Photoreceptor Transplantation in Late Stage Retinal Degeneration

The recent advances in cell-based therapies for the repair of the pigmented epithelium is providing additional impetus for the translation of photoreceptor transplantation to eventual clinical trials. The prospects for transplantation of photoreceptors as a potential therapy for the treatment of photoreceptor degeneration will depend on successfully addressing many critical issues in preclinical studies. Although most of the studies that have carried out transplants of photoreceptors have primarily used normal mice, there have been recent reports that have also shown some success following transplantation to mouse models of retinitis pigmentosa. However, while these results are promising, there are several key issues that require further investigation in order to better understand the optimum timing for transplantation, given the extensive remodeling of the retina that occurs in late stage disease

Identification of Novel Glial Genes by Single-Cell Transcriptional Profiling of Bergmann Glial Cells from Mouse Cerebellum

Bergmann glial cells play critical roles in the structure and function of the cerebellum. During development, their radial processes serve as guides for migrating granule neurons and their terminal endfeet tile to form the glia limitans. As the cerebellum matures, Bergmann glia perform important roles in synaptic transmission and synapse maintenance, while continuing to serve as essential structural elements. Despite growing evidence of the diverse functions of Bergmann glia, the molecular mechanisms that mediate these functions have remained largely unknown. As a step toward identifying the molecular repertoire underlying Bergmann glial function, here we examine global gene expression in individual Bergmann glia from developing (P6) and mature (P30) mouse cerebellum. When we select for developmentally regulated genes, we find that transcription factors and ribosomal genes are particularly enriched at P6 relative to P30; whereas synapse associated molecules are enriched at P30 relative to P6. We also analyze genes expressed at high levels at both ages. In all these categories, we find genes that were not previously known to be expressed in glial cells, and discuss novel functions some of these genes may potentially play in Bergmann glia. We also show that Bergmann glia, even in the adult, express a large set of genes thought to be specific to stem cells, suggesting that Bergmann glia may retain neural precursor potential as has been proposed. Finally, we highlight several genes that in the cerebellum are expressed in Bergmann glia but not astrocytes, and may therefore serve as new, specific markers for Bergmann glia

Generation, Purification and Transplantation of Photoreceptors Derived from Human Induced Pluripotent Stem Cells

Background: Inherited and acquired retinal degenerations are frequent causes of visual impairment and photoreceptor cell replacement therapy may restore visual function to these individuals. To provide a source of new retinal neurons for cell based therapies, we developed methods to derive retinal progenitors from human ES cells. Methodology/Physical Findings: In this report we have used a similar method to direct induced pluripotent stem cells (iPS) from human fibroblasts to a retinal progenitor fate, competent to generate photoreceptors. We also found we could purify the photoreceptors derived from the iPS cells using fluorescence activated cell sorting (FACS) after labeling photoreceptors with a lentivirus driving GFP from the IRBP cis-regulatory sequences. Moreover, we found that when we transplanted the FACS purified iPSC derived photoreceptors, they were able to integrate into a normal mouse retina and express photoreceptor markers. Conclusions: This report provides evidence that enriched populations of human photoreceptors can be derived from iPS cells

Pulmonary giant cells and their significance for the diagnosis of asphyxiation

This study was performed to prove whether the detection of polynuclear giant cells in lungs is useful for the diagnosis of asphyxiation due to throttling or strangulation. Therefore, lung specimens of 54 individuals with different natural and unnatural causes of death were investigated. In most lungs examined numerous alveolar macrophages with 1-2 nuclei were found. Polynuclear giant cells, which were arbitrarily defined as alveolar macrophages containing 3 or more nuclei, were observed in all groups investigated except in the cases of hypoxia due to covering the head with plastic bags. Apparent differences between the other groups in particular an increased number in cases of throttling or strangulation, could not be observed. Immunohistochemical investigations confirmed the hypothesis that the observed polynuclear giant cells were derived from alveolar macrophages. The immunohistochemical analysis of the proliferation marker antigen Ki 67 revealed no positive reaction in the nuclei of polynuclear giant cells indicating that these cells had not developed shortly before death by endomitosis as an adaptative change following reduction in oxygen supply. The results provide evidence that the detection of pulmonary polynuclear giant cells cannot be used as a practical indicator for death by asphyxiation due to throttling or strangulation

Epigenetic Modifiers Are Necessary but Not Sufficient for Reprogramming Non-Myelinating Cells into Myelin Gene-Expressing Cells

Modifications on specific histone residues and DNA methylation play an essential role in lineage choice and cellular reprogramming. We have previously shown that histone modifications or combinatorial codes of transcription factors (TFs) are critical for the differentiation of multipotential progenitors into myelinating oligodendrocytes. In this study we asked whether combining global manipulation of DNA methylation and histone acetylation together with the expression of oligodendrocyte-specific TFs, was sufficient to switch the identity of fibroblasts into myelin gene-expressing cells.Transfection of six oligodendrocyte-specific TFs (Olig1, Olig2, Sox10, Mash1, E47 and Nkx2.2) into NIH3T3 fibroblasts was capable of inducing expression of myelin gene promoter-driven reporters, but did not activate endogenous myelin gene expression. These results suggested the existence of a transcriptionally incompetent chromatin conformation in NIH3T3 fibroblasts. Using chromatin immunoprecipitation (ChIP) analysis, we compared the histone code on the conserved regions of myelin genes (i.e. Mbp and Mag) in differentiating oligodendrocyte progenitors and NIH3T3 fibroblasts. Chromatin at myelin gene loci was characterized by the presence of repressive histone modifications (me3K9H3 and me3K27H3) in NIH3T3 fibroblasts and active histone marks (me3K4H3 and AcH3) in oligodendrocyte lineage cells. To induce a transcriptionally competent chromatin signature, NIH3T3 fibroblasts were treated with 5-azadeoxy-citidine (5-AzaC) to decrease DNA methylation, and trichostatin A (TSA) or sirtinol, to favor histone acetylation. Treatment with 5-AzaC/TSA but not sirtinol, resulted in the detection of endogenous myelin gene transcripts in fibroblasts, although not to the levels detected in myelinating cells. Transfection of oligodendrocyte-specific TFs after 5-AzaC/TSA treatment did not further increase myelin gene expression, nor did it reprogram the transcriptional network of NIH3T3 fibroblasts into that of oligodendrocytes.These results suggest that reprogramming of fibroblasts into myelin gene-expressing cells not only requires transcriptional activation, but also chromatin manipulations that go beyond histone acetylation and DNA methylation

A Quantitative Method to Analyze Drosophila Pupal Eye Patterning

BACKGROUND:The Drosophila pupal eye has become a popular paradigm for understanding morphogenesis and tissue patterning. Correct rearrangement of cells between ommatidia is required to organize the ommatidial array across the eye field. This requires cell movement, cell death, changes to cell-cell adhesion, signaling and fate specification. METHODOLOGY:We describe a method to quantitatively assess mis-patterning of the Drosophila pupal eye and objectively calculate a 'mis-patterning score' characteristic of a specific genotype. This entails step-by-step scoring of specific traits observed in pupal eyes dissected 40-42 hours after puparium formation and subsequent statistical analysis of this data. SIGNIFICANCE:This method provides an unbiased quantitative score of mis-patterning severity that can be used to compare the impact of different genetic mutations on tissue patterning