Two major goals of regenerative medicine are to reproducibly transform adult somatic cells into a pluripotent state and to control their differentiation into specific cell fates. Progress toward these goals would be greatly helped by obtaining a complete picture of the RNA isoforms produced by these cells due to alternative splicing (AS) and alternative promoter selection (APS). To investigate the roles of AS and APS, reciprocal exon-exon junctions were interrogated on a genome-wide scale in differentiating mouse embryonic stem (ES) cells with a prototype Affymetrix microarray. Using a recently released open-source software package named AltAnalyze, we identified 144 genes for 170 putative isoform variants, the majority (67%) of which were predicted to alter protein sequence and domain composition. Verified alternative exons were largely associated with pathways of Wnt signaling and cell-cycle control, and most were conserved between mouse and human. To examine the functional impact of AS, we characterized isoforms for two genes. As predicted by AltAnalyze, we found that alternative isoforms of the gene Serca2 were targeted by distinct microRNAs (miRNA-200b, miRNA-214), suggesting a critical role for AS in cardiac development. Analysis of the Wnt transcription factor Tcf3, using selective knockdown of an ES cell-enriched and characterized isoform, revealed several distinct targets for transcriptional repression (Stmn2, Ccnd2, Atf3, Klf4, Nodal, and Jun) as well as distinct differentiation outcomes in ES cells. The findings herein illustrate a critical role for AS in the specification of ES cells with differentiation, and highlight the utility of global functional analyses of AS.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have demonstrated the ability to improve myocardial function following transplantation into an ischemic heart; however, the functional benefits are transient possibly due to poor cell retention. A diagnostic technique that could visualize transplanted hESC-CMs could help to optimize stem cell delivery techniques. Thus, the purpose of this study was to develop a labeling technique for hESCs and hESC-CMs with the FDA-approved contrast agent indocyanine green (ICG) for optical imaging (OI). hESCs were labeled with 0.5, 1.0, 2.0, and 2.5 mg/ml of ICG for 30, 45, and 60 min at 37 degrees C. Longitudinal OI studies were performed with both hESCs and hESC-CMs. The expression of surface proteins was assessed with immunofluorescent staining. hESCs labeled with 2 mg ICG/ml for 60 min achieved maximum fluorescence. Longitudinal studies revealed that the fluorescent signal was equivalent to controls at 120 h postlabeling. The fluorescence signal of hESCs and hESC-CMs at 1, 24, and 48 h was significantly higher compared to precontrast data (p < 0.05). Immunocytochemistry revealed retention of cell-specific surface and nuclear markers postlabeling. These data demonstrate that hESCs and hESC-CMs labeled with ICG show a significant fluorescence up to 48 h and can be visualized with OI. The labeling procedure does not impair the viability or functional integrity of the cells. The technique may be useful for assessing different delivery routes in order to improve the engraftment of transplanted hESC-CMs or other stem cell progenitors.

PURPOSE: Retinal ganglion cells (RGCs) die as a result of axonal injury in a variety of optic neuropathies, including glaucoma. Reactive oxygen species (ROS) act as intracellular signaling molecules and initiate apoptosis in nerve growth factor-deprived sympathetic neurons and axotomized RGCs. Determination of the role of specific ROS relies on the use of small molecule or protein scavengers with various degrees of specificity. The pro- or anti-cell-death effect of several ROS generating and scavenging systems in cultured RGCs was correlated with their activity in cell-free assays. METHODS: Neonatal rat retinas were dissociated and incubated with ROS-generating systems for hydroxyl radical, superoxide anion (O(2)(-)), and H(2)O(2). Scavengers tested were catalase, polyethylene glycol-superoxide dismutase (PEG-SOD), manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), deferoxamine, and U-74389G. Viability of retrogradely labeled RGCs was determined with calcein-AM 24 hours after plating. O(2)(-) and H(2)O(2) scavenging in cell-free assays was measured with dihydroethidium and Amplex Red (Invitrogen, Carlsbad, CA), respectively. RESULTS: Systematic differences were found between ROS scavenging in cell-free assays and the ability of scavengers to protect RGCs in cell culture. Furthermore, many ROS scavengers lost specificity and protected against various ROS, whereas others failed to protect against their unique ROS target. These activities stray from commonly recognized specificities of individual ROS scavengers or generating systems and are important in understanding ROS biology. In addition, antioxidant defense mechanisms used by RGCs and other retinal cells interfere with responses expected from ROS scavengers in well-defined systems. Last, H(2)O(2) induced intramitochondrial O(2)(-), whereas paraquat produced O(2)(-) outside of the mitochondria, and these areas of generation can mislead interpretations of ROS scavenger activity and effectiveness. CONCLUSIONS: There is discordance between ROS effects in cultured RGCs and cell-free assays, with several mechanisms accounting for this divergence. To identify the roles of ROS signaling in cell death accurately, several approaches should be used. These include using a panel of ROS scavengers and generators, testing the panel in primary neuronal cultures, and quantifying ROS with cell-free assays.

Retinal ganglion cells (RGCs) undergo apoptosis after axonal injury, in part regulated by an intracellular superoxide anion burst, for which the target(s) are unknown. Shifting the RGC redox state towards reduction and preventing sulfhydryl oxidation is neuroprotective in vitro and in vivo, implying that one or more sulfhydryls on one or more critical proteins may be involved. We synthesized novel borane-protected analogues of the reductant tris(2-carboxyethyl)phosphine (TCEP) with the intent of increasing cell permeability and improving chemical stability, and tested their ability to increase RGC survival in vitro. Retinal ganglion cells of postnatal day 2-4 Long-Evans rats were retrogradely labeled with 4',6-diamidino-2-phenylindole (DAPI). At postnatal days 11-13 the animals were sacrificed, the retinas enzymatically dissociated and plated on poly-l-lysine-coated 96-well flat-bottomed tissue culture plates for 72h in Neurobasal-A, B27 supplement lacking antioxidants, and TCEP, bis(3-propionic acid methyl ester)phenylphosphine borane complex (PB1),(3-propionic acid methyl ester)diphenylphosphine borane complex (PB2), or three commercially available phosphines. Viable DAPI-positive RGCs were identified by calcein-AM staining. At 72h, PB1 was effective at rescuing acutely axotomized RGCs at concentrations from 1nM to 100muM. RGC survival with 1nM PB1 was 174+/-12% of control (p=0.002). Another compound, PB2, rescued RGCs at 10pM (177+/-24%; p=0.006) and 10nM (251+/-34%; p=0.004) at 72h. A PAMPA assay demonstrated that PB1 and PB2 were substantially more permeable than TCEP. These data demonstrate that modified reductants are effective RGC neuroprotectants at picomolar-nanomolar concentrations. We propose that these novel molecules may act by inhibiting the sulfhydryl oxidation effect of an intracellular superoxide burst.

PURPOSE: Retinal ganglion cells (RGCs) undergo apoptosis after axonal injury. The time course of cell death is variable and depends in part on the degree of injury sustained. Decreasing reactive oxygen species (ROS) levels or shifting the redox state to reduction promotes the survival of RGCs in tissue culture after axotomy. It was hypothesized that a specific ROS, superoxide anion, acts as an intracellular signaling molecule for RGC death after axotomy. METHODS: Intracellular superoxide levels were measured after dissociation in retrograde-labeled rat RGCs with use of the superoxide-sensitive fluorophores hydroethidium and MitoSOX Red. Having found a significant increase, the effect of axotomy was determined on superoxide levels independent of dissociation with an optic nerve crush model. RESULTS: Optic nerve crush caused RGCs to undergo a superoxide burst. The burst was asynchronous and was manifested in only a fraction of cells at any given time. Neurotrophin deprivation was not responsible for the superoxide burst because it was not prevented by incubation with the neurotrophic factors brain-derived neurotrophic factor, ciliary neurotrophic factor, forskolin, or insulin. Several inhibitors of intracellular superoxide generation were studied, but only antimycin A, which inhibits complex III of the mitochondrial electron transport chain, blocked the increase in superoxide. CONCLUSIONS: These findings suggest that superoxide generated in the mitochondrial electron transport chain could be a parallel system to neurotrophic deprivation for signaling cell death after axonal injury.

PURPOSE: Cell lines are frequently used to elucidate mechanisms of disease pathophysiology. Yet extrapolation of results with cell lines to neurodegenerative disorders is difficult because they are mitotic and usually have other non-neuronal properties. The RGC-5 cell line has many features of retinal ganglion cells (RGCs). Despite its expression of Thy-1 and NMDA receptors, as found in primary RGCs, this line's ability to proliferate and non-neuronal appearance differentiate it from other central neurons, complicating its use for the study of neuronal survival, electrophysiology, or neurite extension. METHODS: A method was identified for differentiating RGC-5 cells using the nonspecific protein kinase inhibitor staurosporine. Cultures were treated with 100 nM to 3.16 muM staurosporine and assessed for a variety of differentiation markers. RESULTS: Differentiated RGC-5 cells expressed numerous neuronal properties, including arrest of proliferation without inducing apoptosis, induction of a neuronal morphology, upregulation of neuronal markers, and establishment of outward rectifying channels. Differentiation was not dependent on a single kinase-dependent pathway, based on profiling multiple kinase phosphorylation targets and attempts to replicate differentiation with multiple specific kinase inhibitors. CONCLUSIONS: This method for producing an RGC-like cell from a proliferating cell line facilitates the following previously impractical techniques: high-throughput screening for agents that are neuroprotective or affect ionic channels; straightforward transduction of gene expression in central neurons by nonviral transfection techniques, including production of stable transfectants; biochemical and other assays of pure RGC-like cells without purification on the basis of cell-surface antigens or anatomic location.

PURPOSE: The signaling of retinal ganglion cell (RGC) death after axotomy is partly dependent on the generation of reactive oxygen species. Shifting the RGC redox state toward reduction is protective in a dissociated mixed retinal culture model of axotomy. The hypothesis for the current study was that tris(2-carboxyethyl)phosphine (TCEP), a sulfhydryl reductant, would protect RGCs in a rat optic nerve crush model of axotomy. METHODS: RGCs of postnatal day 4 to 5 Long-Evans rats were retrogradely labeled with the fluorescent tracer DiI. At approximately 8 weeks of age, the left optic nerve of each rat was crushed with forceps and, immediately after, 4 muL of TCEP (or vehicle alone) was injected into the vitreous at the pars plana to a final concentration of 6 or 60 microM. The right eye served as the control. Eight or 14 days after the crush, the animals were killed, retinal wholemounts prepared, and DiI-labeled RGCs counted. Bandeiraea simplicifolia lectin (BSL-1) was used to identify microglia. RESULTS: The mean number of surviving RGCs at 8 days in eyes treated with 60 microM TCEP was significantly greater than in the vehicle group (1250 +/- 156 vs. 669 +/- 109 cells/mm(2); P = 0.0082). Similar results were recorded at 14 days. Labeling was not a result of microglia phagocytosing dying RGCs. No toxic effect on RGC survival was observed with TCEP injection alone. CONCLUSIONS: The sulfhydryl-reducing agent TCEP is neuroprotective of RGCs in an optic nerve crush model. Sulfhydryl oxidative modification may be a final common pathway for the signaling of RGC death by reactive oxygen species after axotomy.

OBJECTIVES: To describe the optic neuropathy associated with the genetic defect in Friedreich ataxia and suggest a pathophysiologic mechanism. METHODS: An experimental model of retinal ganglion cell death in the presence of metal chelation was used to test a hypothetical mechanism for the optic neuropathy of Friedreich ataxia. RESULTS: Study of cultured rat retinal ganglion cells suggests that abnormal regulation of intracellular iron levels could increase sensitivity to reactive oxygen species and lead to cell death in these metabolically active tissues. CONCLUSION: We hypothesize that decreased expression of frataxin, the mutated gene in Friedreich ataxia, could cause an optic neuropathy by increasing the sensitivity of retinal ganglion cells to oxidative stress.