Showing papers by "Clive N. Svendsen published in 2008"

Long-term vision rescue by human neural progenitors in a rat model of photoreceptor degeneration.

Abstract: The photoreceptor degeneration seen in age-related macular degeneration (AMD) and retinitis pigmentosa (RP) represents the major cause of blindness in developed countries, for which no cure is available1–3 Cell-based therapies have been shown effective in rescuing vision in animal models of retinal degeneration4–9 For donor cells to be suitable in a clinical setting they should be human derived, must be effective in reversing or slowing the degenerative events, must be readily renewable and not senescent, and must be effective over a long period In a series of studies, we have used the Royal College of Surgeons (RCS) rat to explore the efficacy of cell-based therapies in rescuing vision In this animal, the retinal pigment epithelial cell (RPE) fails to phagocytose shed outer segment material at a normal rate because of a mutation in the Mertk gene10 This results in an accumulation of outer segment debris and subsequently leads to photoreceptor cell loss11,12 Although comparable defects have been seen in a cohort of patients with retinitis pigmentosa,13 the animal does serve to some degree as a model for photoreceptor loss caused by defective or dysfunctional RPE and for retinitis pigmentosa in general In previous work,14 we showed that human cortical neural progenitor cells (hNPCctx) rescued vision at near normal levels when injected into the subretinal space of the RCS rat Some-what unexpectedly, they formed an RPE-like layer between photoreceptors and the host RPE layer and migrated to the retina Given the potential for such cells to provide a therapy for degenerative diseases throughout the central nervous system,15 including the retina, we felt it important to evaluate the long-term effects of grafting Critical questions that remained centered on the duration of cell survival and vision rescue and on the long-term behavior of and host response to the transplanted cells To answer these questions, we observed a new group of rats that underwent transplantation at postnatal day (P) 21; functional tests were conducted at approximately P90, P150, and P280 A crucial part of the study was to observe the performance of individual rats at three times Some rats were observed separately for validation of the previous morphologic results at the earlier times and to provide a context for the later data

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

Abstract: 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.

Normal Neurogenesis but Abnormal Gene Expression in Human Fragile X Cortical Progenitor Cells

Abstract: Human stem and progenitor cells offer an innovative way to study early events in development. An exciting new opportunity for these cells is their application to study the underlying developmental ...

A high concentration of epidermal growth factor increases the growth and survival of neurogenic radial glial cells within human neurosphere cultures.

Abstract: Human neural progenitor cells (hNPC) isolated from the fetal cortex can be expanded as aggregates of cells termed neurospheres. Traditional methods have used 20 ng/ml epidermal growth factor (EGF) to drive the proliferation of these cells. Here, we show that 100 ng/ml EGF can significantly increase growth rates of hNPC at later passages. This was through increased survival of dividing cells rather than increased proliferation and associated with prolonged activation of ErbB2 and phosphorylated Akt. High EGF also resulted in a larger proportion of elongated "radial glial"-like cells within the growing neurospheres and increased expression of the radial glial markers. The number of new neurons generated from cultures maintained in 100 ng/ml EGF was significantly higher than from 20 ng/ml EGF. Thus, high concentrations of EGF increase the survival of a highly neurogenic human radial glial cell.

Regulation of Prenatal Human Retinal Neurosphere Growth and Cell Fate Potential by Retinal Pigment Epithelium and Mash1

Abstract: 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.

Isolating, expanding, and infecting human and rodent fetal neural progenitor cells.

Abstract: Neural progenitor cells have tremendous utility for understanding basic developmental processes, disease modeling, and therapeutic intervention. The protocols described in this unit provide detailed information to isolate and expand human and rodent neural progenitor cells in culture for several months as floating aggregates (termed neurospheres) or plated cultures. Detailed protocols for cryopreservation, neural differentiation, exogenous gene expression using lentivirus, and transplantation into the rodent nervous system are also described. Curr. Protoc. Stem Cell Biol. 6:2D.2.1-2D.2.16. © 2008 by John Wiley & Sons, Inc. Keywords: stem cells; brain; in vitro; mouse; rat; embryonic; neural progenitor cells

Encyclopedia of stem cell research

Abstract: ENCYCLOPEDIA OF STEM CELL RESEARCH , ENCYCLOPEDIA OF STEM CELL RESEARCH , کتابخانه مرکزی دانشگاه علوم پزشکی تهران