Expression of CCL2 and its receptor in activation and migration of microglia and monocytes induced by photoreceptor apoptosis.

Purpose: To explore the effect of the CCL2 and CCR2 system on the activation and migration of microglia and monocytes in light-induced photoreceptor apoptosis. Methods: At 1 day, 3 days, 7 days, and 14 days after light exposure, OX42 and ED1 immunostaining were used to label the activation and migration of microglia and monocytes. Double immunostaining of CCL2 with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), OX42, or glial fibrillary acidic protein (GFAP) was applied to explore the relationships among CCL2, apoptotic photoreceptors, activated microglia and monocytes, and macroglial cells (Müller cells and astrocytes). Real-time PCR was used to evaluate the mRNA levels of retinal CCL2 and CCR2 and the proinflammatory factors interleukin (IL)-1 beta and tumor necrosis factor (TNF)-alpha. Results: Real-time PCR analyses showed that CCL2 and CCR2 expression gradually increased after light exposure and peaked at 3 days, coinciding with the infiltration of OX42-positive cells and the expression of IL-1 beta and TNF-alpha in the outer retina. Double immunostaining of CCL2 with TUNEL revealed that CCL2 was expressed robustly in about 30% of the apoptotic photoreceptors at the early stage. As degeneration progressed, immunostaining of CCL2 with OX42 showed that activated and migrated microglia and monocytes expressed CCL2. At the late stage, Müller cells became the main source of CCL2, which was illustrated by CCL2 immunostaining with GFAP. Conclusions: Light exposure led to apoptosis of photoreceptors, which expressed CCL2, accelerating an inflammation-mediated cascade by activating and attracting microglia and monocytes and promoting their secretion of CCL2 in the injured position.

Lineage conversion of mouse fibroblasts to pancreatic α-cells.

α-cells, which synthesize glucagon, also support ß-cell survival and have the capacity to transdifferentiate into ß-cells. However, the role of α-cells in pathological conditions and their putative clinical applications remain elusive due in large part to the lack of mature α-cells. Here, we present a new technique to generate functional α-like cells. α-like cells (iAlpha cells) were generated from mouse fibroblasts by transduction of transcription factors, including Hhex, Foxa3, Gata4, Pdx1 and Pax4, which induce α-cell-specific gene expression and glucagon secretion in response to KCl and Arg stimulation. The cell functions in vivo and in vitro were evaluated. Lineage-specific and functional-related gene expression was tested by realtime PCR, insulin tolerance test (ITT), glucose tolerance test (GTT), Ki67 and glucagon immunohistochemistry analysis were done in iAlpha cells transplanted nude mice. iAlpha cells possess α-cell function in vitro and alter blood glucose levels in vivo. Transplantation of iAlpha cells into nude mice resulted in insulin resistance and increased ß-cell proliferation. Taken together, we present a novel strategy to generate functional α-like cells for the purposes of disease modeling and regenerative medicine.

Transplantation of betatrophin-expressing adipose-derived mesenchymal stem cells induces ß-cell proliferation in diabetic mice.

Recent progress in regenerative medicine has suggested that mesenchymal stem cell (MSC)-based therapy is a novel potential cure for diabetes. Betatrophin is a newly identified hormone that can increase the production and expansion of insulin-secreting ß-cells when administered to mice. In this study, we evaluated the effect of betatrophin overexpression by human adipose-derived MSCs (ADMSCs) by in vitro experiments, as well as following their transplantation into a mice with streptozotocin (STZ)-induced diabetes. The overexpression of betatrophin did not affect the ADMSCs in terms of proliferation, differentiation and morphology. However, the co-culture of human islets with ADMSCs overexpressing betatrophin (ADMSCs-BET) induced islet proliferation, ß-cell specific transcription factor expression, and the islet production of insulin under the stimulation of glucose or KCl and Arg. In addition, ADMSCs-BET enhanced the anti-inflammatory and anti-apoptotic effects of the co-cultured islets compared with ADMSCs cultured alone. In mice with STZ-induced diabetes, the transplantation of ADMSCs-BET ameliorated the hyperglycemia and weight loss associated with STZ-induced diabetes; ADMSCs-BET also significantly enhanced the ratio of ß-cells per islet compared to the transplantation of ADMSCs alone. Thus, our study demonstrates a novel strategy for inducing ß-cell regeneration. ADMSCs-BET may replace insulin injections by increasing the number of endogenous insulin-producing cells in patients with diabetes. This combined strategy of ADMSC transplantation and gene therapy may prove to be a useful therapy for the treatment of diabetes.

Generation of Human Lens Epithelial-Like Cells From Patient-Specific Induced Pluripotent Stem Cells.

Cataractogenesis begins from the dynamic lens epithelial cells (LECs) and adjacent fiber cells. LECs derived from cell lines cannot maintain the crystalline expression as the primary LECs. The current study aimed to efficiently generate large numbers of human LECs from patient-specific induced pluripotent stem cells (iPSCs). Anterior lens capsules were collected from cataract surgery and were used to culture primary hLECs. iPSCs were induced from these primary hLECs by lentiviral transduction of Oct4, Sox2, Klf4, and c-Myc. Then, the generated iPSCs were re-differentiated into hLECs by the 3-step addition of defined factor combinations (Noggin, BMP4/7, bFGF, and EGF) modified from an established method. During the re-differentiation process, colonies of interest were isolated using a glass picking tool and cloning cylinders based on the colony morphology. After two steps of isolation, populations of LEC-like cells (LLCs) were generated and identified by the expression of lens marker genes by qPCR, western blot and immunofluorescence staining. The study introduced a modified protocol to isolate LLCs from iPSCs by defined factors in a short time frame. This technique could be useful for mechanistic studies of lens-related diseases. J. Cell. Physiol. 231: 2555-2562, 2016. © 2016 Wiley Periodicals, Inc.

Generation and characterization of immortalized rat retinal microglial cell lines.

Retinal microglia play an important role as resident immunocompetent and phagocytic cells in the event of injury and disease. Retinal microglia and microglia precursor transplantation show a rescue effect in ischemic retina and retinal degeneration. However, studies of retinal microglia have been hampered by the difficulty of obtaining sufficient numbers of microglia. One way to circumvent this difficulty is to establish permanent retinal microglia cell lines. In the present study, we report the generation of immortalized retinal microglia, T-MG cells, from postnatal day 3 rat retinal tissue using a lentiviral vector encoding SV40 large T antigen. The T-MG cells exhibited cell-type-specific antigens for monocyte/macrophage lineage cells, including CD11b (OX42), ED1 (OX6), and Iba1, and actively phagocytosed latex beads. In addition to primary retinal microglia, T-MG cells also have the ability to recruit into chemokines. Treatment of T-MG cells with lipopolysaccharide (LPS) led to increased levels of tumor necrosis factor-α, interleukin-1ß, and inducible nitric oxide synthase. Genome-wide microarray analysis showed a less than 1% difference in the genes between the T-MG cells and the control primary retinal microglia. The T-MG cells exhibited properties similar to those of the primary retinal microglia and should have considerable utility as an in vitro model for the study of retinal microglia in health and as a curative therapy and an in vivo model for the study of retinal microglia in disease.

Bufalin inhibits the differentiation and proliferation of human osteosarcoma cell line hMG63-derived cancer stem cells.

Cancer stem cells (CSCs) play an important role in drug resistance of tumor and are responsible for high recurrence rates. Agents that can suppress the proliferation and differentiation of CSCs would provide new opportunity to fight against tumor recurrence. In this study, we developed a new strategy to enrich CSCs in human osteosarcoma cell line hMG63. Using these CSCs as model, we tested the effect of bufalin, a traditional Chinese medicine, on the proliferation and differentiation of CSCs. hMG63 cells were cultured in poly-HEMA-treated dish and cancer stem cell-specific medium. In this nonadhesive culture system, hMG63 formed spheres, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every 3 days for five times. The enriched xenograft tumors were cultured in cancer stem cell-specific medium again to form tumor spheres. Expression of cancer stem cell markers of these cells was measured by flow cytometry. These cells were then treated with bufalin, and the proliferation and differentiation ability were indicated by the expression level of molecular markers and the formation of sphere again in vitro. We obtained a low CD133+/CD44 cell population with high-level stem cell marker. When treated with bufalin, the sphere could not get attached to the flask and failed to differentiate, which was indicated by the stable expression of stem cell marker CD133 and OCT-4 in the condition permissive to differentiation. Treatment of bufalin also suppressed the single cells isolated from the sphere to form sphere again in the nonadhesive culture system, and a decreased expression of proliferation marker Ki67 was also detected in these cells. Sphere-formed and chemoresistant colon xenograft tumors in immunodeficient mice could enrich cancer stem cell population. Bufalin could inhibit proliferation and differentiation of CSCs.

Inhibition of LPS-induced retinal microglia activation by naloxone does not prevent photoreceptor death.

Microglia-associated inflammation is closely related to the pathogenesis of retinal degenerative disorders. We have previously shown in vivo that naloxone protected photoreceptors from light-induced apoptosis possibly through inhibiting microglial activation. In this study, we attempted to explore the effect of lipopolysaccharide (LPS)-activated microglia on photoreceptor death and the influence of naloxone treatment using an in vitro retinal microglia and 661 W photoreceptor co-culture system. Immunofluorescent staining and ELISA measurements demonstrated that LPS activated microglia by changing the morphology and increasing the production of proinflammatory factors interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Flow cytometry analysis of annexin V/propidium iodide staining showed that LPS-activated microglia promoted the apoptosis of co-cultured 661 W photoreceptor cells. Naloxone inhibited microglial activation and decreased the release of IL-1beta and TNF-alpha but could not prevent photoreceptors from undergoing apoptosis. Considering the dual role of microglia-associated inflammation in both neurotoxicity and neuroprotection, modulating the function, rather than simply inhibiting their activation, might be a new therapeutic method for preventing photoreceptor degeneration.

[The cell-type-specific HSV-tk gene expression of suicide gene therapy system regulated by lens-specific promoter LEP503].

OBJECTIVE: To explore the specific expression of HSV-tk gene and killing effects on ocular leading cells of the enhanced specific HSV-tk/GCV gene therapy system regulated by lens-specific promoter LEP503. METHODS: Experimental research. The enhanced specific HSV-tk/GCV gene system of two vectors were constructed (Lenti-LEP503-HSVtk-Cre and Lenti-HPGK-Loxp-EGFP-pA-Loxp-HSVtk). The lentiviral vectors were produced by transient transduction of transfering vectors, packaging vectors and enveloping vector into 293T cells. Virus was collected with ultracentrifugation and resuspended with 1 ml phosphate buffered saline and stored at -80°C. The HLEC and RPEC, NIH3T3, 293T cells were transduced with the enhanced specific HSV-tk gene system. The specific expressions of EGFP and HSV-tk were detected by fluorescence microscopy, flow cytometry and RT-PCR. The killing effects of HLEC and RPEC at the concentration of 20 mg/L GCV were assayed and compared by flow cytometry and CCK-8 kit. Difference of RPE cell viability among groups was evaluated by analysis of variance (ANOVA). RESULTS: Expression efficiency of EGFP in RPEC group was 62.3%, 68.3% in NIH3T3 group, 75.8% in 293T group, whereas 17.5% in HLEC group. There was higher expression of HSV-tk at mRNA level in HLEC group than that in RPEC group. The relative intensity of HSV-tk mRNA in HLEC group transduced with the enhanced specific HSV-tk gene system was 4.01, whereas 0.29 in RPEC group. At the concentration of 20 mg/L GCV after 72 hours, the percentage of apoptosis detected by the flow cytometry in HLEC group transduced by the enhanced specific HSV-tk gene system was 76.51%, and 2.44% in RPEC group. There was no significant difference in the RPE cell viability among the enhanced specific HSV-tk gene combination-RPE group, normal-RPE group and negative-RPE control group at the concentration of 20 mg/L GCV after 72 hours (MD(1) = -0.047, P = 0.671; MD(2) = 0.027, P = 0.912). CONCLUSIONS: The enhanced specific HSV-tk gene system express HSV-tk selectively in HLEC. At the concentration of 20 mg/L GCV, it is effective against the proliferation of HLEC in vitro, but has less kill effect on RPEC.

ATRA enhances the bystander effect of suicide gene therapy driven by the specific promoter LEP 503 in human lens epithelial cells.

PURPOSE: To establish a novel, targeted lentivirus-mediated LEP503-HSV-tk/GCV suicide gene therapy system combined with all trans-retinoic acid (ATRA) for the inhibition of human lens epithelial cell (HLEC) proliferation and treatment of posterior capsular opacification (PCO) after cataract surgery; to estimate the enhancement of the bystander effect by ATRA; and to explore the role of Connexin43 (Cx43) mediated gap junctional intercellular communication (GJIC) in the bystander effect of the HSV-K/GCV system. METHODS: A Lenti-LEP503-HSV-tk-EGFP vector was generated by cloning the lens-specific promoter LEP503 (lens specific promoter 503) from genomic DNA of HLECs by PCR. The vector was then inserted into the promoter-less vector from lentivirus-based (CMV)-HSV-tk-EGFP. The expressional specificity of the LEP503 promoter was assessed by investigating the expression of EGFP (enhanced green fluorescent protein) and HSV-tk (herpes simplex virus thymidine kinase) mRNA, both driven by Lenti-LEP503-HSV-tk-EGFP vector, by fluorescence microscopy, RT-PCR, flow cytometry, and western blot assays in HLECs, human adult retinal pigment epithelium cells (RPECs), human adult skin fibroblast cells (ASFCs), and Hela cells. Morphological changes were observed by fluorescence microscopy and cell viability was determined using the Cell Counting kit-8 Cell Proliferation (CCK-8) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays after Lenti-LEP503-HSV-tk/GCV system combined with ATRA treatment on HLECs. Flow cytometry, DNA fragmentation, and western blot assays were employed to analyze the mechanisms of bystander effects. RESULTS: The promoter LEP503-mediated HSV-tk was specifically expressed in HLECs, and ATRA dose-dependently strengthened the bystander effect following LEP503-mediated HSV-tk/GCV gene therapy against lens cells by upregulating the expression of the gap junction protein Cx43. CONCLUSIONS: The Lenti-LEP503-HSV-tk/GCV suicide gene therapy system, combined with ATRA as an adjuvant, may be a feasible supplementary method for PCO treatment that targets residual lens cells.

Efficient generation of lens progenitor cells from cataract patient-specific induced pluripotent stem cells.

The development of a technique to induce the transformation of somatic cells to a pluripotent state via the ectopic expression of defined transcription factors was a transformational event in the field of regenerative medicine. The development of this technique also impacted ophthalmology, as patient-specific induced pluripotent stemcells (iPSCs) may be useful resources for some ophthalmological diseases. The lens is a key refractive element in the eye that focuses images of the visual world onto the retina. To establish a new model for drug screening to treat lens diseases and investigating lens aging and development, we examined whether human lens epithelial cells (HLECs) could be induced into iPSCs and if lens-specific differentiation of these cells could be achieved under defined chemical conditions. We first efficiently reprogrammed HLECs from age-related cataract patients to iPSCs with OCT-4, SOX-2, and KLF-4. The resulting HLEC-derived iPS (HLE-iPS) colonies were indistinguishable from human ES cells with respect to morphology, gene expression, pluripotent marker expression and their ability to generate all embryonic germ-cell layers. Next, we performed a 3-step induction procedure: HLE-iPS cells were differentiated into large numbers of lens progenitor-like cells with defined factors (Noggin, BMP and FGF2), and we determined that these cells expressed lens-specific markers (PAX6, SOX2, SIX3, CRYAB, CRYAA, BFSP1, and MIP). In addition, HLE-iPS-derived lens cells exhibited reduced expression of epithelial mesenchymal transition (EMT) markers compared with human embryonic stem cells (hESCs) and fibroblast-derived iPSCs. Our study describes a highly efficient procedure for generating lens progenitor cells from cataract patient HLEC-derived iPSCs. These patient-derived pluripotent cells provide a valuable model for studying the developmental and molecular biological mechanisms that underlie cell determination in lens development and cataract pathophysiology.

CCR2 overexpression promotes the efficient recruitment of retinal microglia in vitro.

PURPOSE: Retinal microglia can be activated and recruited by chemokines and play a protective role in early retinal degeneration. CC-chemokine ligand 2 (CCL2) and its receptor, CC-chemokine receptor 2 (CCR2), have been implicated as key mediators for the trafficking and accumulation of microglial cells in lesioned tissue. The current study investigates whether the overexpression of CCR2 allows microglia to migrate toward CCL2 more efficiently. METHODS: Primary microglial cells were transduced with lentivirus carrying green fluorescent protein (GFP)-tagged CCR2 (CCR2-GFP). Overexpression of CCR2 was assessed by western blot analysis and fluorescence-assisted cell sorting. The chemotaxis of primary microglia transduced with lentivirus carrying CCR2-GFP was compared to either those transduced with GFP alone or those not transduced, using a chemotaxis chamber assay. RESULTS: Primary microglia showed a high transduction rate following lentivirus application and maintained normal microglial morphology and a significant overexpression of CCR2 protein. We found that CCL2-mediated chemotaxis is concentration and time dependent in microglia. The chemotactic response of microglia cells overexpressing CCR2-GFP was significantly increased compared to that of nontransduced and GFP-expressing microglia. CONCLUSIONS: These findings suggest that microglia can be efficiently transduced with CCR2-GFP lentiviral vectors and that the overexpression of CCR2 in retinal microglia promotes their chemotaxis in response to chemokines, suggesting that these cells may be promising targets for cell-based therapeutic manipulation in retinal disease.

Human amniotic epithelial cells express specific markers of nerve cells and migrate along the nerve fibers in the corpus callosum.

Human amniotic epithelial cells were isolated from a piece of fresh amnion. Using immunocytochemical methods, we investigated the expression of neuronal phenotypes (microtubule-associated protein-2, glial fibrillary acidic protein and nestin) in human amniotic epithelial cells. The conditioned medium of human amniotic epithelial cells promoted the growth and proliferation of rat glial cells cultured in vitro, and this effect was dose-dependent. Human amniotic epithelial cells were further transplanted into the corpus striatum of healthy adult rats and the grafted cells could integrate with the host and migrate 1-2 mm along the nerve fibers in corpus callosum. Our experimental findings indicate that human amniotic epithelial cells may be a new kind of seed cells for use in neurograft.

[Preliminary research on the regeneration of injured rabbit vocal folds after the transplantation of human amniotic epithelial cells].

OBJECTIVE: To investigate the survival, growth and distribution of human amniotic epithelial cells (hAEC) after injected into injured rabbit vocal folds, in addition, to assess the ability of hAEC to affect the components of lamina propria extracellular matrix (ECM) and prevent vocal fold scarring. METHODS: hAEC were isolated from human amnion and marked by Lenti-EGFP. Fifteen New Zealand rabbits were used for this experiment. EGFP-hAEC was injected into the left injured vocal folds in thirteen rabbits, and the contralateral thirteen vocal folds experienced an injured procedure only ("injured untreated control"), and four vocal folds were left as untreated controls. The survival, distribution, differentiation potential and secretion function of hAEC were examined by immunofluorescence method. HE staining and immunohistochemical staining were performed for the evaluation of collagen and fibronectin respectively. RESULTS: hAEC showed a cobblestone-like growth. After implanted into the injured vocal folds, hAEC could survive in vocal fold lamina propria for 2 months. The immunofluorescence analysis showed the evidence of hAEC differentiation into muscle cells as well as secretion the ECM protein. Three months postoperatively, the density of collagen was higher in the injured untreated control folds than that in the injured vocal folds injected with hAEC and the untreated controls. Besides, the content of fibronectin in the injured untreated control group was significantly increased. CONCLUSIONS: hAEC survived in the vocal folds lamina propria, and had the potentiality to differentiate into vocal folds tissue and secret some ECM components. The histological improvement caused by the injected cells demonstrate that hAEC had the ability to promote the repairment and regeneration of injured vocal folds.

Optimization of culture conditions to support undifferentiated growth of human embryonic stem cells.

Human embryonic stem cells (hESCs) are usually maintained in an undifferentiated state by coculture with mitomycin C-treated mouse embryonic fibroblasts (MEFs) as feeder cells in the presence of animal sera such as fetal bovine serum (FBS). Here, we use primary human amnion epithelial cells (hAECs) as feeder cells and human umbilical cord blood serum (CBS) as a replacement for FBS to support undifferentiated growth of hESCs. The 5 approximately 10-fold higher expression levels of ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT were found in hESCs grown on hAECs compared with that on MEFs as measured by quantitative real-time polymerase chain reaction (PCR). By immunofluorescence, the expresisons of Oct-4 and Nanog is also higher in cells grown on hAECs than those on MEFs. Importantly, the ES cells grown on hAECs exhibit normal karyotypes on passage 25, thus ruling out the possibility of transformation. Using flow cytometry analysis, we show that both the ES cells grown on hAECs and MEFs have the same cell cycle distribution pattern. Further, hESCs cultured on hAECs for at least 20 passages could differentiate into three germ layers via teratoma formation. In addition, chromatin immunoprecipitation assay revealed that histone H3 is highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus and the Oct-4 locus in hESCs grown on hAECs. Conversely, hESCs grown on MEFs show histone deacetylation and H3-K4 demethylation. Taken together, these results suggest that hAECs supplemented with 10% CBS are suitable for hESC culture, and that this method may prove to be valuable for use in future regenerative therapies.

Establishment and characterization of multi-drug resistant, prostate carcinoma-initiating stem-like cells from human prostate cancer cell lines 22RV1.

Multi-drug resistance is an important element which leads to ineffectiveness of chemotherapeutics. To identify subpopulations of cancerous prostate cells with multi-drug resistance and cancer stem-cell properties has recently become a major research interest. We identified a subpopulation from the prostate cancer cell line 22RV1, which have high surface expression of both CD117 and ABCG2. We found this subpopulation of cells termed CD117(+)/ABCG2(+) also overexpress stem cells markers such as Nanog, Oct4, Sox2, Nestin, and CD133. These cells are highly prolific and are also resistant to treatment with a variety of chemotherapeutics such as casplatin, paclitaxel, adriamycin, and methotrexate. In addition, CD117(+)/ABCG2(+) cells can readily establish tumors in vivo in a relatively short time. To investigate the mechanism of aggressive tumor growth and drug resistance, we examined the CpG islands on the ABCG2 promoter of CD117(+)/ABCG2(+) cells and found they were remarkably hypomethylated. Furthermore, chromatin immunoprecipitation assays revealed high levels of both histone 3 acetylation and H3K4 trimethylation at the CpG islands on the ABCG2 promoter. Our these data suggest that CD117(+)/ABCG2(+) cells could be reliably sorted from the human prostate cancer cell line 22RV1, and represent a valuable model for studying cancer cell physiology and multi-drug resistance. Furthermore, identification and study of these cells could have a profound impact on selection of individual treatment strategies, clinical outcome, and the design or selection of the next generation of chemotherapeutic agents.

Human amniotic epithelial cell feeder layers maintain mouse embryonic stem cell pluripotency via epigenetic regulation of the c-Myc promoter.

Mouse embryonic stem cells (ESCs) are typically cultured on a feeder layer of mouse embryonic fibroblasts (MEFs), with leukemia inhibitory factor (LIF) added to maintain them in an undifferentiated state. We have previously shown that human amniotic epithelial cells (hAECs) can be used as feeder cells to maintain mouse ESC pluripotency, but the mechanism for this is unknown. In the present study, we found that CpG islands 5' of the c-Myc gene remain hypomethylated in mouse ESCs cultured on hAECs. In addition, levels of acetylation of histone H3 and trimethylation of histone H3K4 in the c-Myc gene promoter were higher in ES cells cultured on hAECs than those in ES cells cultured on MEFs. These data suggested that hAECs can alter mouse ESC gene expression via epigenetic modification of c-Myc, providing a possible mechanism for the hAEC-induced maintenance of ESCs in an undifferentiated state.

Use of human amnion epithelial cells as a feeder layer to support undifferentiated growth of mouse embryonic stem cells.

Mouse and human embryonic stem (ES) cells are usually maintained in an undifferentiated state by coculture with mouse embryonic fibroblasts (MEFs) as feeder cells. In the case of mouse ES cells, addition of leukemia inhibitory factor (LIF) to culture media is also necessary to prevent cell differentiation. Here, we report the use of primary human amnion epithelial cells (hAECs) as feeder cells to culture mouse ES cells using our newly developed protocols. We found that mouse ES cells grown on hAECs express ES cell markers including FGF, Oct-4, Nanog, Sox-2, Rex, and TERT. Interestingly, the expression levels of these genes are three- to five fold higher on hAECs than on MEFs by quantitative real-time PCR. The quicker growing ES cells on hAECs showed a normal 19XY karyotype on passages 25, and ruled out the transformation of ES cells. Using flow cytometry analysis, we show that ES cells grown on hAECs have the same cell cycle distribution pattern as those on MEFs. Further, mouse ES cells cultured on hAECs for at least 20 passages retain the capability of teratoma formation in mice. Finally, we reveal that hAECs express highly LIF that allows for ES growth without the need of addition of commercially obtained LIF. Taken together, our data suggest that hAECs are suitable for mouse ES cell culture and may prove to be a useful alternative to MEFs for human ES cell culture.

Human amnion epithelial cells can be induced to differentiate into functional insulin-producing cells.

Pancreatic islet transplantation has demonstrated that long-term insulin independence may be achieved in patients suffering from diabetes mellitus type 1. However, limited availability of islet tissue means that new sources of insulin-producing cells that are responsive to glucose are required. Here, we show that human amnion epithelial cells (HAEC) can be induced to differentiate into functional insulin-producing cells in vitro. After induction of differentiation, HAEC expressed multiple pancreatic beta-cell genes, including insulin, pancreas duodenum homeobox-1, paired box gene 6, NK2 transcription factor-related locus 2, Islet 1, glucokinase, and glucose transporter-2, and released C-peptide in a glucose-regulated manner in response to other extracellular stimulations. The transplantation of induced HAEC into streptozotocin-induced diabetic C57 mice reversed hyperglycemia, restored body weight, and maintained euglycemia for 30 d. These findings indicated that HAEC may be a new source for cell replacement therapy in type 1 diabetes.

Human amniotic epithelial cells ameliorate behavioral dysfunction and reduce infarct size in the rat middle cerebral artery occlusion model.

Human amniotic epithelial cells (hAECs), having the characteristics of both embryonic and pluripotent stem cells, have the potential to differentiate into various cells. A good deal of research has explored the clinical therapeutic potential of hAECs; rat amniotic epithelial cells have been reported to ameliorate functional deficits after stroke in rats, likely due to neuronal differentiation and cytokine secretion by these cells. We isolated hAECs and transfected them with glial cell line-derived neurotrophic factor (GDNF) or enhanced green fluorescent protein (EGFP) gene using lentiviral vectors. These cells were then transplanted into the brains of rats subjected to a transient middle cerebral artery occlusion. The hAECs survived and migrated to the ischemic area of rats, and some of the transplanted hAECs expressed the neuronal marker MAP2 and the neuronal progenitor marker Nestin, together with the astrocyte marker glial fibrillary acidic protein, and hAEC-EGFP can significantly ameliorate behavioral dysfunction and reduce infarct volume of ischemic rats. By transfecting the cells with lentiviral vectors, GDNF can be stably overexpressed in hAECs, and hAEC-GDNF can more rapidly rescue the deficits of rats after middle cerebral artery occlusion compared with hAEC-EGFP-treated rats. Moreover, the nontransduced cells also had effects comparable to the GDNF-transduced cells on caspase-3 and lesion volume. Because hAECs are in unlimited supply, and their use is not encumbered by ethical arguments, hAECs have a great advantage for stem cell therapy. This model holds tremendous potential for development into wide use in cell-mediated gene therapy in the future.

[Expression and significance of telomerase activity of lens epithelial cells in posterior capsule opacification of rabbits].

OBJECTIVE: To explore the expression of telomerase activity of the lens epithelial cells in posterior capsule opacification of rabbits. METHODS: Clear corneal tunnel phacoemulsification was performed in both eyes of 11 New Zealand rabbits. After the model of posterior capsule opacification succeed in all eyes. Then, telomerase activity of the lens epithelial cells in the equator and posterior capsule opacification of 20 eyes was detected with TRAP-ELISA and TRAP-PAGE techniques. RESULTS: HepG2 cells were used as positive controls. Telomerase activity was detected in the lens epithelial cells in the equator capsule and posterior capsule opacification in rabbits, which showed several dim gradient stripped electrophoresis pattern. A450 - 690 value of telomerase activity in the equator capsule and posterior capsule opacification was 0.85 +/- 0.23 and 0.67 +/- 0.19, respectively, indicating statistically significant difference (t = 2.526, 0.021; P < 0.05). CONCLUSIONS: Telomerase activity exists in the lens epithelial cells of posterior capsule opacification in rabbits. The telomerase activity in this area is lower than that in the equator capsule.