Yuxiao Zeng

Human Bone Marrow Stromal Cells can Differentiate to a Retinal Pigment Epithelial Phenotype when Co-Cultured with Pig Retinal Pigment Epithelium using a Transwell System

Background: There is an increasing interest in generating retinal pigment epithelial (RPE) cells from stem cells for therapy against degenerative eye diseases. Human bone marrow stromal cells (hBMSCs) can be induced to express retinal neuron-specific markers when co-cultured with retinal neurons, however, whether hBMSCs can differentiate into RPE-like cells in a co-culture system has not been clarified. Methods: The induction of hBMSCs into RPE-like cells was performed by combining hBMSCs and pig RPE cells in a transwell system. The biomarkers of hBMSCs-derived RPE cells were determined by quantitative RT-PCR and immunofluorescence. The function of induced cells was assayed by ELISA for secretion of neurotrophic factors. Results: Intracellular pigment granules and many RPE markers existed in hBMSCs-derived RPE cells after co-culturing with pig RPE cells for 14 days. Typical RPE functions, such as phagocytosis of photoreceptor outer segments and secretion of the trophic factors, brain-derived neurotrophic factor (BDNF) and glia-derived neurotrophic factor (GDNF), were observed in these induced cells. Conclusion: hBMSCs can be induced toward functional RPE cells simply by transwell-based co-culture with RPE cells.

Neural stem cells transplanted to the subretinal space of rd1 mice delay retinal degeneration by suppressing microglia activation

BACKGROUND AIMS Retinal degeneration (RD) is an inherited eye disease characterized by irreversible photoreceptor loss. Conventionally, the activation of the resident microglia is secondary to the disease. Stem cell-based therapy has recently made rapid progress in treating RD. Although it has been demonstrated that the effect of stem cell therapy may include immunomodulation, the specific mechanisms have not been clarified. METHODS Immunocytochemistry, terminal deoxynucleotidyl transferase UTP nick end labelling (TUNEL) assay and Western blot were used to analyze the microglia activation and photoreceptor apoptosis in the retina of rd1 mice. GFP-C17.2 neural stem cells (NSCs) were transplanted into the subretinal space to study the immunomodulatory and neuroprotective effects. The transwell co-culture of BV2 cells with GFP-C17.2 was performed to study the proliferation, apoptosis and secretion levels of inflammatory factors. Real time-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were performed to explore the gene and protein level of factors secreted by NSCs and microglia. RESULTS TUNEL-positive cells were primarily distributed in the inner nuclear layer (INL) of rd1 mice on P8d, appeared in the outer nuclear layer (ONL) on P10d and peaked on P14d. Meanwhile, microglia migrated to the ONL and reached the maximum level, accompanied by the changes in the levels of fractalkine and its unique receptor CX3CR1 protein. After transplantation of NSCs on P7d into the subretinal space of rd1 mice, the activated microglia were inhibited and the degeneration of ONL was delayed. In addition, microglia activation was suppressed by co-cultured NSCs in vitro. The gene and protein level of tissue inhibitor of metalloproteinase (TIMP1) in NSCs was elevated, whereas that of matrix metalloproteinase (MMP9) in BV2 microglia was markedly suppressed in this co-culture system. CONCLUSIONS Transplanted NSCs in the retina exerted immunomodulatory effects on microglia, thus delaying the degeneration of photoreceptors.

Overexpression of Fibulin-5 in Retinal Pigment Epithelial Cells Inhibits Cell Proliferation and Migration and Downregulates VEGF, CXCR4, and TGFB1 Expression in Cocultured Choroidal Endothelial Cells

Purpose of the study: Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss. Fibulin-5 (FBLN5) plays a pleiotropic role in the pathogenesis of AMD. We examined whether the in vitro overexpression of FBLN5 in retinal pigment epithelial (RPE) cells alters the proliferation and migration of cocultured choroidal endothelial cells (CECs) and explored the possible mechanisms involved. Materials and methods: A recombinant lentiviral vector carrying the Fbln5 gene was generated to transduce rat RPE cells. The expression of FBLN5 in transduced RPE cells was detected by quantitative real-time PCR and Western blot. The transduced RPE cells were then cocultured with rhesus macaque CECs in a Transwell coculture system. The impact of overexpression of FBLN5 in RPE cells on CEC proliferation and migration was assessed, as well as the impact on the mRNA expressions of vascular endothelial growth factor (VEGF), C-X-C chemokine receptor type 4 (CXCR4), and transforming growth factor β1 (TGFB1). Results: Our results showed that a recombinant lentivirus carrying the Fbln5 gene, which could induce overexpression of FBLN5 in RPE cells, was successfully generated. Overexpression of FBLN5 in RPE cells inhibited cell proliferation and migration and downregulated the mRNA expressions of VEGF, CXCR4, and TGFB1 in cocultured CECs. Conclusions: These findings suggest that FBLN5 may interfere with choroidal neovascularization by downregulating VEGF, CXCR4, and TGFB1 expression and inhibiting CEC proliferation and invasion, intensifying interest in FBLN5 as a target for therapeutic intervention in neovascular AMD.

A Cell Electrofusion Chip for Somatic Cells Reprogramming.

Cell fusion is a potent approach to explore the mechanisms of somatic cells reprogramming. However, previous fusion methods, such as polyethylene glycol (PEG) mediated cell fusion, are often limited by poor fusion yields. In this study, we developed a simplified cell electrofusion chip, which was based on a micro-cavity/ discrete microelectrode structure to improve the fusion efficiency and to reduce multi-cell electrofusion. Using this chip, we could efficiently fuse NIH3T3 cells and mouse embryonic stem cells (mESCs) to induce somatic cells reprogramming. We also found that fused cells demethylated gradually and 5-hydroxymethylcytosine (5hmC) was involved in the demethylation during the reprogramming. Thus, the cell electrofusion chip would facilitate reprogramming mechanisms research by improving efficiency of cell fusion and reducing workloads.

Intermittent high oxygen influences the formation of neural retinal tissue from human embryonic stem cells.

The vertebrate retina is a highly multilayered nervous tissue with a large diversity of cellular components. With the development of stem cell technologies, human retinas can be generated in three-dimensional (3-D) culture in vitro. However, understanding the factors modulating key productive processes and the way that they influence development are far from clear. Oxygen, as the most essential element participating in metabolism, is a critical factor regulating organic development. In this study, using 3-D culture of human stem cells, we examined the effect of intermittent high oxygen treatment (40% O2) on the formation and cellular behavior of neural retinas (NR) in the embryonic body (EB). The volume of EB and number of proliferating cells increased significantly under 40% O2 on day 38, 50, and 62. Additionally, the ratio of PAX6+ cells within NR was significantly increased. The neural rosettes could only develop with correct apical-basal polarity under 40% O2. In addition, the generation, migration and maturation of retinal ganglion cells were enhanced under 40% O2. All of these results illustrated that 40% O2 strengthened the formation of NR in EB with characteristics similar to the in vivo state, suggesting that the hyperoxic state facilitated the retinal development in vitro.

Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures — a new donor for cell therapy

Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases.

Combined transplantation of human mesenchymal stem cells and human retinal progenitor cells into the subretinal space of RCS rats

Retinitis pigmentosa (RP) is one of hereditary retinal diseases characterized by the loss of photoreceptors. Cell transplantation has been clinically applied to treat RP patients. Human retinal progenitor cells (HRPCs) and human bone marrow-derived mesenchymal stem cells (HBMSCs) are the two commonly and practically used stem cells for transplantation. Since combined transplantation could be a promising way to integrate the advantages of both stem cell types, we transplanted HRPCs and HBMSCs into the subretinal space (SRS) of Royal College of Surgeons (RCS) rats. We report that HRPCs/HBMSCs combined transplantation maintains the electroretinogram results much better than HRPCs or HBMSCs single transplantations. The thickness of outer nuclear layer also presented a better outcome in the combined transplantation. Importantly, grafted cells in the combination migrated better, both longitudinally and latitudinally, than single transplantation. The photoreceptor differentiation of grafted cells in the retina of RCS rats receiving combined transplantation also showed a higher ratio than single transplantation. Finally, activation of microglia and the gliosis of Müller cells were more effectively suppressed in combined transplantation, indicating better immunomodulatory and anti-gliosis effects. Taken together, combining the transplantation of HRPCs and HBMSCs is a more effective strategy in stem cell-based therapy for retinal degenerative diseases.

Bone Marrow CD133 Stem Cells Ameliorate Visual Dysfunction in Streptozotocin-induced Diabetic Mice with Early Diabetic Retinopathy.

Diabetic retinopathy (DR), one of the leading causes of vision loss worldwide, is characterized by neurovascular disorders. Emerging evidence has demonstrated retinal neurodegeneration in the early pathogenesis of DR, and no treatment has been developed to prevent the early neurodegenerative changes that precede detectable microvascular disorders. Bone marrow CD133+ stem cells with revascularization properties exhibit neuroregenerative potential. However, whether CD133+ cells can ameliorate the neurodegeneration at the early stage of DR remains unclear. In this study, mouse bone marrow CD133+ stem cells were immunomagnetically isolated and analyzed for the phenotypic characteristics, capacity for neural differentiation, and gene expression of neurotrophic factors. After being labeled with enhanced green fluorescent protein, CD133+ cells were intravitreally transplanted into streptozotocin (STZ)-induced diabetic mice to assess the outcomes of visual function and retina structure and the mechanism underlying the therapeutic effect. We found that CD133+ cells co-expressed typical hematopoietic/endothelial stem/progenitor phenotypes, could differentiate to neural lineage cells, and expressed genes of robust neurotrophic factors in vitro. Functional analysis demonstrated that the transplantation of CD133+ cells prevented visual dysfunction for 56 days. Histological analysis confirmed such a functional improvement and showed that transplanted CD133+ cells survived, migrated into the inner retina (IR) over time and preserved IR degeneration, including retina ganglion cells (RGCs) and rod-on bipolar cells. In addition, a subset of transplanted CD133+ cells in the ganglion cell layer differentiated to express RGC markers in STZ-induced diabetic retina. Moreover, transplanted CD133+ cells expressed brain-derived neurotrophic factors (BDNFs) in vivo and increased the BDNF level in STZ-induced diabetic retina to support the survival of retinal cells. Based on these findings, we suggest that transplantation of bone marrow CD133+ stem cells represents a novel approach to ameliorate visual dysfunction and the underlying IR neurodegeneration at the early stage of DR.

Transplanted olfactory ensheathing cells restore retinal function in a rat model of light-induced retinal damage by inhibiting oxidative stress

There is still not an effective treatment for continuous retinal light exposure and subsequent photoreceptor degeneration. Olfactory ensheathing cell (OEC) transplantation has been shown to be neuroprotective in spinal cord, and optic nerve injury and retinitis pigmentosa. However, whether OECs protect rat photoreceptors against light-induced damage and how this may work is unclear. Thus, to elucidate this mechanism, purified rat OECs were grafted into the subretinal space of a Long-Evans rat model with light-induced photoreceptor damage. Light exposure decreased a- and b- wave amplitudes and outer nuclear layer (ONL) thickness, whereas the ONL of rats exposed to light for 24 h after having received OEC transplants in their subretinal space was thicker than the PBS control and untreated groups. A- and b- wave amplitudes from electroretinogram of OEC-transplanted rats were maintained until 8 weeks post OEC transplantation. Also, transplanted OECs inhibited formation of reactive oxygen species in retinas exposed to light. In vitro experiments showed that OECs had more total antioxidant capacity in a co-cultured 661W photoreceptor cell line, and cells were protected from damage induced by hydrogen-peroxide. Thus, transplanted OECs preserved retinal structure and function in a rat model of light-induced degeneration by suppressing retinal oxidative stress reactions.

Lin28b stimulates the reprogramming of rat Müller glia to retinal progenitors

ABSTRACT In lower‐order vertebrates, Müller glia exhibit characteristics of retinal progenitor cells, while in higher vertebrates, such as mammals, the regenerative capacity of Müller glia is limited. Recently, we reported that Lin28b promoted the trans‐differentiation of Müller cells to rod photoreceptor and bipolar cells in the retina of retinitis pigmentosa rat model, whereas it is unclear whether Lin28b can stimulate the reprogramming of Müller glia in vitro for transplantation into a damaged retina. In the present study, Long‐Evens rat Müller glia were infected with Adeno‐Lin28b or Adeno‐GFP. Over‐expression of Lin28b in isolated rat Müller glia resulted in the suppression of GFAP expression, enhancement of cell proliferation and a significant increase of the expression of retinal progenitor markers 5 days after infection. Moreover, Lin28b caused a significant reduction of the Let‐7 family of microRNAs. Following sub‐retinal space transplantation, Müller glia‐derived retinal progenitors improved b‐wave amplification of 30d Royal College of Surgeons retinitis pigmentosa model (RCS‐P+) rats, as detected by electroretinography (ERG) recordings. Taken together, these data suggest that the up‐regulation of Lin28b expression facilitated the reprogramming of Müller cells toward characteristics of retinal progenitors. HIGHLIGHTSLin28b reprograms Müller glia to retinal progenitors.Let‐7 micrRNAs are suppressed by Lin28b.Transplantation of reprogrammed Müller glia restores retinal function.