Retinal stem/progenitor properties of iris pigment epithelial cells

Neural stem cells/progenitors that give rise to neurons and glia have been identified in different regions of the brain, including the embryonic retina and ciliary epithelium of the adult eye. Here, we first demonstrate the characterization of neural stem/progenitors in postnatal iris pigment epithelial (IPE) cells. Pure isolated IPE cells could form spheres that contained cells expressing retinal progenitor markers in non-adherent culture. The spheres grew by cell proliferation, as indicated by bromodeoxyuridine incorporation. When attached to laminin, the spheres forming IPE derived cells were able to exhibit neural phenotypes, including retinal-specific neurons. When co-cultured with embryonic retinal cells, or grafted into embryonic retina in vivo, the IPE cells could also display the phenotypes of photoreceptor neurons and Muller glia. Our results suggest that the IPE derived cells have retinal stem/progenitor properties and neurogenic potential without gene transfer, thereby providing a novel potential source for both basic stem cell biology and therapeutic applications for retinal diseases.     

Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells

We previously reported the differentiation of mouse embryonic stem (ES) cells into retinal progenitors. However, these progenitors rarely differentiate into photoreceptors unless they are cultured with embryonic retinal tissues. Here we show the in vitro generation of putative rod and cone photoreceptors from mouse, monkey and human ES cells by stepwise treatments under defined culture conditions, in the absence of retinal tissues. With mouse ES cells, Crx+ photoreceptor precursors were induced from Rx+ retinal progenitors by treatment with a Notch signal inhibitor. Further application of fibroblast growth factors, Shh, taurine and retinoic acid yielded a greater number of rhodopsin+ rod photoreceptors, in addition to default cone production. With monkey and human ES cells, feeder- and serum-free suspension culture combined with Wnt and Nodal inhibitors induced differentiation of Rx+ or Mitf+ retinal progenitors, which produced retinal pigment epithelial cells. Subsequent treatment with retinoic acid and taurine induced photoreceptor differentiation. These findings may facilitate the development of human ES cell-based transplantation therapies for retinal diseases.     

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.     

Differences in the character of interaction of normal and transformed human keratinocytes with laminin isoforms

Laminins constitute a family of heterotrimeric glycoproteins of basement membranes. Laminins promote cell adhesion, migration, growth, and differentiation. So far, at least 12 different isoforms of laminin have been known. However, no sufficient knowledge is available on the nature of cell response on different laminins. The study was aimed to compare adhesive properties of two laminin isoforms, laminin-1 and laminin-2/4, with respect to normal (freshly isolated keratinocytes) and transformed (A-431) human skin cells. We have used the following assays: cell adhesion to the substrate covered with laminin isoformes, interaction of latex beads (D = 1 micron) coated with the same proteins with cells in suspension, and a comparative study of the cytoskeleton structure of cells spread on the immobilized laminins. It was demonstrated that laminin-2/4 is a more effective potent promotor of adhesion for both normal keratinocytes and transformed A-431 cells, compared with laminin-1. A comparison of many attached protein-covered beads allowed to estimate a relative quantity of cell surface receptors to laminin isoforms in different cell types. The relative number of receptors to laminin-2/4 on the keratinocyte surface is 7 times higher than that to laminin-1 after a 30 min incubation with cells, and is 6 times higher after 1 hour. As for A-431 cells, their attachment to laminin-2/4 beads is 5 times higher than that to laminin-1-beads after a 1 min incubation, but as early as after 5 min this distinction disappeared, owing to bead internalization. The presence of a specific receptor to laminin-2/4 but not to laminin-1 on the keratinocyte surface has been suggested. Keratin differences in cytoskeleton organization in normal and transformed skin cells spread on the substrates covered with laminin-1 and laminin-2/4 were demonstrated.     

Selective binding of mouse and human spermatozoa to beads coated with extracellular matrix components

The ability of mouse epididymal and human ejaculated spermatozoa to bind to beads coated with various extracellular matrix components was examined. Mouse spermatozoa preferentially bound to beads coated with heparin (average values ranging between 6.2 and 8.8 sperm per bead were obtained in different experiments) and with chondroitinsulfate (6.2-7.0), and also, although with significant differences across replicate experiments, to beads coated with laminin (7.9-15.6 sperm per bead) and with collagen type I (6.1-18.5). Human spermatozoa bound to collagen-coated beads (15.4-22.6 sperm per bead) and, to a much lower extent, to chondroitin-sulfate-coated beads (3.2-4.7); they were also able to bind heparin-coated beads, although with ample differences between individual sperm donors (ranging between 0.8 and 18.7 sperm per bead). Very few human and mouse sperm bound fibronectin-coated beads; beads coated with albumin, hyaluronic acid, and chondronectin were always totally free of adhering sperm. The possible physiological role of the interactions between spermatozoa and extracellular matrix components are discussed.     

Accumulation of Rhodopsin in Late Endosomes Triggers Photoreceptor Cell Degeneration

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.     

Calnexin is essential for rhodopsin maturation, Ca2+ regulation, and photoreceptor cell survival

In sensory neurons, successful maturation of signaling molecules and regulation of Ca2+ are essential for cell function and survival. Here, we demonstrate a multifunctional role for calnexin as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca2+ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration that is enhanced by light, suggesting that calnexin's function as a Ca2+ buffer is important for photoreceptor cell survival. Our results illustrate a critical role for calnexin in Rh1 maturation and Ca2+ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration.     

Acidic fibroblast growth factor stimulates opsin levels in retinal photoreceptor cells in vitro

It is demonstrated that newborn rat retinal photoreceptor cells can differentiate in monolayer culture, and synthesize de novo photoreceptor-specific proteins such as opsin. When maintained in serum supplemented medium on a laminin substrate, these cells survive for up to 3 weeks. The addition of acidic fibroblast growth factor stimulates an increase in the levels of opsin of 5-10-fold control values, and prolongs cell survival by up to 6 days.     

Murine Müller cells are progenitor cells for neuronal cells and fibrous tissue cells

Mammalian Müller cells have been reported to possess retinal progenitor cell properties and generate new neurons after injury. This study investigates murine Müller cells under in vitro conditions for their capability of dedifferentiation into retinal progenitor cells. Müller cells were isolated from mouse retina, and proliferating cells were expanded in serum-containing medium. For dedifferentiation, the cultured cells were transferred to serum-replacement medium (SRM) at different points in time after their isolation. Interestingly, early cell passages produced fibrous tissue in which extracellular matrix proteins and connective tissue markers were differentially expressed. In contrast, aged Müller cell cultures formed neurospheres in SRM that are characteristic for neuronal progenitor cells. These neurospheres differentiated into neuron-like cells after cultivation on laminin/ornithine cell culture substrate. Here, we report for the first time that murine Müller cells can be progenitors for both, fibrous tissue cells and neuronal cells, depending on the age of the cell culture.     

In vitro growth properties of Xenopus retinal neurons undergo developmental modulation

To determine whether Xenopus retinal neurons undergo intrinsic developmental changes in growth properties, retinal explants from embryos and tadpoles of different stages were grown on laminin, fibronectin, and collagen I in serum-free media. Growth was assayed in terms of a neurite growth index (NGI) and the appearance of clockwise bundles, or a clockwise growth index (CGI). The first neurites from stage 25 optic vesicles are pioneers and display a unique growth phenotype; they emerge rapidly, survive for a short time, show little substrate preferences for growth (they grow almost as well on BSA as they do on laminin and fibronectin), and form no clockwise bundles under any conditions. Neurites from progressively older retinas (stages 32-37) share with stage 25 neurites the rapid outgrowth pattern, but begin to show substrate preferences and clockwise growth. From stage 40 to 50, the mature growth pattern is expressed; a lag in initial outgrowth, long-term survival, distinct substrate preferences (they grow 10 times better on laminin and fibronectin than on BSA) and display robust clockwise growth patterns on laminin and fibronectin. The acquisition of clockwise growth is independent of optic fiber contact with the tectum or exposure to diffusible factors from mature brain tissues. The results suggest that retinal neurons undergo developmental modulation of surface adhesive properties and/or cytoskeletal organization.     

Generation of three-dimensional retinal organoids expressing rhodopsin and S- and M-cone opsins from mouse stem cells

Purpose

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

细胞外间质-整合素在牵张刺激心肌细胞肥大中的作用

应用牵张刺激培养细胞的模型,观察原原、纤维连接蛋白、层粘连素对牵张刺激心肌细胞肥大的影响,探讨细胞外间质－融洽纱受体在超负荷心肌肥大的跨膜信号传导机制中的作用。发现,胶原、纤维连接蛋白、层粘连素明显有助于培养心肌细胞的贴壁、伸展。牵张刺激后,胶原、纤维连接蛋白基质组心肌细胞的＾３Ｈ－亮氨酸掺入率和心肌细胞表面积均显著大于对照组,而层粘连素组无显著变化;可溶性纤维连接蛋白、ＲＧＤ肽均可显著抑制牵张刺     

细胞外间质-整合素在牵张刺激心肌细胞肥大中的作用

应用牵张刺激培养细胞的模型 ,观察胶原、纤维连接蛋白、层粘连素对牵张刺激心肌细胞肥大的影响 ,探讨细胞外间质 -整合素受体在超负荷心肌肥大的跨膜信号传导机制中的作用。结果发现 ,胶原、纤维连接蛋白、层粘连素明显有助于培养心肌细胞的贴壁、伸展。牵张刺激后 ,胶原、纤维连接蛋白基质组心肌细胞的 3H -亮氨酸掺入率和心肌细胞表面积均显著大于对照组 ,而层粘连素组无显著变化 ;可溶性纤维连接蛋白、RGD肽均可显著抑制牵张刺激诱导的培养心肌细胞 (胶原为粘附基质 )的3H -亮氨酸掺入率升高和心肌细胞表面积增大 ,而层粘连素无明显作用。结果表明 ,特异的细胞外间质 -整合素在超负荷心肌肥大机制中发挥了跨膜信号传导作用。     

Phagocytosis of lectin-coated beads by dystrophic and normal retinal pigment epithelium

In the dystrophic pigmented Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) has a diminished capacity to phagocytose shed photoreceptor outer segments (ROS). An alteration in phagocytic recognition or ligand-receptor interactions between the RPE and ROS's could contribute to this defect. To this end, we have examined whether or not RPE lectin receptors are implicated in phagocytosis in the normal and dystrophic rat RPE by comparing differences in phagocytic uptake of lectin-coated beads. To test this, the following lectins were bound either indirectly to sugar-coated latex beads or directly to activated beads: Concanavalin A (conA), specific for mannose; Ulex europeus (ULEX), specific for fucose; Lens culinaris (LcH), specific for mannose; and wheat germ agglutinin (WGA), specific for N-acetyl glucosamine and sialic acid. The distribution of the lectin binding around beads was visualized and confirmed using lectin-Ferritin conjugates. Lectin-coated beads were fed to normal and dystrophic pigmented RPE tissue explants to determine differences in phagocytic uptake. We found that whether beads were directly or indirectly coated, similar results were obtained, but that there were differences in uptake of two types of lectin-coated beads by dystrophic as compared with normal animals. The dystrophic RPE phagocytosed greater numbers of conA-mannose beads (6.9/cell) than the normal RPE (3.6/cell). LcH-mannose beads were also phagocytosed by dystrophic (2.7/cell) but not by the normal (0/cell). A similar number of ULEX-fucose beads were taken up by dystrophic (3.8/cell) and normal (3.4/cell) RPE and neither took up WGA-N-acetyl glucosamine beads (0/cell). These results showing that the dystrophic RPE takes up greater numbers of conA and LcH-coated beads than the normal RPE suggest that a ligand-receptor interaction involving mannose may contribute to this difference in phagocytic uptake.     

Laminin-2/4 from human placenta is a better adhesion agent for primary keratinocytes than laminin-1 from EHS sarcoma

A comparison of the adhesion of human primary keratinocytes to laminin-1 from murine EHS sarcoma and laminin-2/4 from human placenta was carried out using two methods, cell adhesion to substrates covered with the laminin isoforms, and interaction of keratinocytes from suspension with latex beads coated with the proteins. Laminin-2/4 was considerably more potent as a promoter of attachment of primary human keratinocytes than laminin-1 (and fibronectin), with increased attachment of cells correlating well with the number of latex bead binding sites. Only small cells of diameter of less than 20 microm bound more than 5 beads. Staining of keratinocytes with involucrin antibodies confirmed the existence of an inverse relationship between laminin-2/4-coated bead binding and differentiation.     

Oxygen transport and consumption by suspended cells in microgravity: a multiphase analysis

A rotating bioreactor for the cell/tissue culture should be operated to obtain sufficient nutrient transfer and avoid damage to the culture materials. Thus, the objective of the present study is to determine the appropriate suspension conditions for the bead/cell distribution and evaluate oxygen transport in the rotating wall vessel (RWV) bioreactor. A numerical analysis of the RWV bioreactor is conducted by incorporating the Eulerian-Eulerian multiphase and oxygen transport equations. The bead size and rotating speed are the control variables in the calculations. The present results show that the rotating speed for appropriate suspensions needs to be increased as the size of the bead/cell increases: 10 rpm for 200 microm; 12 rpm for 300 microm; 14 rpm for 400 microm; 18 rpm for 600 microm. As the rotating speed and the bead size increase from 10 rpm/200 microm to 18 rpm/600 microm, the mean oxygen concentration in the 80% midzone of the vessel is increased by approximately 85% after 1-h rotation due to the high convective flow for 18 rpm/600 microm case as compared to 10 rpm/200 microm case. The present results may serve as criteria to set the operating parameters for a RWV bioreactor, such as the size of beads and the rotating speed, according to the growth of cell aggregates. In addition, it might provide a design parameter for an advanced suspension bioreactor for 3-D engineered cell and tissue cultures.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

An improved rhodopsin/EGFP fusion protein for use in the generation of transgenic Xenopus laevis

Previous studies by Papermaster and coworkers introduced the use of rhodopsin-green fluorescent protein (rho-GFP) fusion proteins in the construction of transgenic Xenopus laevis with retinal rod photoreceptor cell-specific transgene expression [Moritz et al., J. Biol. Chem. 276 (2001) 28242-28251]. These pioneering studies have helped to develop the Xenopus system not only for use in the investigation of rhodopsin biosynthesis and targeting, but for studies of the phototransduction cascade as well. However, the rho-GFP fusion protein used in the earlier work had only 50% of the specific activity of wild-type rhodopsin for activation of transducin and only 10% of the activity of wild-type in rhodopsin kinase assays. While not a problem for the biosynthesis studies, this does present a problem for investigation of the phototransduction cascade. We report here an improved rhodopsin/EGFP fusion protein in which placement of the EGFP domain at the C-terminus of rhodopsin results in wild-type activity for activation of transducin, wild-type ability to serve as a substrate for rhodopsin kinase, and wild-type localization of the protein to the rod photoreceptor cell outer segment in transgenic X. laevis.