Mengfei Chen

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.

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.

Endoplasmic reticulum stress promotes amyloid-beta peptides production in RGC-5 cells

Endoplasmic reticulum (ER) stress has been implicated in various neurodegenerative diseases, including Alzheimer’s disease. We have previously observed amyloid production in the retina of the Tg2576 transgenic mouse model of Alzheimer’s disease. In this study, we used tunicamycin-induced ER stress in RGC-5 cells, a cell line identical to the photoreceptor cell line 661W, to investigate the effect of ER stress on production of amyloid-beta (Abeta) peptides. We found that the mRNA level of amyloid-beta precursor protein (APP) remained stable, while the protein level of amyloid-beta precursor protein (APP) was decreased, the amyloid-beta precursor protein cleaving enzymes beta-site APP-cleaving enzyme 1 and presenilin 1 were upregulated, Abeta1–40 and Abeta1–42 production were increased, and reactive oxygen species production and apoptosis markers were elevated following induction of ER stress. The protein level of Abeta degradation enzymes, neprilysin, endothelin-converting enzyme 1, and endothelin-converting enzyme 2 remained unchanged during the prolonged ER stress, showing that the generation of Abeta did not result from reduction of proteolysis by these enzymes. Inclusion of group II caspase inhibitor, Z-DEVD-FMK, increased the ER stress mediated Abeta production, suggesting that they are generated by a caspase-independent mechanism. Our findings provided evidence of a role of ER stress in Abeta peptide overproduction and apoptotic pathway activation in RGC-5 cells.

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.