Immunoreactivity of Pluripotent Markers SSEA-5 and L1CAM in Human Tumors, Teratomas, and Induced Pluripotent Stem Cells

Pluripotent stem cell markers can be useful for diagnostic evaluation of human tumors. The novel pluripotent marker stage-specific embryonic antigen-5 (SSEA-5) is expressed in undifferentiated human induced pluripotent cells (iPSCs), but little is known about SSEA-5 expression in other primitive tissues (e.g., human tumors). We evaluated SSEA-5 immunoreactivity patterns in human tumors, cell lines, teratomas, and iPS cells together with another pluripotent cell surface marker L1 cell adhesion molecule (L1CAM). We tested two hypotheses: (1) SSEA-5 and L1CAM would be immunoreactive and colocalized in human tumors; (2) SSEA-5 and L1CAM immunoreactivity would persist in iPSCs following retinal differentiating treatment. SSEA-5 immunofluorescence was most pronounced in primitive tumors, such as embryonal carcinoma. In tumor cell lines, SSEA-5 was highly immunoreactive in Capan-1 cells, while L1CAM was highly immunoreactive in U87MG cells. SSEA-5 and L1CAM showed colocalization in undifferentiated iPSCs, with immunopositive iPSCs remaining after 20 days of retinal differentiating treatment. This is the first demonstration of SSEA-5 immunoreactivity in human tumors and the first indication of SSEA-5 and L1CAM colocalization. SSEA-5 and L1CAM warrant further investigation as potentially useful tumor markers for histological evaluation or as markers to monitor the presence of undifferentiated cells in iPSC populations prior to therapeutic use

Human embryonic and neuronal stem cell markers in retinoblastoma

Retinoblastoma (RB) is the most common intraocular tumor of early childhood. The early onset of RB, coupled with our previous findings of cancer stem cell characteristics in RB, led us to hypothesize that subpopulations of RB tumors harbor markers and behaviors characteristic of embryonic and neuronal origin. Our RB sources included: human pathological tissues, and the human RB cell lines Y79 and WERI-RB27. Microarray screening, single and dual-label immunocytochemistry and RT-PCR were performed to detect embryonic and neuronal stem cell markers, such as Oct3/4, Nanog, CD133, and Musashi-1. To test for functional evidence of stem cell behavior, we examined RB cells for their ability to form neurospheres and retain BrdU label as indicators of self-renewal and slow cell cycling, respectively. Microarray comparisons of human RB tumors with normal retinal tissue detected upregulation of a number of genes involved in embryonic development that were also present in Y79 cells, including Oct3/4, Nanog, Musashi-1 and Musashi-2, prominin-1 (CD133), Jagged-2, Reelin, Thy-1, nestin, Meis-1,NCAM, Patched, and Notch4. Expression of Musashi-1, Oct3/4 and Nanog was confirmed by immunostaining and RT-PCR analyses of RB tumors and RB cell lines. CD133 expression was confirmed by PCR analysis. Y79 and WERI-RB27 contained populations of Hoechst-dim/ABCG2-positive cells that co-localized with embryonic stem cell markers Oct3/4-ABCG2 and Nanog-ABCG2. Subpopulations of Y79 and WERI-RB27 cells were label-retaining (as seen by BrdU incorporation) and were able to generate neurospheres, both hallmarks of a stem cell phenotype. Small subpopulation(s) of RB cells express human embryonic and neuronal stem cell markers. There are also subpopulations that demonstrate functional behavior (label retention and self-renewal) consistent with cancer stem cells. These findings support the hypothesis that RB is a heterogeneous tumor comprised of subpopulation(s) with stem cell-like properties

High-contrast enzymatic immunohistochemistry of pigmented tissues

Historically, standard enzyme immunohistochemistry has been accomplished with brown (DAB, diaminobenzidine) substrate. This can become problematic in pigmented tissues, such as the retina, where brown pigment of retinal pigmented epithelial (RPE) cells can be easily confounded with brown substrate.  Although immunofluorescence detection methods can overcome this challenge, fluorescence may fade over a period of weeks, while enzyme substrates allow for more long-lasting, archival results. In this report, we will describe a high-contrast enzyme immunohistochemistry method ideal for pigmented tissues that utilizes purple (VIP) substrate. We compared brown (DAB) and purple (VIP) substrates in enzyme immunohistochemistry experiments using human retina (paraffin sections) and monkey retinal pigmented epithelial cells (frozen sections), both containing brown pigmented cells. We compared substrates using several primary antibodies against markers that can be detected in the retina, including GFAP, VEGF, CD147 (EMMPRIN), RHO (rhodopsin) and PAX6.  Methyl green was used as a counterstain for paraffin sections. A side-by-side comparison between DAB and VIP immunohistochemistry showed excellent contrast between pigmented cells and the purple VIP substrate in both human retinal tissue and monkey pigmented epithelial cells for all of the markers tested. This was a marked improvement over DAB staining in pigmented cells and tissues. For both paraffin sections and frozen sections of pigmented tissues, purple VIP substrate is an excellent alternative to brown DAB substrate and non-permanent immunofluorescence methods

Effects of Cyclooxygenase Inhibitors on Apoptotic Neuroretinal Cells

Glaucoma is characterized by a loss of retinal ganglion cells (RGC) which is associated with a decrease of visual function. Neuroprotective agents as a new therapeutic strategy could prevent the remaining neurons from apoptotic cell death. Previous studies have shown the involvement of the Cyclooxygenase (COX)-2 signalling in the apoptotic death of neurons. Herein we investigated the neuroprotective effect of COX-1/COX-2- and selective COX-2- inhibitors on apoptotic. R28, a neuroretinal cell line and determined the PGE 2 levels by ELISA. Furthermore we investigated differences in protein expression in the cells after exposure to elevated pressure compared to untreated cells by ProteinChip analysis. In addition, a protein profiling study of the cells after exposure to elevated pressure was performed. The protein expression profiles were measured by SELDI-TOF (Surface Enhanced Laser Desorption/Ionization-time of flight) Protein Chips. The protein identification was performed by mass spectrometry (MS). It could be shown that COX-2 inhibition significantly prevented the cells from apoptosis and reduced the PGE 2 concentrations. Selective COX-2 inhibitors were significant more potent than non-selective inhibitors or COX-1 inhibitors. We found differently expressed protein patterns in neuroretinal cells cultured at atmospheric pressure compared to those cells exposed to elevated pressure with or without celecoxib respectively. We identified three biomarkers, ubiquitin, HSP10 and NDKB, which were differently expressed in the groups. However, our data indicates a distinct neuroprotective effect of COX-2 inhibition. The local treatment with selective COX-2 inhibitors might provide an innovative strategy of therapeutic interventio

Lithium chloride regulates the proliferation of stem-like cells in retinoblastoma cell lines: a potential role for the canonical Wnt signaling pathway

PURPOSE: Cancer stem cells are found in many tumor types and are believed to lead to regrowth of tumor mass due to their chemoresistance and self-renewal capacity. We previously demonstrated small subpopulations of cells in retinoblastoma tissue and cell lines that display cancer stem cell-like activities, including expression of stem cell markers, Hoechst dye exclusion, slow cycling, and self-renewal ability. Identifying factors regulating stem cell proliferation will be important for selectively targeting stem cells and controlling tumor growth. Wingless and Int1 (Wnt) signaling is an essential cellular communication pathway that regulates proliferation and differentiation of non-neoplastic stem/progenitor cells in the retina and other tissues, but its role in cancer stem cells in the retinal tumor retinoblastoma is unknown. In this study, we investigated whether the Wnt pathway activator lithium chloride (LiCl) regulates proliferation of retinoblastoma cancer stem-like cells. METHODS: The number of stem-like cells in Weri and Y79 retinoblastoma cell line cultures was measured by 5-bromo-2-deoxyuridine (BrdU) pulse-chase, immunohistochemistry, and quantitative polymerase chain reaction (PCR) for stem cell marker genes. The cell lines were sorted into stem-like and non-stem-like populations by fluorescence-activated cell sorting (FACS), using an antibody against the stem cell marker ATP-binding cassette, subfamily G, member 2 (ABCG2). Activated Wnt signaling was measured in the sorted cells by western blotting and immunolocalization of the central mediator β-catenin. RESULTS: LiCl increased the number of stem-like cells, measured by BrdU retention and elevated expression of the stem cell marker genes Nanog, octamer transcription factor 3 and 4 (Oct3/4), Musashi 1 (Msi1), and ABCG2. Sorted ABCG2-positive stem-like cells had higher levels of β-catenin than ABCG2-negative non-stem cells, suggesting elevated canonical Wnt signaling. Furthermore, stem cell marker gene expression increased after small interfering RNA (siRNA) knock-down of the Wnt inhibitor secreted frizzled-related protein 2 (SFRP2). CONCLUSIONS: These results indicate that the cancer stem-like cell population in retinoblastoma is regulated by canonical Wnt/β-catenin signaling, which identifies the Wnt pathway as a potential mechanism for the control of stem cell renewal and tumor formation in retinoblastoma tumors in vivo