.

포도당 농도가 망막상피세포의 반응성 산소 생성량과 세포활성에 미치는 영향

양유리,김성일,고재웅 대한안과학회 2006 대한안과학회지 Vol.47 No.7

Purpose: To investigate the effects of glucose concentrations on formation of reactive oxygen products and cellular activity in human retinal pigment epithelial cells. Methods: Human retinal pigment epithelial cells were cultured with high glucose (200 mg/100 ml, 300 mg/100 ml, 400 mg/100 ml) and normal glucose (100 mg/100 ml). The amounts of reactive oxygen products were assayed with dihydroethidium (DHE). Paraquat-induced cellular activity was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5 -diphenyl tetrazolium bromide (MTT) method. Results: Reactive oxygen products of human retinal pigment epithelial cells were increased 120%, 250% and 390% in high glucose (200 mg/100 ml, 300 mg/100 ml, 400 mg/100 ml) media compared to those of normal glucose (100 mg/100 ml) media. Paraquat-induced cell toxicity was increased by high glucose concentrations. Conclusions: High glucose increased formation of reactive oxygen products in human retinal pigment epithelial cells. These results suggest that high glucose can make human retinal pigment epithelial cells more sensitive to oxidative cellular injury.

Synthesized TGF-βs in RPE regulates cellular proliferation

Sung Chul Lee,Gong Je Seong,Soon Hyun Kim,Oh Woong Kwon 대한안과학회 1999 Korean Journal of Ophthalmology Vol.13 No.1

Retinal pigment epithelial (RPE) cells transdifferentiate in culture, a transition which is accompanied by a shift in biological activity. The present study investigates whether transforming growth factor (TGF)-β has the same effects on morphologically transformed RPE cells that it has on primary RPE cells. It also evaluates the autocrine and paracrine activities of TGF-βs synthesized by RPE cells as well as the anti-TGF-β effect of mannose-6-phosphate (M-6-P). RPE cells were subcultured at the sixth passage to induce morphological change. The effect of second passaged RPE-conditioned medium (CM) on DNA synthesis was evaluated by the incorporation of ³H-thymidine in rabbit subconjunctival fibroblasts (SCFs) and primary RPE cells. The presence of TGF-β in RPE-CM was determined using immunoblotting analysis. And the inhibitory effect of M-6-P on cell proliferation mediated by RPE-CM was also analyzed using ³H-thymidine incorporation into DNA. TGF-β1, TGF-β2, and TGF-β3 inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, but the spindle-shaped sixth passaged RPE cells were not inhibited by these growth factors. The medium conditioned by RPE cells stimulated the proliferation of SCFs and inhibited the proliferation of primary RPE cells, in a manner similar to TGF-β. When this medium was precipitated with either anti-TGF-β1, anti-TGF-β2, or anti-TGF-β3 antibodies, all three TGF-βs, with an apparent molecular size of 25 kDa, were detected. Mannose-6-phosphate significantly blocked the effect of RPE-CM on cell proliferation. These findings indicate that RPE cells produce biologically functional TGF-βs and that M-6-P can block the inhibitory effect of RPE-CM on cell proliferation.

Therapeutic Extracellular Vesicles from Tonsil-Derived Mesenchymal Stem Cells for the Treatment of Retinal Degenerative Disease

Choi Seung Woo,Seo Sooin,Hong Hye Kyoung,Yoon So Jung,Kim Minah,Moon Sunghyun,Lee Joo Yong,Lim Jaeseung,Lee Jong Bum,Woo Se Joon 한국조직공학과 재생의학회 2023 조직공학과 재생의학 Vol.20 No.6

BACKGROUND: Retinal degenerative disease (RDD), one of the most common causes of blindness, is predominantly caused by the gradual death of retinal pigment epithelial cells (RPEs) and photoreceptors due to various causes. Cell-based therapies, such as stem cell implantation, have been developed for the treatment of RDD, but potential risks, including teratogenicity and immune reactions, have hampered their clinical application. Stem cell-derived extracellular vesicles (EVs) have recently emerged as a cell-free alternative therapeutic strategy; however, additional invasiveness and low yield of the stem cell extraction process is problematic. METHODS: To overcome these limitations, we developed therapeutic EVs for the treatment of RDD which were extracted from tonsil-derived mesenchymal stem cells obtained from human tonsil tissue discarded as medical waste following tonsillectomy (T-MSC EVs). To verify the biocompatibility and cytoprotective effect of T-MSC EVs, we measured cell viability by co-culture with human RPE without or with toxic all-trans-retinal. To elucidate the cytoprotective mechanism of T-MSC EVs, we performed transcriptome sequencing using RNA extracted from RPEs. The in vivo protective effect of T-MSC EVs was evaluated using Pde6b gene knockout rats as an animal model of retinitis pigmentosa. RESULTS: T-MSC EVs showed high biocompatibility and the human pigment epithelial cells were significantly protected in the presence of T-MSC EVs from the toxic effect of all-trans-retinal. In addition, T-MSC EVs showed a dosedependent cell death-delaying effect in real-time quantification of cell death. Transcriptome sequencing analysis revealed that the efficient ability of T-MSC EVs to regulate intracellular oxidative stress may be one of the reasons explaining their excellent cytoprotective effect. Additionally, intravitreally injected T-MSC EVs had an inhibitory effect on the destruction of the outer nuclear layer in the Pde6b gene knockout rat. CONCLUSIONS: Together, the results of this study indicate the preventive and therapeutic effects of T-MSC EVs during the initiation and development of retinal degeneration, which may be a beneficial alternative for the treatment of RDD.

Sensitivity of CD95-induced apoptosis in different proliferative status of human retinal pigment epithelial cells

Jin Hee Chang,Se Woong Kang,Don Il Ham 대한안과학회 2001 Korean Journal of Ophthalmology Vol.15 No.2

It is known that CD95 (APO-1/Fas) is expressed on the cell surface, and apoptotic cell death can be induced by the CD95 ligation in the cultured, proliferating human retinal pigment epithelial (RPE) cells. However, little is known about CD95 on the non-proliferating RPE cells. In this study, human RPE cells were cultured up to 4 weeks after they reached the confluence, to simulate the non-proliferating RPE cells in situ. There was no significant difference in CD95 expression on the cell surface between the predominantly proliferating, preconfluent cells and predominantly non-proliferating, postconfluent cells in flow cytometric assays. However, unlike proliferating cells, no cellular death occurred in the predominantly non-proliferating cells after the treatment of agonistic anti-CD95 antibody with cycloheximide, pretreated with interferon-γ. Our results suggest that the CD95/CD95L system probably plays a physiologic role in vivo to remove the abnormal, proliferating RPE cells, and factors other than the surface expression of CD95 may determine the sensitivity to the CD95 signals.

녹차의 포름알데히드에 의한 인간 망막 색소 상피세포 사멸 차단 효과

박수현(Soo-Hyun Park) 한국차학회 2012 한국차학회지 Vol.18 No.4

Green tea (GT) is consumed throughout the world because of its beneficial effects on preventing the onset of many diseases in humans. The major components of GT are epigallocatechin-3-gallate (EGCG), epigallocatechin (EGC), epicatechin gallate (ECG) and epicatechin (EC). Formaldehyde (FA) is a volatile substance that is found in many environmental conditions, such as pesticides and new buildings. The eye is a very sensitive organ to a range of insults. Retinal pigment epithelial cells are one of the components of the eye, whose dysfunction is implicated in diseases, such as diabetic retinopathy and macular degeneration. This study examined the preventive effect of green tea extracts on FA-induced dysfunction of ARPE cells, a human retinal epithelial cell line. FA induced a decrease in cell viability, stimulated lactate dehydrogenase (LDH) activity and increased lipid peroxide (LPO) formation. A treatment with the GT extracts (GTE, 10 mg/ml) prevented FA-induced cell death and LDH activity in ARPE cells. In addition, the treatment of EGCG, EGC, ECG, and EC also prevented the FA-induced decrease in cell viability, increased LPO formation and decreased the GSH contents. Overall, GT prevented FA-induced cell death mainly by decreasing oxidative stress in ARPE cells. In conclusion, GTE prevents the FA-induced decrease in cell viability by decreasing the level of oxidative stress in ARPE cells.

A New Rapid and Non-radioactive Assay for Monitoring and Determining the Proliferation of Retinal Pigment Epithelial Cells

Hyeong Gon Yu,Hum Chung,Young Suk Yu,Jong Mo Seo,Jang Won Heo 대한안과학회 2003 Korean Journal of Ophthalmology Vol.17 No.1

AlamarBlue is used to induce color and fluorescence in the microenvironment of activated cells. The alamarBlue assay was studied to determine if it could be used as a method of evaluating the number of retinal pigment epithelial (RPE) cells. A series of two-fold dilutions of RPE cells were placed into 96-well culture plates. The alamarBlue was added to the culture media after attaching the cells. The absorbance and fluorescence were measured consecutively at various intervals over a period of 24 hr. Cell viability were evaluated by means of the trypan blue exclusion method and flow cytometry using a combination of propidium iodide and annexin V was done to prove the safety of alamarBlue assay to the cells. Both the absorbance and the fluorescence had a linear relationship with the number of RPE cells. Exposing the RPE cells to alamarBlue was not detrimental to the cells. In conclusion, the alamarBlue assay constitutes a one-step, extremely simple, reproducible, economical and non-toxic procedure for evaluating the number of viable RPE cells.

Effects of Ranibizumab, Bevacizumab, and Aflibercept on Senescent Retinal Pigment Epithelial Cells

Jae-Byoung Chae,Chang-Rae Rho,Jeong-Ah Shin,Jungmook Lyu,Seungbum Kang 대한안과학회 2018 Korean Journal of Ophthalmology Vol.32 No.4

Purpose: Anti-vascular endothelial growth factor (VEGF) agents have been used for the last 10 years, but their safety profile, including cytotoxicity against various ocular cells such as retinal pigment epithelial (RPE) cells, remains a serious concern. Safety studies of VEGF agents conducted to date have primarily relied on healthy RPE cells. In this study, we assessed the safety of three anti-VEGF agents, namely, ranibizumab, bevacizumab, and aflibercept, on senescent RPE cells. Methods: Senescent human induced pluripotent stem cell-derived RPE cells were generated by continuous replication and confirmed with senescence biomarkers. The viability, proliferation, protein expression, and phagocytosis of the senescent RPE cells were characterized 3 days after anti-VEGF treatment with clinical doses of ranibizumab, bevacizumab, or aflibercept. Results: Clinical doses of ranibizumab, bevacizumab, or aflibercept did not decrease the viability or alter proliferation of senescent RPE cells. In addition, the anti-VEGF agents did not induce additional senescence, impair the protein expression of zonula occludens-1 and RPE65, or reduce the phagocytosis capacity of senescent RPE cells. Conclusions: Clinical dosages of ranibizumab, bevacizumab, or aflibercept do not induce significant cytotoxicity in senescent RPE cells.

Tunicamycin-induced Endoplasmic Reticulum Stress Upregulates the Expression of Pentraxin 3 in Human Retinal Pigment Epithelial Cells

황나래,권민영,차재봉,정수월,우제문 대한안과학회 2016 Korean Journal of Ophthalmology Vol.30 No.6

Purpose: To investigate the production of long pentraxin 3 (PTX3) in response to tunicamycin-induced endoplasmicreticulum (ER) stress and its role in ER stress-associated cell death, PTX3 expression was evaluatedin the human retinal pigment epithelial cell line, ARPE-19. Methods: PTX3 production in ARPE-19 cells was analyzed in the absence or presence of tunicamycin treatmentby enzyme-linked immunosorbent assay. PTX3 protein and mRNA levels were estimated using western blotanalysis and real-time reverse transcription-polymerase chain reaction, respectively. Protein and mRNA levelsof CCAAT-enhancer-binding protein homologous protein (CHOP) and ARPE-19 cell viability were measuredin the presence of tunicamycin-induced ER stress in control or PTX3 small hairpin RNA (shRNA)-transfectedARPE-19 cells. Results: The protein and mRNA levels of PTX3 were found to be significantly increased by tunicamycin treatment. PTX3 production was significantly decreased in inositol-requiring enzyme 1α shRNA-transfected ARPE-19 cells compared to control shRNA-transfected cells. Furthermore, pretreatment with the NF-kB inhibitorabolished tunicamycin-induced PTX3 production. Decreased cell viability and prolonged protein and mRNAexpression of CHOP were observed under tunicamycin-induced ER stress in PTX3 shRNA transfected ARPE-19 cells. Conclusions: These results suggest that PTX3 production increased in the presence of tunicamycin-inducedER stress. Therefore, PTX3 could be an important protector of ER stress-induced cell death in human retinalpigment epithelial cells. Inositol-requiring enzyme 1α and the NF-kB signaling pathway may serve as potentialtargets for regulation of PTX3 expression in the retina. Therefore, their role in PTX3 expression needs to befurther investigated.