Fetal Human Eyes Research Articles

Sheets of human retinal progenitor transplants improve vision in rats with severe retinal degeneration

Loss of photoreceptors and other retinal cells is a common endpoint in retinal degenerate (RD) diseases that cause blindness. Retinal transplantation is a potential therapy to replace damaged retinal cells and improve vision. In this study, we examined the development of human fetal retinal sheets with or without their retinal pigment epithelium (RPE) transplanted to immunodeficient retinal degenerate rho S334ter-3 rats. Sheets were dissected from fetal human eyes (11–15.7 weeks gestation) and then transplanted to the subretinal space of 24–31 d old RD nude rats. Every month post surgery, eyes were imaged by high-resolution spectral-domain optical coherence tomography (SD-OCT). SD-OCT showed that transplants were placed into the subretinal space and developed laminated areas or rosettes, with clear development of plexiform layers first seen in OCT at 3 months post surgery. Several months later, as could be expected by the much slower development of human cells compared to rat cells, transplant photoreceptors developed inner and later outer segments. Retinal sections were analyzed by immunohistochemistry for human and retinal markers and confirmed the formation of several retinal subtypes within the retinal layers. Transplant cells extended processes and a lot of the cells could also be seen migrating into the host retina. At 5.8–8.6 months post surgery, selected rats were exposed to light flashes and recorded for visual responses in superior colliculus, (visual center in midbrain). Four of seven rats with transplants showed responses to flashes of light in a limited area of superior colliculus. No response with the same dim light intensity was found in age-matched RD controls (non-surgery or sham surgery). In summary, our data showed that human fetal retinal sheets transplanted to the severely disturbed subretinal space of RD nude rats develop mature photoreceptors and other retinal cells, integrate with the host and induce vision improvement.

Development of the hyaloid, choroidal and retinal vasculatures in the fetal human eye.

The development of the ocular vasculatures is perfectly synchronized to provide the nutritional and oxygen requirements of the forming human eye. The fetal vasculature of vitreous, which includes the hyaloid vasculature, vasa hyaloidea propria, and tunica vasculosa lentis, initially develops around 4-6 weeks gestation (WG) by hemo-vasculogenesis (development of blood and blood vessels from a common progenitor, the hemangioblast). This transient fetal vasculature expands around 12 WG by angiogenesis (budding from primordial vessels) and remains until a retinal vasculature begins to form. The fetal vasculature then regresses by apoptosis with the assistance of macrophages/hyalocytes. The human choroidal vasculature also forms by a similar process and will supply nutrients and oxygen to outer retina. This lobular vasculature develops in a dense collagenous tissue juxtaposed with a cell constitutively producing vascular endothelial growth factor (VEGF), the retinal pigment epithelium. This epithelial/endothelial relationship is critical in maintaining the function of this vasculature throughout life and maintaining it's fenestrated state. The lobular capillary system (choriocapillaris) develops first by hemo-vasculogenesis and then the intermediate choroidal blood vessels form by angiogenesis, budding from the choriocapillaris. The human retinal vasculature is the last to develop. It develops by vasculogenesis, assembly of CXCR4+/CD39+ angioblasts or vascular progenitors perhaps using Muller cell Notch1 or axonal neuropilinin-1 for guidance of semaphorin 3A-expressing angioblasts. The fovea never develops a retinal vasculature, which is probably due to the foveal avascular zone area of retina expressing high levels of antiangiogenic factors. From these studies, it is apparent that development of the mouse ocular vasculatures is not representative of the development of the human fetal, choroidal and retinal vasculatures.

New medium used in the differentiation of human pluripotent stem cells to retinal cells is comparable to fetal human eye tissue

Human pluripotent stem cells (hPSCs) have the potential to differentiate along the retinal lineage. However, most induction systems are dependent on multiple small molecular compounds such as Dkk-1, Lefty-A, and retinoic acid. In the present study, we efficiently differentiated hPSCs into retinal cells using a retinal differentiation medium (RDM) without the use of small molecular compounds. This novel differentiation system recapitulates retinal morphogenesis in humans, i.e. hPSCs gradually differentiate into optic vesicle-shaped spheres, followed by optic cup-shaped spheres and, lastly, retinal progenitor cells. Furthermore, at different stages, hPSC-derived retinal cells mirror the transcription factor expression profiles seen in their counterparts during human embryogenesis. Most importantly, hinge epithelium was found between the hPSC-derived neural retina (NR) and retinal pigment epithelium (RPE). These data suggest that our culture system provides a new method for generating hPSC-derived retinal cells that, for the first time, might be used in human transplantation.

From Blood Islands to Blood Vessels: Morphologic Observations and Expression of Key Molecules during Hyaloid Vascular System Development

The mode of development of the human hyaloid vascular system (HVS) remains unclear. Early studies suggested that these blood vessels formed by vasculogenesis, while the current concept seems to favor angiogenesis as the mode of development. We examined embryonic and fetal human HVS using a variety of techniques to gain new insights into formation of this vasculature. Embryonic and fetal human eyes from 5.5 to 12 weeks gestation (WG) were prepared for immunohistochemical analysis or for light and electron microscopy. Immunolabeling of sections with a panel of antibodies directed at growth factors, transcription factors, and hematopoietic stem cell markers was employed. Light microscopic examination revealed free blood islands (BI) in the embryonic vitreous cavity (5.5-7 WG). Giemsa stain revealed that BI were aggregates of mesenchymal cells and primitive nucleated erythroblasts. Free cells were also observed. Immunolabeling demonstrated that BI were composed of mesenchymal cells that expressed hemangioblast markers (CD31, CD34, C-kit, CXCR4, Runx1, and VEGFR2), erythroblasts that expressed embryonic hemoglobin (Hb-ε), and cells that expressed both. Few cells were proliferating as determined by lack of Ki67 antigen. As development progressed (12 WG), blood vessels became more mature structurally with pericyte investment and basement membrane formation. Concomitantly, Hb-ε and CXCR4 expression was down-regulated and von Willebrand factor expression was increased with the formation of Weibel-Palade bodies. Our results support the view that the human HVS, like the choriocapillaris, develops by hemo-vasculogenesis, the process by which vasculogenesis, erythropoiesis, and hematopoiesis occur simultaneously from common precursors, hemangioblasts.

VEGF 165b in the developing vasculatures of the fetal human eye

VEGF(165) b is an anti-angiogenic form of VEGF(165) produced by alternative splicing. The localization of pro-angiogenic VEGF(165) and anti-angiogenic VEGF(165) b was investigated during development of the vasculatures in fetal human eyes from 7 to 21 weeks gestation (WG). The fetal vasculature of vitreous, which includes tunica vasculosa lentis (TVL), had moderate VEGF(165) immunoreactivity at 7WG and very little VEGF(165) b. Both forms were elevated at 12WG. VEGF(165) then decreased around 17WG when the TVL regresses but VEGF(165) b remained elevated. In choroid, VEGF(165) was present in forming choriocapillaris (CC) and retinal pigment epithelium (RPE) at 7WG while VEGF165b was present in CC and mesenchymal precursors within the choroidal stroma. By 21WG, both forms were elevated in RPE and choroidal blood vessels but VEGF(165) b was apical and VEGF(165) basal in RPE. Diffuse VEGF(165) immunoreactivity was prominent in 12WG innermost retina where blood vessels will form while VEGF(165) b was present in most CXCR4(+) progenitors in the inner neuroblastic layer and migrating angioblasts in the putative nerve fiber layer. By 21WG, VEGF(165) was present in nerve fibers and VEGF(165) b in the inner Muller cell process. The localization of VEGF(165) b was distinctly different from VEGF(165) both spatially and temporally and it was often associated with nucleus in progenitors.

Cotransport of water by the Na+-K+-2Cl−cotransporter NKCC1 in mammalian epithelial cells

Water transport by the Na+-K+-2Cl(-) cotransporter (NKCC1) was studied in confluent cultures of pigmented epithelial (PE) cells from the ciliary body of the fetal human eye. Interdependence among water, Na+ and Cl(-) fluxes mediated by NKCC1 was inferred from changes in cell water volume, monitored by intracellular self-quenching of the fluorescent dye calcein. Isosmotic removal of external Cl(-) or Na+ caused a rapid efflux of water from the cells, which was inhibited by bumetanide (10 μm). When returned to the control solution there was a rapid water influx that required the simultaneous presence of external Na+ and Cl(-). The water influx could proceed uphill, against a transmembrane osmotic gradient, suggesting that energy contained in the ion fluxes can be transferred to the water flux. The influx of water induced by changes in external [Cl(-)] saturated in a sigmoidal fashion with a Km of 60 mm, while that induced by changes in external [Na+] followed first order kinetics with a Km of about 40 mm. These parameters are consistent with ion transport mediated by NKCC1. Our findings support a previous investigation, in which we showed water transport by NKCC1 to be a result of a balance between ionic and osmotic gradients. The coupling between salt and water transport in NKCC1 represents a novel aspect of cellular water homeostasis where cells can change their volume independently of the direction of an osmotic gradient across the membrane. This has relevance for both epithelial and symmetrical cells.

Ezrin, a membrane-organizing protein, as a polarization marker of the retinal pigment epithelium in vertebrates.

Immunoreactivity for ezrin, a membrane-organizing phosphoprotein that tethers actin microfilaments to cell membrane proteins, was evaluated as a polarization marker in the intraocular neuroepithelial cells of vertebrates, especially in the retinal pigment epithelium (RPE). Six fetal human eyes representing the 14th-28th gestational weeks, 9 normal adult eyes, 12 eyes with intraocular tumors, and 26 eyes from 15 other vertebrate species were analyzed by immunohistochemistry using the avidin-biotinylated peroxidase complex (ABC) method and monoclonal antibody (mAb) 3C12 to ezrin. The apical cytoplasm and microvilli of the human RPE always reacted with mAb 3C12, but the basal cytoplasm was labeled in reactive RPE only. In autopsy eyes and if fixation was delayed, ezrin immunoreactivity in RPE was more diffuse. Developing RPE became gradually immunoreactive from the 14th week of gestation onward. The microvilli of the baboon, pig, raccoon dog, cow, and rat RPE cells were likewise labeled, and their basal cytoplasm was variably immunoreactive as well, but the microvilli of the avian RPE did not react with the antibody used. In all six mammals mentioned, both layers of the ciliary epithelium and the anterior iris epithelium reacted for ezrin, and the posterior epithelium was weakly labeled in pig, cow, and rat eyes. Normal peripheral and reactive human retina, and normal baboon, pig, raccoon dog, cow, rat, black grouse, and jay eyes, showed immunoreaction for ezrin in Muller cells, usually in their microvilli. Ezrin is widely found in RPE and anterior segment neuroepithelia of the mammalian eye, in which it may segregate membrane proteins to specific membrane surfaces, especially to the apical microvilli of the RPE, which intimately interact with outer segments of photoreceptor cells. The ezrin gene on human chromosome 6q25-26 is consequently a candidate gene for causing retinal degenerations.

Type XIII Collagen is Widely Expressed in the Adult and Developing Human Eye and Accentuated in the Ciliary Muscle, the Optic Nerve and the Neural Retina

The distribution of mRNAs coding for type XIII collagen, a novel nonfibril-forming collagen, was studied by Northern and in situ hybridizations of adult and fetal human eyes and the corresponding protein was localized by indirect immunofluorescence in frozen sections of 12 and 17 week human fetal eyes using a polyclonal antipeptide antibody to type XIII collagen.Type XIII collagen was found to be widely expressed in ocular tissues when studied at both the mRNA and protein levels in fetal and adult human tissues. No major differences were observed in the expression patterns between fetal and adult tissues. Surprizingly, the strongest signals seen in in situ hybridizations and immunofluorescence stainings occurred in the optic nerve bundles and in the ganglion cell layer of the retina. Other notable locations containing type XIII collagen included the developing ciliary smooth muscle, the posterior two-thirds of the corneal stroma and the striated extraocular muscles. Low level signals were also detected in the blood vessel walls and mesenchymal cells of the other ocular tissues. All immunosignals detected were adherent to cells, and the extracellular matrices appeared to be devoid of type XIII collagen.Our results are in concert with the presumed plasma membrane location of type XIII collagen, and it is hypothesized that this molecule could be involved in cell-matrix and perhaps cell–cell interactions. The wide expression of type XIII collagen in the eye, and especially in the neural structures, warrants future studies on type XIII collagen in other nerve structures and in pathological conditions affecting the eye. Due to its wide expression, type XIII collagen is likely to be an important factor for the normal development and functioning of the eye.

Retinal Pigment Epithelial Cells Are Heterogeneous in Their Expression of MHC-II After Stimulation with Interferon-γ

The pattern of interferon-gamma-induced major histocompatibility complex Class II antigen expression was evaluated on the retinal pigment epithelium. Experiments were performed in vitro using explant cultures of aged and fetal human eyes and in vivo in albino rabbits. The human explants were stimulated with 50 U ml−1interferon-gamma for 3 days prior to immunostaining for Class II. The rabbit eyes were subretinally injected in vivo with 50 μl of interferon-gamma (500 U ml−1) and analyzed immunohistochemically 3 days later. A heterogeneous pattern of Class II expression was present in the interferon-gamma-stimulated retinal pigment epithelial cells, in both the in vivo and the in vitro experiments. In aged human eyes the percent of Class-II positive cells was higher in the periphery than in the posterior pole (macular region) after interferon-gamma stimulation (P<0.01). No such difference was found in the fetal eyes. These data demonstrate that retinal pigment epithelial cells are heterogeneous in their response to interferon-gamma. The results are supportive of previous studies demonstrating the structural and proliferative heterogeneity of the retinal pigment epithelium. Together, these studies provide support for the possibility of functional retinal pigment epithelial heterogeneity.

Cytoskeleton of the intraocular epithelial and muscle cells of the fetal human eye

Cytoskeleton of the intraocular epithelial and muscle cells of the fetal human eye

4236 Cytoskeleton of the intraocular epithelial and muscle cells of the fetal human eye

4236 Cytoskeleton of the intraocular epithelial and muscle cells of the fetal human eye

An improved method of isolating fetal human retinal pigment epithelium.

The purpose of this study was to develop an improved method of isolating fetal human retinal pigment epithelium (RPE) for tissue culture or transplantation. Fetal human eyes ranging from 8 to 20 wks of gestation were collected and stored in Optisol solution. Under a dissecting microscope, an incision was made behind the ora serrata and extended circumferentially to remove the anterior segment. The vitreous was withdrawn, and the neural retina was carefully detached from the RPE. The sclera then was teased away from the choroid-RPE. The choroid-RPE was treated with 2% dispase in DMEM + 20 mM HEPES at 37 degrees C for 25 min. While still in dispase, the RPE was separated from the choroid using a pair of fine tipped jeweler's forceps under dark-field. An intact sheet of RPE could be separated from the choroid after treatment with dispase. No choroidal contamination was present as determined by light microscopy or cell culture. In vitro, the isolated RPE cells demonstrated classic cobblestone phenotype and expressed cytokeratin. This technique provides an easy and reliable method for isolating pure sheets of fetal human RPE. It also allows utilization of the neural retina of the same eye for other purposes, as the neural retina is not exposed to the enzymatic digestion. These features make this method especially useful for RPE and retinal transplantation; such an application is already underway.

Cytoskeleton, adhesion, and extracellular matrix of fetal human retinal pigmented epithelial cells in culture.

Small sheets of human retinal pigmented epithelial (RPE) cells isolated from 6- to 9-week-old fetal human eyes were used to establish RPE cells in culture. After 4-5 days in culture the RPE cells in various parts of a colony were examined by immunofluorescence and fluorescence microscopy to determine the distribution of the cytoskeletal proteins tubulin, myosin, F-actin, vimentin, and cytokeratin and the extracellular matrix proteins laminin and type IV collagen. The extracellular matrix components laminin and type IV collagen, which are abundant underneath the RPE cells in the centre of the colony, progressively decrease in amount towards the edge of the colony. Microtubules labeled by antisera to tubulin were present in the cytoplasm of RPE cells throughout the colony. F-actin and myosin were found in stress fibres in the RPE cells at the edge of the colony and in compact circumferential rings circumscribing the apical ends in RPE cells in the centre of the colony. Examination of cell-substratum adhesion patterns by interference reflection microscopy showed that focal contacts were present only in RPE cells at the edge of the colony where they were located at the ends of the stress fibres. The large focal adhesions previously observed in colonies of chick RPE cells, however, were not seen in human RPE cells. Human RPE cells throughout the colony had cytokeratin-containing intermediate filaments as well as vimentin-containing intermediate filaments, whereas only the latter were observed in chick RPE cells. These results indicate that in the general organization of the cytoskeleton and in the distribution of the extracellular matrix components, laminin and type IV collagen, colonies of human RPE cells are similar but not identical to colonies of chick RPE cells.