Sequential changes in the gene expression profile of murine retinal progenitor cells during the induction of differentiation

PurposeFollowing transplantation, cultured retinal progenitor cells (RPCs) integrate into the diseased host retina and exhibit morphologies and markers indicative of local cellular phenotypes. In vitro analysis of cultured RPCs allows detailed examination of marker gene expression during the initial phase of differentiation and can provide insight into the variables influencing this process.MethodsUsing cultured murine RPCs, this study compares the effects of fetal bovine serum (FBS) with those of ciliary neurotrophic factor (CNTF), individually or in combination with epidermal growth factor (EGF). Differentiation was assessed by way of the relative expression of 17 genes using quantitative PCR (qPCR) at five time points over a seven-day period.ResultsBoth CNTF and FBS rapidly altered the gene expression of RPCs, with very marked upregulation of glial fibrillary acidic protein (GFAP; FBS>CNTF) and marked down-regulation of the proliferation marker Ki-67, consistent with the induction of differentiation. The evidence supports a preponderantly pro-glial influence for both the FBS and CNTF, however, neuronal markers were also upregulated to a lesser extent. Immunocytochemistry confirmed subpopulations labeling with neuronal markers, including rhodopsin. In the presence of sustained EGF stimulation, the differentiating influences of both FBS and CNTF remained perceptible as transient peaks of relative gene expression, but were markedly diminished overall.ConclusionsThis study shows that it is possible to compare the relative efficacy of in vitro differentiation protocols using murine RPCs and qPCR. The differentiating influences of both serum and CNTF were confirmed, but shown to be powerfully moderated by EGF. This suggests that EGF withdrawal is the dominant feature of these differentiation protocols and that exposure to either serum or CNTF is insufficient to irreversibly commit a cultured RPC population to terminal differentiation unless accompanied by concomitant cessation of mitogenic stimulation

Feline Neural Progenitor Cells II: Use of Novel Plasmid Vector and Hybrid Promoter to Drive Expression of Glial Cell Line-Derived Neurotrophic Factor Transgene

Sustained transgene expression is required for the success of cell transplant-based gene therapy. Most widely used are lentiviral-based vectors which integrate into the host genome and thereby maintain sustained transgene expression. This requires integration into the nuclear genome, and potential risks include activation of oncogenes and inactivation of tumor suppressor genes. Plasmids have been used; however lack of sustained expression presents an additional challenge. Here we used the pCAG-PyF101-eGFP plasmid to deliver the human GDNF gene to cat neural progenitor cells (cNPCs). This vector consists of a CAGG composite promoter linked to the polyoma virus mutant enhancer PyF101. Expression of an episomal eGFP reporter and GDNF transgene were stably maintained by the cells, even following induction of differentiation. These genetically modified cells appear suitable for use in allogeneic models of cell-based delivery of GDNF in the cat and may find veterinary applications should such strategies prove clinically beneficial

Xenotransplantation of Human Neural Progenitor Cells to the Subretinal Space of Nonimmunosuppressed Pigs

To investigate the feasibility of transplanting human neural progenitor cells (hNPCs) to the retina of nonimmunosuppressed pigs, cultured hNPCs were injected into the subretinal space of 5 adult pigs after laser burns were applied to promote donor cell integration. Postoperatively, the retinal vessels appeared normal without signs of exudation, bleeding, or subretinal elevation. Eyes were harvested at 10–28 days. H&E consistently showed mild retinal vasculitis, depigmentation of the RPE, and marked mononuclear cell infiltrate in the choroid adjacent to the site of transplantation. Human-specific antibodies revealed donor cells in the subretinal space at 10–13 days and smaller numbers within the retina on days 12 and 13, with evidence suggesting a limited degree of morphological integration; however, no cells remained at 4 weeks. The strong mononuclear cell reaction and loss of donor cells indicate that modulation of host immunity is likely necessary for prolonged xenograft survival in this model

Retinal tissue engineering using mouse retinal progenitor cells and a novel biodegradable, thin-film poly(e-caprolactone) nanowire scaffold

Retinal progenitor cells (RPCs) can be combined with nanostructured polymer scaffolds to generate composite grafts in culture. One strategy for repair of diseased retinal tissue involves implantation of composite grafts of this type in the subretinal space. In the present study, mouse retinal progenitor cells (RPCs) were cultured on laminin-coated novel nanowire poly(e-caprolactone)(PCL) scaffolds, and the survival, differentiation, and migration of these cells into the retina of C57bl/6 and rhodospsin −/− mouse retinal explants and transplant recipients were analyzed. RPCs were cultured on smooth PCL and both short (2.5 μm) and long (27 μm) nanowire PCL scaffolds. Scaffolds with adherent mRPCs were then either co-cultured with, or transplanted to, wild-type and rhodopsin −/− mouse retina. Robust RPC proliferation on each type of PCL scaffold was observed. Immunohistochemistry revealed that RPCs cultured on nanowire scaffolds increased expression of mature bipolar and photoreceptor markers. Reverse transcription polymerase chain reaction revealed down-regulation of several early progenitor markers. PCL-delivered RPCs migrated into the retina of both wild-type and rhodopsin knockout mice. The results provide evidence that RPCs proliferate and express mature retinal proteins in response to interactions with nanowire scaffolds. These composite grafts allow for the migration and differentiation of new cells into normal and degenerated retina

Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors Restores Retinal Structure and Function in Degenerative Mice

This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs), could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease

Between seas and continents: aspects of the scientific career of Hermann Von Ihering, 1850-1930

This paper covers some periods in Hermann von Ihering’s scientific trajectory: his training in zoology in Germany and Naples, his international activities based in Brazil, and his return to Germany. It deals with aspects of the formulation of his theories on land bridges. It focuses on the network of contacts he maintained with German émigrés like himself, and primarily with Florentino Ameghino, which allowed him to interact in international scientific circles. It mentions excerpts of his letters and his publications in the periods when he began corresponding with Ameghino (1890), when he travelled to Europe in search of support for his theories (1907), and when he published his book on the history of the Atlantic Ocean (1927).Facultad de Ciencias Naturales y Muse

A genomic catalog of Earth’s microbiomes

The reconstruction of bacterial and archaeal genomes from shotgun metagenomes has enabled insights into the ecology and evolution of environmental and host-associated microbiomes. Here we applied this approach to >10,000 metagenomes collected from diverse habitats covering all of Earth’s continents and oceans, including metagenomes from human and animal hosts, engineered environments, and natural and agricultural soils, to capture extant microbial, metabolic and functional potential. This comprehensive catalog includes 52,515 metagenome-assembled genomes representing 12,556 novel candidate species-level operational taxonomic units spanning 135 phyla. The catalog expands the known phylogenetic diversity of bacteria and archaea by 44% and is broadly available for streamlined comparative analyses, interactive exploration, metabolic modeling and bulk download. We demonstrate the utility of this collection for understanding secondary-metabolite biosynthetic potential and for resolving thousands of new host linkages to uncultivated viruses. This resource underscores the value of genome-centric approaches for revealing genomic properties of uncultivated microorganisms that affect ecosystem processes

2017 Research & Innovation Day Program

A one day showcase of applied research, social innovation, scholarship projects and activities.https://first.fanshawec.ca/cri_cripublications/1004/thumbnail.jp