Xuerong Sun

Generation of Retinal Ganglion–like Cells from Reprogrammed Mouse Fibroblasts

PURPOSE Somatic cells can be reprogrammed into an embryonic stem cell-like pluripotent state by Oct-3/4, Sox2, c-Myc, and Klf4. Sox2 as an essential reprogramming factor also contributes to the development of the eye and the retina. This study was conducted to determine whether induced pluripotent stem (iPS) cells express retinal progenitor cell (RPC)-related genes and whether iPS cells can directly differentiate into retinal ganglion cells (RGCs). METHODS Mouse iPS cells were induced by the ectopically expressed four factors in tail-tip fibroblasts (TTFs). The expression of RPC-related genes in iPS cells was analyzed by RT-PCR and immunofluorescence. iPS cells were induced to differentiate into RGCs by the addition of Dkk1 + Noggin (DN) + DAPT and overexpression of Math5. iPS-derived retinal ganglion (RG)-like cells were injected into the retina, and the eyes were analyzed by immunohistochemistry. RESULTS iPS cells inherently express RPC-related genes such as Pax6, Rx, Otx2, Lhx2, and Nestin. Overexpression of Math5 and addition of DN can directly differentiate iPS into retinal ganglion-like cells. These iPS-derived RG-like cells display long synapses and gene expression patterns, including Math5, Brn3b, Islet-1, and Thy1.2. Furthermore, inhibiting Hes1 by DAPT increases the expression of RGC marker genes. In addition, iPS-derived RG-like cells were able to survive but were unable to be integrated into the normal retina after transplantation. CONCLUSIONS The four factor iPS cell inherently expressed RPC-related genes, and the iPS cell could be further turned into RG-like cells by the regulation of transcription factor expression. These findings demonstrate that iPS cells are valuable for regeneration research into retinal degeneration diseases.

Differentiation of mouse induced pluripotent stem cells into corneal epithelial-like cells.

Somatic cells can be reprogrammed into a pluripotent ES‐cell‐like state (termed induced pluripotent stem cells, iPS) by transcription factors, which have enormous therapeutic potential for regenerative medicine. We have investigated whether iPS can directly differentiate into corneal epithelium‐like cells. Mouse iPS cells were co‐cultured with corneal limbal stroma. RT‐PCR, immunohistochemistry and scanning electron microscopy analysis were used to detect differentiated iPS. Undifferentiated iPS cells expressed ES cells related genes. Co‐culture with corneal limbal stroma, in the presence of additional factors bFGF, EGF and NGF, activated keratin expression 12 (K12, a marker of corneal epithelial cells) and downregulated Nanog. These data suggest that mouse iPS cells can differentiate into corneal epithelial‐like cells by replication of a corneal epithelial stem cell niche.

Role of MEF feeder cells in direct reprogramming of mousetail-tip fibroblasts

Pluripotent stem cells can be induced from somatic cells by the transcription factors Oct3/4, Sox2, c‐Myc and Klf4 when co‐cultured with mouse embryonic fibroblast (MEF) feeder cells. To date, the role of the feeder cells in the reprogramming process remains unclear. In this study, using a comparative analysis, we demonstrated that MEF feeder cells did not accelerate reprogramming or increase the frequency of induced pluripotent stem (iPS) cell colonies. However, feeder conditions did improve the growth of primary iPS colonies and were necessary for passaging the primary colonies after reprogramming was achieved. We further developed a feeder‐free culture system for supporting iPS growth and sustaining pluripotency by adding bFGF and activin A (bFA) to the medium. These data will facilitate the generation of human iPS cells without animal feeders for regenerative medicine.

Hypoxia induces beta-amyloid in association with death of RGC-5 cells in culture

Beta-amyloid (Aβ) derived from amyloid precursor protein (APP) has been associated with retinal degeneration in Alzheimers disease (AD) and glaucoma. This study examined whether hypoxia exposure induces Aβ accumulation in RGC-5 cells. While levels of APP mRNA and protein significantly increased in the cells, elevated abundance of Aβ was also observed in cells and culture medium between 12 or 24 and 48h after 5% O(2) hypoxia treatment. Additionally, there is a close relationship between induction of APP and Aβ and intracellular accumulation of ROS along with loss of mitochondrial membrane potential followed by the death of RGC-5 cells in culture under hypoxia. These results suggest a possible involvement of APP and Aβ in the death of RGCs challenged by hypoxia.

Adult peripheral blood mononuclear cells transdifferentiate in vitro and integrate into the retina in vivo

Adult peripheral blood‐derived cells are able to differentiate into a variety of cell types, including nerve cells, liver‐like cells and epithelial cells. However, their differentiation into retina‐like cells is controversial. In the present study, transdifferentiation potential of human adult peripheral blood mononuclear cells into retina‐like cells and integration into the retina of mice were investigated. Freshly isolated adult peripheral blood mononuclear cells were divided into two groups: cells in group I were cultured in neural stem cell medium, and cells in group II were exposed to conditioned medium from rat retinal tissue culture. After 5 days, several distinct cell morphologies were observed, including standard mononuclear, neurons with one or two axons and elongated glial‐like cells. Immunohistochemical analysis of neural stem cell, neuron and retina cell markers demonstrated that cells in both groups were nestin‐, MAP2 (microtubule‐associated protein)‐ and GFAP (glial fibrillary acidic protein)‐positive. Flow cytometry results suggested a significant increase in nestin‐, MAP2‐ and CD16‐positive cells in group I and nestin‐, GFAP‐, MAP2‐, vimentin‐ and rhodopsin‐positive cells in group II. To determine survival, migration and integration in vivo, cell suspensions (containing group I or group II cells) were injected into the vitreous or the peritoneum. Tissue specimens were obtained and immunostained 4 weeks after transplantation. We found that cells delivered by intravitreal injection integrated into the retina. Labelled cells were not detected in the retina of mice receiving differentiated cells by intraperitoneal injection, but cells (groups I and II) were detected in the liver and spleen. Our findings revealed that human adult peripheral blood mononuclear cells could be induced to transdifferentiate into neural precursor cells and retinal progenitor cells in vitro, and the differentiated peripheral blood mononuclear cells can migrate and integrate into the retina in vivo.

Cholinergic neuronal differentiation of bone marrow mesenchymal stem cells in rhesus monkeys

The purpose of the present study was to determine the best cholinergic neuronal differentiation method of rhesus monkey bone marrow mesenchymal stem cells (BMSCs). Four methods were used to induce differentiation, and the groups were assigned accordingly: basal inducing group (culture media, bFGF, and forskolin); SHH inducing group (SHH, inducing group); RA inducing group (RA, basal inducing group); and SHH+RA inducing group (SHH, RA, and basal inducing group). All groups displayed neuronal morphology and increased expression of nestin and neuron-specific enolase. The basal inducing group did not express synapsin, and cells from the SHH inducing group did not exhibit neuronal resting membrane potential. In contrast, results demonstrated that BMSCs from the RA and SHH+RA inducing groups exhibited neuronal resting membrane potential, and cells from the SHH+RA inducing group expressed higher levels of synapsin and acetylcholine. In conclusion, the induction of cholinergic differentiation through SHH+RA was determined to be superior to the other methods.

Cyclic intensive light exposure induces retinal lesions similar to age-related macular degeneration in APPswe/PS1 bigenic mice

BackgroundIntensive light exposure and beta-amyloid (Aβ) aggregates have been known as a risk factor for macular degeneration and an important component in the pathologic drusen structure involved in this disorder, respectively. However, it is unknown whether Aβ deposition mediates or exacerbates light exposure-induced pathogenesis of macular degeneration. Several studies including the one from us already showed accumulation of Aβ deposits in the retina in Alzheimers transgenic mice. Using histopathological analysis combined with electroretinographic functional assessment, we investigated the effects of cyclic intensive light exposure (CILE) on the architecture of retina and related function in the APPswe/PS1bigenic mouse.ResultsHistopathological analysis has found significant loss of outer nuclear layer/photoreceptor outer segment and outer plexiform layer along with abnormal hypo- and hyper-pigmentation in the retinal pigment epithelium (RPE), remarkable choroidal neovascularization (CNV), and exaggerated neuroinflammatory responses in the outer retina of APPswe/PS1 bigenic mice following cyclic intensive light exposure (CILE), whereas controls remained little change contrasted with age-matched non-transgenic littermates. CILE-induced degenerative changes in RPE are further confirmed by transmission electron microcopy and manifest as formation of basal laminar deposits, irregular thickening of Bruchs membrane (BrM), deposition of outer collagenous layer (OCL) in the subretinal space, and vacuolation in the RPE. Immunofluorescence microscopy reveals drusenoid Aβ deposits in RPE as well as neovessels attached which are associated with disruption of RPE integrity and provoked neuroinflammatory response as indicated by markedly increased retinal infiltration of microglia. Moreover, both immunohistochemistry and Western blots detect an induction of vascular endothelial growth factor (VEGF) in RPE, which corroborates increased CNV in the outer retina in the bigenic mice challenged by CILE.ConclusionsOur findings demonstrate that degenerative changes in the outer retina in the APPswe/PS1 bigenic mouse induced by CILE are consistent with these in AMD. These results suggest that an Alzheimers transgenic animal model with accumulation of Aβ deposits might be an alternative animal model for AMD, if combined with other confounding factors such as intensive light exposure for AMD.

Induced differentiation of bone marrow mesenchymal stem cells towards retinal ganglion precursor cells

Induced differentiation of bone marrow mesenchymal stem cells towards retinal ganglion precursor cells

A simple and effective pressure culture system modified from a transwell cell culture system

Mechanical pressure plays an important role in many physiological and pathological processes. Mimicking the mechanical pressure present in vitro is necessary for related research, but usually requires expensive and complicated equipment. In this study we created a simple pressure culture system based on the transwell culture system. By cutting off the top rim of the transwell insert, the cells were compressed between the insert membrane and the well floor. The new pressure culture system was proven effective in that it induced cell morphological change, integrin β1 upregulation, actin polymerization and growth change in rat retinal ganglion cells, human nasopharyngeal carcinoma cells and mice embryonic fibroblasts. Though the pressure value is immeasurable and inhomogeneous, the easily available culture system still provides a choice for the laboratories that do not have access to the better, but much more expensive pressure culture equipment.

Retinoid acid and taurine promote NeuroD1-induced differentiation of induced pluripotent stem cells into retinal ganglion cells

Induced pluripotent stem cells (iPSCs) possess the capacity to differentiate into multiple cell types including retinal neurons. Despite substantial progress in the transcriptional regulation of iPSC differentiation process, the efficiency of generation of retinal neurons from iPSCs is still low. In this study, we investigated the role of transcription factor NeuroD1 in the differentiation of iPSCs into retinal neurons. We observed that retrovirus-mediated NeuroD1 overexpression in iPSCs increased the efficiency of neuronal differentiation. Immunostaining analysis showed that NeuroD1 overexpression increased the expression of retina ganglion cell markers including Islet-1, Math5, Brn3b, and Thy1.2. Retinoid acid (RA) and taurine further improved the differentiation efficiency of iPSCs overexpressing NeuroD1. However, RA and taurine did not promote differentiation in the absence of NeuroD1 overexpression. Together, our study provides new evidence in transcription factor-regulated stem cell differentiation in vitro.