Effects of leptin on proliferation and differentiation of hypoxic rat retinal progenitor cells in vitro / 南方医科大学学报

OBJECTIVE@#To investigate the the effects of leptin on the proliferation, differentiation and PTEN expression of rat retinal progenitor cells (RPCs) cultured under hypoxic condition.@*METHODS@#SD rat RPCs were cultured in normoxic conditions or exposed to hypoxia in the presence of 0, 0.3, 1.0, 3.0, 10, and 30 nmol/L leptin for 12, 48 and 72 h, and the cell viability was assessed using cell counting kit 8 (CCK 8) assay. The RPCs in primary culture were divided into control group, hypoxia group, and hypoxia+leptin group, and after 48 h of culture, the cell medium was replaced with differentiation medium and the cells were further cultured for 6 days. Immunofluorescence staining was employed to detect the cells positive for β-tubulin III and GFAP, and Western blotting was used to examine the expression of PTEN at 48 h of cell culture.@*RESULTS@#The first generation of RPCs showed suspended growth in the medium with abundant and bright cellular plasma and formed mulberry like cell spheres after 2 days of culture. Treatment with low-dose leptin (below 3.0 nmol/L) for 48 h obviously improved the viability of RPCs cultured in hypoxia, while at high concentrations (above 10 nmol/L), leptin significantly suppressed the cell viability (P < 0.05). The cells treated with 3.0 nmol/L leptin for 48 h showed the highest viability (P < 0.05). After treatment with 3.0 nmol/L leptin for 48 h, the cells with hypoxic exposure showed similar GFAP and β-tubulin Ⅲ positivity with the control cells (P>0.05), but exhibited an obvious down-regulation of PTEN protein expression compared with the control cells (P < 0.05).@*CONCLUSION@#In rat RPCs with hypoxic exposure, treatment with low dose leptin can promote the cell proliferation and suppress cellular PTEN protein expression without causing significant effects on cell differentiation.

Clinical studies using stem cells for treatment of retinal diseases: state of the art / Estudos clínicos com uso de células-tronco para tratamento de doenças da retina: estado da arte

ABSTRACT Degenerative retinal diseases such as retinitis pigmentosa, Stargardt's macular dystrophy, and age-related macular degeneration are characterized by irreversible loss of vision due to direct or indirect photoreceptor damage. No effective treatments exist, but stem cell studies have shown promising results. Our aim with this review was to describe the types of stem cells that are under study, their effects, and the main clinical trials involving them.

RESUMO As doenças degenerativas da retina, como retinose pigmentar, distrofia macular de Stargardt e degeneração macular relaciona à idade, são caracterizadas por perda irre versível da visão devido a danos diretos ou indiretos aos fotorreceptores. Não existem tratamentos eficazes, porém os estudos com células-tronco mostraram resultados promissores. Nosso objetivo com esta revisão foi descrever os tipos de células-tronco em estudo, seus efeitos e os principais ensaios clínicos que as envolvem.

Hypoxia-induced changes of retinal progenitor cells migration by chemotaxis factor 4 / 中华实验眼科杂志

Background In vitro study showed that chemotaxis consist of chemotaxis factor 4(CXCR4)and stromal cells derived factor-1(SDF-1)and may play a role in the orientation and migration of retinal progenitor cells (RPCs)toward lesion.Overexpression of CXCR4 in RPCs can enhance the chemotaxis activity.Objective This work was to explore the feasibility and underlying mechanism of up-regulation of CXCR4 on RPCs induced by hypoxia.Methods RPCs were retained in an incubator with normal O2volume(16％)or hypoxia condition(10％ O2)for 12 hours and 24 hours respectively.Flow cytometer cell analysis screening(FACS)was conduced to measure the proportion of CXCR4-expressing cells,and CXCR4,HIF-1 mRNA were analyzed by reverse transcription-polymerse chain reaction(RT-PCR).The chemotical effect of 30 mg/L SDF-1 to RPCs cultured under the hypoxia condition was assessed using Boyden chamber.Results The expression level of CXCR4(CXCR4 mRNA/β-actin mRNA)inRPCs cultured by 10％ O2 for 12 and 24 hours were 0.28+0.07and 0.48+0.17 and increased by 1.75 and 3.00 fold more than that of 16％ O2 culture group(0.16+0.02)(P＜0.01).The expression level of HIF-1 mRNA(HIF-1 mRNA/β-actin mRNA)in RPCs cultured by 10％ O2 for 12 and 24 hours were 0.18 ±0.07and 0.38 ±0.13 and increased by 3.00 and 6.30 fold more than that of 16％ O2 culture group(0.06±0.01)(P＜0.01).The chemotical effect of 30 μg/L SDF-1 to RPCs increased from 13.00％ in 16％ O2 culture group to 36.00％ and 46.00％ in the cells cultured by 10％ O2for 12 and 24 hours.FACS revealed that the proportion of CXCR4+ cells in hypoxia-exposure for 12 and 24 hours were 26.90％ and 46.10％,respectively,but that in 16％ O2 culture group was 9.10％,showing a statistically significant difference(P ＜ 0.01).Conclusions RPCs induced by hypoxia can enhance the expression of CXCR4 in RPE cells and the chemotaxia to SDF-1.The overexpression of H1F-1 in RPCs may be involved in the up-regulation of CXCR4 expression.

Trasplante de tejido retiniano y de células progenitoras retinianas: una promesa terapéutica para pacientes con enfermedad de la retina / Retinal tissue transplantation and retinal progenitor cells: a therapeutic promise for patients with retinal disease

La retinopatía diabética, la degeneración macular relacionada con la edad y la retinitis pigmentosason las enfermedades retinianas más frecuentes en todo el mundo. A pesar de no contar consuficientes estudios que demuestren resultados funcionales positivos en cuanto a recuperar lafunción visual, el uso de células madre y células progenitoras retinianas y el trasplante de retinafetal parecen bastante promisorios. Hasta el momento no se han podido obtener resultadospositivos sobre la funcionalidad de las células trasplantadas, pero sí se ha demostrado que elprocedimiento para transferir el tejido retiniano es seguro y confiable. Aún no se ha intentadoen seres humanos el trasplante de células progenitoras retinianas, pero dicho trasplante ha dadoresultados satisfactorios en modelos múridos. Los estudios con células progenitoras retinianashan logrado demostrar en modelos múridos que se activan y expresan los fotorreceptores. Existenalgunas barreras de disponibilidad para el uso de células progenitoras retinianas, que se debensuperar con el fin de adelantar estudios que permitan aumentar las posibilidades de integracióny diferenciación de dichas células hacia fotorreceptores.

Retinal tissue transplantation and retinal progenitor cells: A therapeutic promise for patients with retinal diseaseWorldwide, diabetic retinopathy, age-related macular degeneration, and retinitis pigmentosahave the highest incidence rate among retinal diseases. Despite the lack of enough trialsdemonstrating positive functional results on eyesight recovery, the use of stem cells, retinalprogenitor cells, and fetal retinal tissue transplantation seem very promising. So far positiveresults on the functionality of the transplanted cells have not been obtained. However, the safetyand reliability of the procedure to transfer retinal tissue have been demonstrated. Transplantationof retinal progenitor cells has not been tried on human beings, but there have been satisfactory results with it in murine models. Trials with retinalprogenitor cells have demonstrated activation andexpression of photoreceptors in murine models. Somebarriers of availability exist for the use of retinalprogenitor cells that must be overcome in order tocarry out studies to increase the possibility of theirintegration and differentiation towards photoreceptors.

PROPERTIES OF PROLIFERATION AND DIFFERENTIATION OF NEONATAL RAT RETINAL PROGENITOR CELLS IN VITRO / 药物分析学报

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.

Properties of proliferation and differentiation of neonatal rat retinal progenitor cells in vitro / 西安交通大学学报·英文版

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.

CULTIVATION OF RETINAL PROGENITOR CELLS OF DIFFERENT AGES / 解剖学报

Objective To study the cultivation of retinal progenitor cells of different ages. Methods Retinal progenitor cells of E14 and E18 SD rats were isolated,cultivated in suspension in modified DMEM/F12 serum-free medium and then differentiation was induced in vitro.Cells were observed under phase-contrast microscopy daily and identified by immunocytochemistry,scanning electron microscopy and transmission electron microscopy. Results Cultivated in DMEM/F12 serum-free medium,retinal progenitor cells formed cell spheres.After being plated,isolated cells migrated outwards and differentiated.Scanning electron microscopy demonstrated the morphology of the cell spheres and differentiated cells.Transmission electron microscopy demonstrated that there were stem cell-like cells in cell spheres and neuron-and glia-like cells after the plating.Immunocytochemistry demonstrated that most cells in spheres expressed neural stem cell marker nestin and cell division marker BrdU.After the plating,retinal progenitor cells could be induced to differentiate into various retinal cells,including Thy1.1-positive retinal ganglion cells.The percentage of retinal ganglion cells was 16.91%?4.05% at E14 and 4.65%?1.88% at E18.The differences were statistically significant(t=15.04,P