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Organoid-derived human retinal progenitor cells promote early dedifferentiation of Müller glia in Royal College of Surgeons rats

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International Journal of Ophthalmology(English edition) 2023年第4期16卷 483-498页

作者： Qiang Guo Yu-Xiao Zeng Shu-Dong Huang Ting Zou Zheng-Qin Yin Southwest Hospital/Southwest Eye Hospital Third Military Medical University(Army Medical University)Chongqing 400038China Key Lab of Visual Damage and Regeneration&Restoration of Chongqing Chongqing 400038China

AIM:To explore whether the subretinal transplantation of retinal progenitor cells from human embryonic stem cell-derived retinal organoid(h ERO-RPCs)could promote Müller glia dedifferentiation and transdifferentiation,thus improving visual function and delaying retinal degenerative ***:h ERO-RPCs were subretinally transplanted into Royal College of Surgeons(RCS)***(ERG)recording was performed at 4 and 8wk postoperation to assess retinal *** immunofluorescence,the changes in outer nuclear layer(ONL)thickness and retinal Müller glia were explored at 2,4,and 8wk *** verify the effect of h ERO-RPCs on Müller glia in vitro,we cocultured h ERO-RPCs with Müller glia with a Transwell *** coculture,Ki67 staining and quantitative polymerase chain reaction(q PCR)were performed to measure the proliferation and m RNA levels of Müller glia *** migration experiment was used to detect the effect of h ERO-RPCs on Müller glial *** between two groups were performed by the unpaired Student’s t-test,and comparisons among multiple groups were made with one-way ANOVA followed by Tukey’s multiple comparison ***:The visual function and ONL thickness of RCS rats were significantly improved by transplantation of h ERO-RPCs at 4 and 8wk *** addition to inhibiting gliosis at 4 and 8wk postoperation,h ERO-RPCs significantly increased the expression of dedifferentiation-associated transcriptional factor in Müller glia and promoted the migration at 2,4 and 8wk postoperation,but not the transdifferentiation of these cells in RCS *** vitro,using the Transwell system,we found that h ERO-RPCs promoted the proliferation and migration of primary rat Müller glia and induced their dedifferentiation at the m RNA ***:These results show that h ERO-RPCs might promote early dedifferentiation of Müller glia,which may provide novel insights into the mechanisms of stem cell

关键词： retinal degeneration retinal organoid retinal progenitor cells subretinal transplantation Muller glia dedifferentiation

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Anti-apoptotic effect of retinoic acid on retinal progenitor cells mediated by a protein kinase A-dependent mechanism

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Cell Research 2007年第2期17卷 151-162页

作者： RomanKholodenko IrinaKholodenko Viktor Sorokin AnnaTolmazova OlgaSazonova AntonBuzdin Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry 16/10 Miklukho-MaklayaMoscow 117997Russia Russian State Medical University Ostrovityanova 1Moscow 117312Russia

retinal progenitor cells (RPCs) are neural stem cells able to differentiate into any normal adult retinal cell type,except for pigment epithelial cells. Retinoic acid (RA) is a powerful growth/differentiation factor that generallycauses growth inhibition,differentiation and/or *** this study,we demonstrate that RA not only affectsmouse RPC differentiation but also improves cell survival by reducing spontaneous apoptotic rate without affectingRPC *** enhanced cell survival was accompanied by a significant upregulation of the expression ofprotein kinase A (PKA) and several protein kinase C (PKC) *** of cells grown in RA-free mediawith 8-bromoadenosine3′,5′-cyclic monophosphate,a known activator of PKA,resulted in an anti-apoptotic effectsimilar to that caused by RA;whereas the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesul-fonamide dihydrochloride led to a significant (～32%) increase in *** contrast,treatment of RPCs withany of two PKC selective inhibitors,2,2′,3,3′,4,4′-hexahydroxy-1,1′-biphenyl-6,6′-dimethanol dimethyl ether andbisindolylmaleimide Ⅺ,led to diminished apoptosis;while a PKC activator,phorbol 12-myristate 13-acetate,in-creased *** and other data suggest that the effect of RA on RPC survival is mostly due to the increasedanti-apoptotic activity elicited by PKA,which might in turn be antagonized by *** a mechanism is a newexample of tight regulation of important biological processes triggered by *** the detailed mechanismsremain to be elucidated,we provide evidence that the pro-survival effect of RA on RPCs is not mediated by changedexpression of p53 or bcl-2,and appears to be independent of β-amyloid,Fas ligand,TNF-α,ganglioside GM1 andceramide C 16-induced apoptotic pathways.

关键词： retinal progenitor cells apoptosis retinoic acid protein kinase A protein kinase C

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Effects of Low-dose Triamcinolone Acetonide on Rat retinal progenitor cells under Hypoxia Condition

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Chinese Medical Journal 2016年第13期129卷 1600-1606页

作者： Yao Xing Li-Jun Cui Qian-Yan Kang Department of Ophthalmology First Affiliated Hospital of Medical School of Xi'an Jiaotong University Xi'an Shaanxi 710061 China

Background： retinal degenerative diseases are the leading causes of blindness in developed world. retinal progenitor cells （RPCs） play a key role in retina restoration. Triamcinolone acetonide （TA） is widely used for the treatment of retinal degenerative diseases. In this study, we investigated the role of TA on RPCs in hypoxia condition. Methods： RPCs were primary cultured and identified by immunofluorescence staining. cells were cultured under normoxia, hypoxia 6 h, and hypoxia 6 h with TA treatment conditions, For the TA treatment groups, after being cultured under hypoxia condition for 6 h, RPCs were treated with different concentrations of TA for 48-72 h. Cell viability was measured by cell counting kit-8 （CCK-8） assay. Cell cycle was detected by flow cytometry. Western blotting was employed to examine the expression ofcyclin D 1, Akt, p-Akt, nuclear factor （N F）-KB p65, and caspase-3. Results： CCK-8 assays indicated that the viability of RPCs treated with 0.01 mg/ml TA in hypoxia group was improved after 48 h, comparing with control group （P 〈 0.05）. After 72 h, the cell viability was enhanced in both 0.0l mg/ml and 0.02 mg/ml TA groups compared with control group （all P 〈 0.05）. Flow cytometry revealed that there were more cells in S-phase in hypoxia 6 h group than in normoxia control group （P 〈 0.05）. RPCs in S and G2/M phases decreased in groups given TA, comparing with other groups （all P 〈 0.05）. There was no significant difference in the total Akt protein expression among different groups, whereas upregulation of p-Akt and NF-KB p65 protein expression and downregulation of caspase-3 and cyclin DI protein expression were observed in 0.01 mg/ml TA group, comparing with hypoxia 6 h group and control group （all P 〈 0.05）. Conclusion： Low-dose TA has anti-apoptosis effect on RPCs while it has no stimulatory effect on cell proliferation.

关键词： Apoptosis Cell Cycle Hypoxia retinal progenitor cells Triamcinolone Acetonide

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Wharton's jelly mesenchymal stem cells differentiate into retinal progenitor cells

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Neural Regeneration Research 2013年第19期8卷 1783-1792页

作者： Ying Hu Jun Liang Hongping Cui Xinmei Wang Hua Rong Bin Shao Hao Cui Department of Ophthalmology First Affiliated Hospital Harbin Medical University Department of Ophthalmology Shanghai East Hospital Affiliated to Tongji University Department of Histology and Embryology Mudanjiang Medical University Department of Ophthalmology Fourth Affiliated Hospital Harbin Medical University Department of Head-Neck and Breast Tumor Mudanjiang Tumor Hospital

Human Wharton＇s jelly mesenchymal stem cells were isolated from fetal umbilical cord. cells were cultured in serumfree neural stem cellconditioned medium or neural stem cellconditioned medium supplemented with Dkk1, a Wnt/13 catenin pathway antagonist, and LeftyA, a Nodal signaling pathway antagonist to induce differentiation into retinal progenitor cells. Inverted microscopy showed that after induction, the spindleshaped or fibroblastlike Wharton＇s jelly mesenchymal stem cells changed into bulbous cells with numerous processes. Immunofluorescent cytochemical stain ing and reversetranscription PCR showed positive expression of retinal progenitor cell markers, Pax6 and Rx, as well as weakly downregulated nestin expression. These results demonstrate that Wharton＇s jelly mesenchymal stem cells are capable of differentiating into retinal progenitor cells in vitro.

关键词： neural regeneration stem cells Wharton's jelly mesenchymal stem cells microenvironment induc-tion reagent induction retinal progenitor cells nerve cells retinal disease grants-supported paper neuroregeneration

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Influence of hypoxia on retinal progenitor and ganglion cells in human induced pluripotent stem cell-derived retinal organoids

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国际眼科杂志:英文版 2023年第10期16卷 1574-1581页

作者： Jin-Lin Du Li-Xiong Gao Tao Wang Zi Ye Hong-Yu Li Wen Li Quan Zeng Jia-Fei Xi Wen Yue Zhao-Hui Li Medical School of Chinese PLA Beijing 100853China Senior Department of Ophthalmology the Third Medical Center of Chinese PLA General HospitalBeijing 100039China Departement of Ophthalmology the 6^(th)Medical Center of PLA General HospitalBeijing 100048China Stem Cell and Regenerative Medicine Lab Beijing Institute of Radiation MedicineBeijing 100850China

AIM:To observe the effect of low oxygen concentration on the neural retina in human induced pluripotent stem cell(hiPSC)-derived retinal organoids(ROs).METHODS:The hiPSC and a three-dimensional culture method were used for the *** embryoid bodies(EBs)were randomly and equally divided into hypoxic and normoxic *** of the EBs were taken on days 38,45,and 52,and the corresponding volume of EBs was ***,samples were collected at these three timepoints,followed by fixation,sectioning,and ***:The proportion of Ki67-positive proliferating cells increased steadily on day 38;this proliferationpromoting effect tended to increase tissue density rather than tissue *** days 45 and 52,the two groups had relatively similar ratios of Ki67-positive *** immunofluorescence analysis showed that the ratio of SOX2-positive cells significantly increased within the neural retina on day 52(Pcells significantly decreased following hypoxia treatment at all three timepoints(P0.05).Moreover,the proportion of PAX6-/TUJ1+cells within the neural retinas increased considerably(Pretinal progenitor cell pool in hiPSC-derived ROs.

关键词： hypoxia retinal organoid retinal progenitor cells retinal ganglion cells

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PROPERTIES OF PROLIFERATION AND DIFFERENTIATION OF NEONATAL RAT retinal progenitor cells IN VITRO

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Journal of Pharmaceutical Analysis 2006年第2期18卷 174-178页

作者：康前雁刘勇赵建军邱芬陈新林田玉梅胡明 Department of Ophthalmology of the First Affiliated Hospital Xi'an Jiaotong University School of Medicine Xi'an 710061 China Research Center for Neuroscience Department of Human Anatomy Histology and Embryology Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education Xi'an Jiaotong University School of Medicine Xi'an 710061 China

Objective To investigate the properties of proliferation and differentiation of neonatal rat retinal progenitor cells (RPCs) in vitro. Methods RPCs were isolated from neonatal SD rats neural retina and cultured in DMEM/F12+N2 with EGF and bFGF (suspension medium )or 10%FBS without EGF and bFGF (differentiation medium). The cells grew as suspended spheres or adherent monolayers, depending on different culture conditions. The neural stem cells or retinal progenitors, neurons, astrocytes, retinal ganglion cells, rod photoreceptors and the proliferating cells were evaluated with immunofluorescence analysis by Nestin or Pax6, Map2, GFAP, Thy-1, Rhodopsin and BrdU antibodies respectively. Results RPCs could propagate and differentiate in suspension or differentiation medium and express the markers of Nestin (92.86%) or Pax6 (86.75%), Map2 (38.54%), GFAP (20.93%), Thy-1 (27.66%) and Rhodopsin(13.33%)in suspension medium; however, Nestin (60.27%), Pax6 (52%), Map2 (34.94%), GFAP (38.17%), Thy-1(30.84%) and Rhodopsin (34.67%) in differentiation medium. 96.4% of the population in the neurospheres was BrdU-positive cells. The cells could spontaneously adherent forming some subspheres and retinal specific cell types. Conclusion Neonatal rat RPCs possess the high degree of proliferation and can differentiate into neurons, astrocytes, retinal ganglion cells and rod photoreceptors in vitro. There are different proportions for RPCs to differentiate into specific cell types.

关键词： retinal progenitor cells proliferation differentiation cell culture

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Postnatal development of rat retina:a continuous observation and comparison between the organotypic retinal explant model and in vivo development

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Neural Regeneration Research 2025年第3期20卷 900-912页

作者： Baoqi Hu Rui Wang Hanyue Zhang Xiou Wang Sijia Zhou Bo Ma Yan Luan Xin Wang Xinlin Chen Zhichao Zhang Qianyan Kang Department of Ophthalmology The First Affiliated Hospital of Xi’an Jiaotong UniversityXi’anShaanxi ProvinceChina Department of Ophthalmology The First Affiliated Hospital of Northwest UniversityXi’anShaanxi ProvinceChina Institute of Neurobiology Xi’an Jiaotong University Health Science CenterXi’anShaanxi ProvinceChina

The organotypic retinal explant culture has been established for more than a decade and offers a range of unique advantages compared with in vivo experiments and cell ***,the lack of systematic and continuous comparison between in vivo retinal development and the organotypic retinal explant culture makes this model controversial in postnatal retinal development ***,we aimed to verify the feasibility of using this model for postnatal retinal development studies by comparing it with the in vivo *** this study,we showed that postnatal retinal explants undergo normal development,and exhibit a consistent structure and timeline with retinas in ***,we used SOX2 and PAX6 immunostaining to identify retinal progenitor *** then examined cell proliferation and migration by immunostaining with Ki-67 and doublecortin,***-67-and doublecortin-positive cells decreased in both in vivo and explants during postnatal retinogenesis,and exhibited a high degree of similarity in abundance and distribution between ***,we used Ceh-10 homeodomain-containing homolog,glutamate-ammonia ligase(glutamine synthetase),neuronal nuclei,and ionized calcium-binding adapter molecule 1 immunostaining to examine the emergence of bipolar cells,Müller glia,mature neurons,and microglia,*** timing and spatial patterns of the emergence of these cell types were remarkably consistent between in vivo and explant *** study showed that the organotypic retinal explant culture model had a high degree of consistency with the progression of in vivo early postnatal retina *** findings confirm the accuracy and credibility of this model and support its use for long-term,systematic,and continuous observation.

关键词： bipolar cells differentiation in vivo microglia Müller glia organotypic retinal explant culture postnatal retina development proliferation retinal progenitor cells

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