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Sjögren’s syndrome (SS) is a systemic autoimmune disease delineated by chronic lymphocytic infiltrates into the lacrimal or salivary glands, leading to severe dry eye and dry mouth. Mesenchymal stem cells have been shown to be effective in treating numerous autoimmune diseases. This study aimed to illustrate the effects of mesenchymal stem cells on the attenuation of dry eyes (DE) through the inhibition of autophagy markers in a SS mouse model. NOD/ShiLtJ female mice with developed DE were treated with either subconjunctival or lacrimal gland injections of hMSCs (Catholic MASTER Cells). After maintenance for 14 days, clinical DE markers such as tear secretion and corneal staining were observed, as well as goblet cell counts in the conjunctiva, infiltration of inflammatory foci, B and T cells, and autophagy markers in the lacrimal glands. Proinflammatory cytokine expressions of the cornea and conjunctiva, as well as the lacrimal glands, were examined. Clinical markers, such as tear secretion and corneal stain scores, goblet cell counts in the conjunctiva, and foci infiltrations in the lacrimal glands were attenuated in mice treated with subconjunctival or lacrimal gland injections of hMSCs compared to the PBS-treated control group. B cell marker B220 decreased in the lacrimal glands of hMSCs-treated mice, as well as reduced proinflammatory cytokine expressions in the lacrimal glands and cornea. Notably, expression of autophagy markers ATG5 and LC3B-II, as well as HIF-1α and mTOR which play roles in the pathways of autophagy modulation, were shown to be attenuated in the lacrimal glands of hMSCs-treated mice compared to the PBS-treated control mice. Treatment with hMSCs by lacrimal gland or subconjunctival injection demonstrated the alleviation of DE through the repression of autophagy markers, suggesting the therapeutic potentials of hMSCs in a SS mouse model.

Allergic conjunctivitis is mediated by eosinophilic infiltration and Th2 type immune responses. This study aims to elucidate the role of rapamycin, mTOR inhibitor, on OVA-induced experimental allergic conjunctivitis (EAC). Rapamycin administration intraperitoneally markedly reduced clinical signs, total and OVA-specific IgE and IgG1/G2a ratio in serum, and conjunctival eosinophilic infiltration. Infiltrations of CD11c+ dendritic cells and CD4+ T cells, and the expressions of chemokines and adhesion molecules in the conjunctiva were attenuated in rapamycin-treated mice, as well as decreased Th1 and Th2 cytokines in the cervical lymph nodes compared to non-treated mice. The expression of mTOR signaling proteins was increased in EAC and reduced by rapamycin treatment. Topical application of rapamycin was also proved to show reduced clinical signs, eosinophil infiltration, and Th2 type immune responses comparable to those from intraperitoneal injection of rapamycin. These findings suggest the therapeutic implications of rapamycin in the attenuation of allergic conjunctivitis.

Autophagy is known to be implicated in the pathogenesis of Sjögren syndrome (SS), but evidences are limited. We aimed to examine the levels of autophagy markers in tear film and conjunctival epithelial cells from SS dry eye patients, and analyze their correlations with clinical features. Patients with SS dry eye exhibited lower Schirmer values, lower tear breakup time, and higher ocular staining scores. In tears, ATG5 and LC3B-II/I levels were significantly higher in SS dry eye. ATG5 and LC3B-II mRNA in the conjunctiva were also elevated in SS dry eye compared with non-SS dry eye. The immunostaining of conjunctival epithelium showed a punctate pattern of ATG5 and LC3B-II in SS dry eye. These staining patterns were also observed in the lacrimal gland of SS animal models. ATG5 levels in tears and the conjunctival epithelium strongly correlated with ocular staining scores, and one month of topical corticosteroid treatment reduced both ATG5 and LC3B-II/I levels in tear film and the conjunctival epithelium of patients with SS dry eye. Our results suggest that autophagy is enhanced or dysregulated in SS and autophagy markers may be serve as both diagnostic and therapeutic biomarkers in SS dry eye.

Allergic conjunctivitis is an inflammatory eye disease mediated by Th2 type immune response. The role of extracellular superoxide dismutase 3 (SOD3) in immune response and allergic conjunctival inflammation was examined in a murine model for experimental allergic conjunctivitis (EAC). Allergic conjunctivitis was induced in mice by allergen challenge with ovalbumin in alum via the conjunctival sac. SOD3 was topically applied and allergy indicators were compared. Clinical signs associated with conjunctivitis, such as OVA-specific IgE production, IgG1/G2a ratio and eosinophil infiltration, were drastically reduced in mice treated with SOD3. They also had less dendritic cells and CD4+ T cells in conjunctiva than controls. Attenuated allergic inflammation was accredited to reduced Th2 type cytokine responses and increased Treg cytokine in draining lymph node. The characteristics of EAC were attributed to the absence of SOD3. Our findings suggest that SOD3 might be considered as a potential target for Th2-driven allergic conjunctival inflammation.

Photo and video microscopy expand the utility of microscopic observations in education, but attachments needed for this have been prohibitively expensive or too fragile to allow students individual access to these techniques. We describe a do-it-yourself method using inexpensive materials that allows students to build an adaptor that will allow them to turn their cell phones into “CellCams” that they can use to capture microscopic images and videos. Activities are presented that give students ways to learn about concepts of microscopy using their self-collected images.

Aim: To show that human embryonic stem cells (hESCs) can be efficiently differentiated into oligodendrocyte precursor cells (OPCs) in a xeno-free medium with a specific medium supplement and specific human recombinant growth factors. Materials & methods: The xeno-free OPC-differentiation medium for pluripotent stem cells was developed by using StemPro®neural stem cell xeno-free medium supplement together with human recombinant growth factors SHH, PDGF-AA, IGF-1, EGF, basic FGF and CNTF, in addition to RA, T3, human laminin and ascorbic acid. We analyzed the differentiated hESC-derived OPCs and oligodendrocytes with quantitative real-time (RT)-PCR, RT-PCR, flow cytometry and immunocytochemistry, and we performed NG2-positive selection for OPC cultures with fluorescence-activated cell sorting. Results: Based on quantitative RT-PCR analysis, OPCs after 9 weeks of differentiation in xeno-free medium expressed OLIG2, SOX10 and NKX2.2 at elevated levels compared with control conditions. According to the flow cytometric analysis, the cells expressed A2B5 (>70%) and NG2 (40–60%) at 5 weeks time point whereas maturing oligodendrocytes expressed O4 (60-80%) at 11 weeks time point. In addition, hESC-derived OPC populations were purified based on NG2-positive selection using fluorescence-activated cell sorting. NG2-positive OPC populations survived and differentiated further into O4 expressing oligodendrocytes in xeno-free medium, and the sorted cell populations were free from pluripotent Tra1–81 and Oct-4 -positive cells. Conclusions: This study confirms that the xeno-free culturing method can support the differentiation and purification of hESC-derived OPC populations and provides an initial step toward safe cell graft production for the future clinical applications.

Human embryonic stem (ES) cells are promising resources for developing new treatments for neurodegenerative diseases. Spinal muscular atrophy (SMA) is one of the leading causes of childhood paralysis and infant mortality. SMA is caused by inactivation of the survival motor neuron-1 (SMN1) gene. The nearly identical SMN2 gene contains a silent polymorphism that disrupts splicing and as a result cannot compensate for loss of SMN1. The SMA Project was established by the National Institute of Neurological Disorders and Stroke (NINDS) as a pilot effort to establish a fully transparent coalition between academics, industry, and government to create a centralized network of shared resources and information to identify and test new SMA therapeutics. As one of the funded projects, the work described here tested the feasibility of generating a SMA cell-based assay using neural lineages derived from human ES cells approved for National Institutes of Health (NIH)-funded research. Minigene cassettes were constructed, employing firefly luciferase or green fluorescent protein (GFP) as reporters for splicing efficiency of SMN1 and/or SMN2 under the control of the SMN1, SMN2, or cytomegalovirus (CMV) promoters. Transient transfection of proliferating neuroprogenitors in a 96-well format with plasmid DNA or adenoviral vectors showed differential levels that correlated with the splicing minigene and the promoter used; luciferase activities with SMN1 splicing minigenes were higher than SMN2, and the CMV promoter generated higher levels of activity than the SMN1 and SMN2 promoters. Our results indicate that human ES cell-derived neuroprogenitors provide a promising new primary cell source for assays of new therapeutics for neurodegenerative diseases.

Multipotent neural stem cells (NSC) have been derived from human embryonic stem cells (hESC) as well as isolated from fetal tissues. However, there have been few exclusive markers of NSC identified to date, and the differences between NSC from various sources are poorly understood. Although cells isolated from these two sources share many important characteristics, it is not clear how closely they are related in terms of gene expression. Here, we compare the gene expression profiles of 11 lines of NSC derived from hESC (ES_NSC), four lines of NSC isolated from fetus (F_NSC), and two lines of restricted progenitors in order to characterize these cell populations and identify differences between NSC derived from these two sources. We showed that ES_NSC were clustered together with high transcriptional similarities but were distinguished from F_NSC, oligodendrocyte precursor cells, and astrocyte precursor cells. There were 17 genes expressed in both ES_NSC and F_NSC whose expression was not identified in restricted neural progenitors. Furthermore, the major differences between ES_NSC and F_NSC were mostly observed in genes related to the key neural differentiation pathways. Here, we show that comparison of global gene expression profiles of ES_NSC, F_NSC, and restricted neural progenitor cells makes it possible to identify some of the common characteristics of NSC and differences between similar stem cell populations derived from hESCs or isolated from fetal tissue. Disclosure of potential conflicts of interest is found at the end of this article.

An understanding of the regulatory mechanisms responsible for pluripotency in embryonic stem cells (ESCs) is critical for realizing their potential in medicine and science. Significant similarities exist among ESCs harvested from different species, yet major differences have also been observed. Here, by cross-species analysis of a large set of functional categories and all transcription factors and growth factors, we reveal conserved and divergent functional landscapes underlining fundamental and species-specific mechanisms that regulate ESC development. Global transcriptional trends derived from all expressed genes, instead of differentially expressed genes alone, were examined, allowing for a higher discriminating power in the functional portrait. We demonstrate that cross-species correlation of transcriptional changes that occur upon ESC differentiation is a powerful predictor of ESC-important biological pathways and functional cores within a pathway. Hundreds of functional modules, as defined by Gene Ontology, were associated with conserved expression patterns but bear no overt relationship to ESC development, suggestive of new mechanisms critical to ESC pluripotency. Yet other functional modules were not conserved; instead, they were significantly up-regulated in ESCs of either species, suggestive of species-specific regulation. The comparisons of ESCs across species and between human ESCs and embryonal carcinoma stem cells suggest that while pluripotency as an essential function in multicellular organisms is conserved throughout evolution, mechanisms primed for differentiation are less conserved and contribute substantially to the differences among stem cells derived from different tissues or species. Our findings establish a basis for defining the “stemness” properties of ESCs from the perspective of functional conservation and variation. The data and analyses resulting from this study provide a framework for new hypotheses and research directions and a public resource for functional genomics of ESCs.

Although embryonic stem (ES) cells are capable of forming any cell type in the body, the mechanisms that control cell type-specific differentiation are largely unknown. In the present study, we examined the process of differentiation to motor neurons and oligodendrocytes from mouse (Olig2GFP) ES cells. Mouse ES cells undergo a sequential process of differentiation over a 3-week period to generate motor neurons and oligodendrocytes. At day 7 of differentiation, Olig2-expressing cells are biased to a neuronal lineage. However, further differentiation (day 32) resulted in the majority of Olig2-expressing cells exhibiting an oligodendrocyte phenotype as well as a reduced ability to make motor neurons. Exposure of human ES cells to Sonic hedgehog (Shh) likewise resulted in enhanced motor neuron differentiation. Our results establish the requirements for directing ES cells to become motor neurons and oligodendrocytes and show that ES cell-derived Olig2 + cells can give rise to both motor neurons and oligodendrocytes, depending on the time at which differentiation is initiated.

The past few years have seen remarkable progress in our understanding of stem cell biology. The wealth of genomic data and the multiplicity of sources have enabled researchers to begin to profile stem cells in detail. In this paper we describe the biological and technical controls necessary to obtain reliable data and the relative merits of various large-scale analytical techniques including microarray, expressed sequence tag enumeration, serial analysis of gene expression and massively parallel signature sequencing. We suggest that while much has been learned, additional information remains to be gleaned by meta-analysis of existing data.

Background Human embryonic stem (ES) cells hold great promise for medicine and science. The transcriptome of human ES cells has been studied in detail in recent years. However, no systematic analysis has yet addressed whether gene expression in human ES cells may be regulated in chromosomal domains, and no chromosomal domains of coexpression have been identified. Results We report the first transcriptome coexpression map of the human ES cell and the earliest stage of ES differentiation, the embryoid body (EB), for the analysis of how transcriptional regulation interacts with genomic structure during ES self-renewal and differentiation. We determined the gene expression profiles from multiple ES and EB samples and identified chromosomal domains showing coexpression of adjacent genes on the genome. The coexpression domains were not random, with significant enrichment in chromosomes 8, 11, 16, 17, 19, and Y in the ES state, and 6, 11, 17, 19 and 20 in the EB state. The domains were significantly associated with Giemsa-negative bands in EB, yet showed little correlation with known cytogenetic structures in ES cells. Different patterns of coexpression were revealed by comparative transcriptome mapping between ES and EB. Conclusion The findings and methods reported in this investigation advance our understanding of how genome organization affects gene expression in human ES cells and help to identify new mechanisms and pathways controlling ES self-renewal or differentiation.