Modeling early retinal development with human embryonic and induced pluripotent stem cells.

Human pluripotent stem cells have the potential to provide comprehensive model systems for the earliest stages of human ontogenesis. To serve in this capacity, these cells must undergo a targeted, stepwise differentiation process that follows a normal developmental timeline. Here we demonstrate the ability of both human embryonic stem cells (hESCs) and induced pluripotent stem (iPS) cells to meet these requirements for human retinogenesis. Upon differentiation, hESCs initially yielded a highly enriched population of early eye field cells. Thereafter, a subset of cells acquired features of advancing retinal differentiation in a sequence and time course that mimicked in vivo human retinal development. Application of this culture method to a human iPS cell line also generated retina-specific cell types at comparable times in vitro. Lastly, altering endogenous signaling during differentiation affected lineage-specific gene expression in a manner consistent with established mechanisms of early neural and retinal cell fate determination. These findings should aid in the investigation of the molecular events governing retinal specification from human pluripotent stem cells.

Regulation of prenatal human retinal neurosphere growth and cell fate potential by retinal pigment epithelium and Mash1.

During development of the central nervous system, stem and progenitor cell proliferation and differentiation are controlled by complex inter- and intracellular interactions that orchestrate the precise spatiotemporal production of particular cell types. Within the embryonic retina, progenitor cells are located adjacent to the retinal pigment epithelium (RPE), which differentiates prior to the neurosensory retina and has the capacity to secrete a multitude of growth factors. We found that secreted proteinaceous factors in human prenatal RPE conditioned medium (RPE CM) prolonged and enhanced the growth of human prenatal retinal neurospheres. The growth-promoting activity of RPE CM was mitogen-dependent and associated with an acute increase in transcription factor phosphorylation. Expanded populations of RPE CM-treated retinal neurospheres expressed numerous neurodevelopmental and eye specification genes and markers characteristic of neural and retinal progenitor cells, but gradually lost the potential to generate neurons upon differentiation. Misexpression of Mash1 restored the neurogenic potential of long-term cultures, yielding neurons with phenotypic characteristics of multiple inner retinal cell types. Thus, a novel combination of extrinsic and intrinsic factors was required to promote both progenitor cell proliferation and neuronal multipotency in human retinal neurosphere cultures. These results support a pro-proliferative and antiapoptotic role for RPE in human retinal development, reveal potential limitations of human retinal progenitor culture systems, and suggest a means for overcoming cell fate restriction in vitro.

A novel serum-free method for culturing human prenatal retinal pigment epithelial cells.

PURPOSE: Established techniques for culturing primary human retinal pigment epithelial (RPE) cells have facilitated the laboratory investigation of this multipurpose retinal cell layer. However, most culture methods involve the use of animal serum to establish and maintain RPE monolayers, which can complicate efforts to define and study factors involved in the maturation and function of these cells. Therefore, this study was conducted to develop a simple, serum-free system to propagate and sustain human RPE in vitro. METHODS: RPE was dissected from human prenatal donor eyes and cultured in serum-free defined medium containing the commercially formulated supplement B27 or N2. Cultures were grown initially as adherent tissue sections or suspended spherical aggregates and later expanded and maintained as monolayers. PCR, Western blot analysis, and immunocytochemistry were used to monitor gene and protein expression in established cultures, followed by examination of secretory products in RPE conditioned medium by ELISA and mass spectrometric analysis. RESULTS: In medium supplemented with B27, but not N2, RPE could be expanded up to 40,000-fold over six passages and maintained in culture for more than 1 year. In long-term cultures, typical cellular morphology and pigmentation were observed, along with expression of characteristic RPE markers. RPE monolayers also retained proper apical-basal orientation and secreted multiple factors implicated in the maintenance of photoreceptor health and the pathogenesis of age-related macular degeneration. CONCLUSIONS: Monolayer cultures of human prenatal RPE can be grown and maintained long term in the total absence of serum and still retain the phenotype, gene and protein expression profile, and secretory capacity exhibited by mature RPE cells.

Protection of visual functions by human neural progenitors in a rat model of retinal disease.

BACKGROUND: A promising clinical application for stem and progenitor cell transplantation is in rescue therapy for degenerative diseases. This strategy seeks to preserve rather than restore host tissue function by taking advantage of unique properties often displayed by these versatile cells. In studies using different neurodegenerative disease models, transplanted human neural progenitor cells (hNPC) protected dying host neurons within both the brain and spinal cord. Based on these reports, we explored the potential of hNPC transplantation to rescue visual function in an animal model of retinal degeneration, the Royal College of Surgeons rat. METHODOLOGY/PRINCIPAL FINDINGS: Animals received unilateral subretinal injections of hNPC or medium alone at an age preceding major photoreceptor loss. Principal outcomes were quantified using electroretinography, visual acuity measurements and luminance threshold recordings from the superior colliculus. At 90-100 days postnatal, a time point when untreated rats exhibit little or no retinal or visual function, hNPC-treated eyes retained substantial retinal electrical activity and visual field with near-normal visual acuity. Functional efficacy was further enhanced when hNPC were genetically engineered to secrete glial cell line-derived neurotrophic factor. Histological examination at 150 days postnatal showed hNPC had formed a nearly continuous pigmented layer between the neural retina and retinal pigment epithelium, as well as distributed within the inner retina. A concomitant preservation of host cone photoreceptors was also observed. CONCLUSIONS/SIGNIFICANCE: Wild type and genetically modified human neural progenitor cells survive for prolonged periods, migrate extensively, secrete growth factors and rescue visual functions following subretinal transplantation in the Royal College of Surgeons rat. These results underscore the potential therapeutic utility of hNPC in the treatment of retinal degenerative diseases and suggest potential mechanisms underlying their effect in vivo.

T47D breast cancer cell growth is inhibited by expression of VACM-1, a cul-5 gene.

Vasopressin-activated calcium-mobilizing (VACM-1), a cul-5 gene, is localized on chromosome 11q22-23 close to the gene for Ataxia Telangiectasia in a region associated with a loss of heterozygosity in breast cancer tumor samples. To examine the biological role of VACM-1, we studied the effect of VACM-1 expression on cellular growth and gene expression in T47D breast cancer cells. Immunocytochemistry studies demonstrated that VACM-1 was expressed in 0.6-6% of the T47D cells and localized to the nucleus of mitotic cells. Overexpressing VACM-1 significantly attenuated cellular proliferation and MAPK phosphorylation when compared to the control cells. In addition, VACM-1 decreased egr-1 and increased Fas-L mRNA levels. Further, egr-1 protein levels were significantly lower in the nuclear fraction from VACM-1 transfected cells when compared to controls. These data indicate that VACM-1 is involved in the regulation of cellular growth.

Expression of the receptor tyrosine kinase Axl promotes ocular melanoma cell survival.

Metastatic tumor cells originating from cancers of a variety of tissues such as breast, skin, and prostate may remain dormant for long periods of time. In the case of uveal melanoma, the principal malignancy of the eye, complete removal of the primary tumor by enucleation can nonetheless be followed by metastatic tumor growth in distant organs months, years, or even decades later. This suggests that tumor cells have already spread to secondary sites at the time of treatment and remain dormant as micrometastases. Identifying factors that govern long-lived survival of metastatic tumor cells is therefore key to decreasing mortality associated with this and other diseases. While investigating factors differentially expressed in melanoma cells and normal melanocytes, we identified the receptor tyrosine kinase Axl and found up-regulation of Axl in uveal melanomas and melanoma cell lines by RNase protection, Western analysis, and immunohistochemistry. Axl has been shown to mediate cell growth and survival through its ligand Gas6 in non-transformed cells. To test whether stimulation of Axl can enhance survival of uveal melanoma cells, we assessed the degree of mitogenesis and cell survival by bromodeoxyuridine incorporation and trypan blue exclusion, respectively, upon stimulation of Mel 290 uveal melanoma cells with Gas6 in vitro. We show that Gas6 mediates mitogenesis and cell survival in Mel 290 cells. We further demonstrate that these effects occur specifically through the Axl receptor by modulating the expression of Axl with an antisense construct. cDNA microarray analysis of 12,687 genes then revealed that Gas6 stimulation of Axl in Mel 290 cells results primarily in the down-regulation of Cyr61, a member of the CCN protein family involved in tumor progression. These data show that the Axl pathway mediates increased survival of uveal melanoma cells, potentially advantageous during cancer dormancy, and that Axl may function in part through regulation of Cyr61.