Histology of tendon and enthesis - suitable techniques for specific research questions.

The musculoskeletal system consists of different components comprising a wide range of tissue types, with tendons being one part. Tendon degeneration or rupture have a high prevalence in all age groups, often with poor outcomes of surgical treatment such as chronic pain and high re-tear rates. Therefore, much effort has been directed to further develop diagnostic and therapeutic methods as well as reconstruction techniques, including using adequate placeholders or implants. Diagnostic approaches and advanced stages of preclinical studies will inevitably include histological examination of the pathologically affected tissue. The present study presents adequate tendon-related, histological techniques, including the embedding of soft- and hard-tissue samples in different media. Consideration is also given to samples containing residual implant materials or having been subjected to standard staining protocols and immunohistochemical procedures. The study further examines cells and tendon structure to detect degenerative, fibrotic or inflammatory conditions and possible foreign-body responses to implanted materials. Infraspinatus tendons from preclinical studies carried on rat and sheep samples, as well as human biceps tendon samples, have been used as example materials.

Do biomarkers allow a differentiation between osteonecrosis of the femoral head and osteoarthritis of the hip? - a biochemical, histological and gene expression analysis.

OBJECTIVE: Osteonecrosis of the femoral head (ONFH) is a devastating disease of the hip joint. Its early diagnosis is crucial to increase the chances of joint preserving, yet difficult due to similarities with osteoarthritis (OA) of the hip in its clinical appearance. The purpose of this study was to enhance the understanding of ONFH and its pathologic processes in contrast to OA and to identify serum biomarkers helping to improve the diagnosis of the disease. DESIGN: Bone and bone marrow samples were collected from 24 patients diagnosed with OA and 25 patients with ONFH during total hip replacement surgery. RNA was isolated, histological examination, determination of free reactive oxygen species as well as gene expression and biomarker analysis were performed. RESULTS: Histological analysis revealed differences in the structural and cellular pattern between the groups. Gene expression analysis revealed a significant upregulation for the genes ASPN, COL1A1, COL2A1 and IL6 and a significant downregulation for HIF1A in ONFH compared to OA group. Analysis of serum biomarkers showed significant differences between the groups for asporin and adiponectin. A final logistical regression model including the parameters adiponectin, asporin and HIF 1α was overall significant, explained 34.5 % of variance and classified 74.5 % of the cases correctly. CONCLUSION: The combination of adiponectin, asporin and HIF 1α as serum biomarkers revealed a classification accuracy of 74.5 %. The information provided in this study may help to enhance the understanding of pathologic processes in ONFH and to elaborate further aspects of prediction and treatment.

In vitro and in vivo evaluation of biodegradable, open-porous scaffolds made of sintered magnesium W4 short fibres.

A cytocompatible and biocompatible, degradable, open-porous, mechanically adaptable metal scaffold made of magnesium alloy W4 melt-extracted short fibres was fabricated by liquid phase sintering. Cylindrical samples (3×5 mm) of sintered W4 short fibres were evaluated under in vitro (L929, HOB, eudiometer, weight loss) and in vivo conditions (rabbits: 6 and 12 weeks). The in vitro corrosion environment (e.g., temperature, flow, composition of corrosion solution, exposure time) significantly influenced the corrosion rates of W4 scaffolds compared with corrosion in vivo. Corrosion rates under cell culture conditions for 72 h varied from 1.05 to 3.43 mm y(-1) depending on the media composition. Corrosion rates measured in eudiometric systems for 24 h were ~24-27 times higher (3.88-4.43 mm y(-1)) than corrosion in vivo after 6 weeks (0.16 mm y(-1)). Moreover, it was found that the cell culture media composition significantly influences the ionic composition of the extract by selectively dissolving ions from W4 samples or their corrosion products. A pilot in vivo study for 6 and 12 weeks demonstrated active bone remodelling, no foreign body reaction and no clinical observation of gas formation during W4 scaffold implantation. Long-term in vivo studies need to be conducted to prove complete degradation of the W4 scaffold and total replacement by the host tissue.

Magnesium hydroxide temporarily enhancing osteoblast activity and decreasing the osteoclast number in peri-implant bone remodelling.

Repeated observations of enhanced bone growth around various degradable magnesium alloys in vivo raise the question: what is the major mutual origin of this biological stimulus? Several possible origins, e.g. the metal surface properties, electrochemical interactions and biological effects of alloying elements, can be excluded by investigating the sole bone response to the purified major corrosion product of all magnesium alloys, magnesium hydroxide (Mg(OH)(2)). Isostatically compressed cylinders of pure Mg(OH)(2) were implanted into rabbit femur condyles for 2-6 weeks. We observed a temporarily increased bone volume (BV/TV) in the vicinity of Mg(OH)(2) at 4 weeks that returned to a level that was equal to the control at 6 weeks. The osteoclast surface (OcS/BS) was significantly reduced during the first four weeks around the Mg(OH)(2) cylinder, while an increase in osteoid surface (OS/BS) was observed at the same time. At 6 weeks, the OcS/BS adjacent to the Mg(OH)(2) cylinder was back within the same range of the control. The mineral apposition rate (MAR) was extensively enhanced until 4 weeks in the Mg(OH)(2) group before matching the control. Thus, the enhanced bone formation and temporarily decreased bone resorption resulted in a higher bone mass around the slowly dissolving Mg(OH)(2) cylinder. These data support the hypothesis that the major corrosion product Mg(OH)(2) from any magnesium alloy is the major origin of the observed enhanced bone growth in vivo. Further studies have to evaluate if the enhanced bone growth is mainly due to the local magnesium ion concentration or the local alkalosis accompanying the Mg(OH)(2) dissolution.

Biodegradable magnesium scaffolds: Part II: peri-implant bone remodeling.

In this study, histomorphometrical parameters of the peri-implant bone remodeling around degrading open-porous scaffolds made of magnesium alloy AZ91D were investigated and compared with the peri-implant bone remodeling around an autologous bone transplant in the contralateral side in a rabbit model after 3 and 6 months. Osteoblast activity was displayed by collagen I (alpha 2) mRNA in situ hybridization. Major scaffold degradation was completed within 3 months after implantation showing no osteolysis around the scaffolds, both after 3 and 6 months. Enhanced formation of unmineralized extracellular matrix and an enhanced mineral apposition rate adjacent to the degrading magnesium scaffolds were accompanied by an increased osteoclastic bone surface, which resulted in higher bone mass and a tendency to a more mature trabecular bone structure around the magnesium scaffolds compared to the control. These results show that even fast-degrading magnesium scaffolds induce extended peri-implant bone remodeling with a good biocompatibility. In summary, this study shows that degrading magnesium scaffolds promote both bone formation and resorption in a rabbit model and are therefore very promising candidates for the development of novel implants in musculoskeletal surgery.

Biodegradable magnesium scaffolds: Part 1: appropriate inflammatory response.

Current tissue engineering strategies focus on the replacement of pathologically altered tissues by the transplantation of cells in combination with supportive biocompatible scaffolds. Scaffolds for tissue engineering strategies in musculoskeletal research require an appropriate mechanical stability. In recent studies, considerable attention has thus been given to magnesium alloys as biodegradable implants. The aim of this study was to characterize the biocompatibility of magnesium scaffolds by the inflammatory host response. Open porous scaffolds made of the magnesium alloy AZ91D were implanted into the distal femur condyle of rabbits and were compared to autologous bone, which was transplanted into the contralateral condyle in a 3 and 6 months follow-up group. After 3 months, magnesium scaffolds were already largely degraded and most of the original magnesium alloy has disappeared. Concomitantly, a fibrous capsule enclosed the operation site. Histological analysis revealed that the magnesium scaffolds caused no significant harm to their neighboring tissues. This study shows that even fast degrading magnesium scaffolds show a good biocompatibility and react in vivo with an appropriate inflammatory host response. Magnesium alloy based implants are therefore a very promising approach in the development of mechanically suitable and open porous scaffolds for the replacement of subchondral bone in cartilage tissue engineering.

Highly sensitive biosafety model for stem-cell-derived grafts.

BACKGROUND: The recent success in the derivation of differentiated cell types from stem cells has raised prospects for the application of regenerative cell therapy. In particular, embryonic stem cells are attractive sources for cell transplantation, due to their immortality and rapid growth. These cells, however, also possess tumorigenic properties, which raises serious safety concerns and makes biosafety testing mandatory. Our goal was to establish a highly sensitive animal model for testing the proliferative potential of stem-cell grafts. METHODS: BALB/c nude mice received cell grafts of non-neoplastic MRC-5 cells containing defined numbers of mouse embryonic stem cells. We either injected 1 million viable cells into the kidney capsule, or mixed 2 million cells with Matrigel for s.c. transplantation. To analyze the possible impact of an intact immune response on tumor development, we also transplanted the cells into immunocompetent mice. Animals were sacrificed when the tumors became >1 cm and were analyzed in detail. RESULTS: The nude mouse model reproducibly allowed detection of 20 tumorigenic cells, and even as few as 2 ES cells were found to form teratoma. Interestingly, the administration of cell grafts at two different application sites resulted in different growth kinetics and tumor phenotypes. The highest level of sensitivity (100% detection of 20 tumorigenic ES cells) was achieved by s.c. injection of cells mixed with Matrigel. The influence of the immune system on tumor-cell development was demonstrated by a higher tumor rate of transplants in immunodeficient nude mice compared with immunocompetent mice. DISCUSSION: We have established a reliable animal model for routine assessment of the biosafety profile of stem-cell-derived cell transplants. This model will facilitate the generation of homogenous non-tumorigenic cell populations, and will help to integrate standardized safety systems into the application of stem-cell-derived grafts for clinical purposes.

Generation of confluent cardiomyocyte monolayers derived from embryonic stem cells in suspension: a cell source for new therapies and screening strategies.

BACKGROUND: Cellular cardiomyoplasty is evolving as a new strategy to treat cardiac diseases. A prerequisite is a reliable source of pure cardiomyocytes, which could also help in the exploitation of recent advances in genomics and drug screening. Our goal was to establish a robust lab-scale process for the generation of embryonic stem (ES)-cell-derived cardiomyocytes in suspension. METHODS: A 71 ES cell clone carrying a construct consisting of the alpha-cardiac myosin heavy chain (alphaMHC) promoter driving the neomycin resistance gene was used for antibiotic-driven cardiomyocyte enrichment. Rotating suspension culture was established to initiate embryoid body (EB) formation. To track growth and differentiation kinetics, cell count and flow cytometry for SSEA-I, E-cadherin (stem-cell marker)and sarcomeric myosin (cardiomyocytes marker) was performed. Oct4 expression was measured via real time (RT)-PCR. RESULTS: Cultures comprising 2.5-8 x 10(6) differentiating FS cells/mL were obtained after 9 days in rotating suspension. Upon G418 addition,vigorous contracting spheres, termed cardiac bodies (CB), developed. These cultures consisted of about 2.1 x 10(5) enriched cardiomyocytes/mL after 6- 10 days of selection. Suspensions comprising 90- 95%viable single cells were generated using an improved dissociation method. Seeding of cardiomyocytes with 7 x 10(4) cell/cm(2) resulted in a homogeneous monolayer of synchronously contracting cells. Myocyte specific immunohistochemistry indicated purity of > 99%. DISCUSSION: We have established a reliable lab-scale protocol to generate cultures of highly enriched cardiomyocytes in suspension. This will facilitate development of larger-scale processes for stem-cell based cardiomyocyte supply. An improved method is provided to derive vital suspensions of cardiomyocytes, which could be utilized for transplantation as well as for drug screening purposes.

From stem cells towards neural layers: a lesson from re-aggregated embryonic retinal cells.

Cells from dissociated embryonic avian retinae have the capacity to re-aggregate in rotation culture and form cellular spheres reconstituting a complete arrangement of all retinal layers. This exquisite phenomenon is based upon in vitro proliferation of multipotent precursor stem cells and spatial organization of their differentiating descendants. The addition of soluble factors from cultured retinal pigmented epithelial (RPE) or radial glial cells is essential to revert inside-out spheres (rosetted retinal spheres) into correctly laminated outside-out spheres (stratified spheres). Such complete restoration of a laminated brain tissue by cell re-aggregation has been achieved only for the embryonic avian retina, but not the mammalian retina, nor for other brain parts. This review summarises the history of the re-aggregation approach, presents avian retinal re-aggregate models, and analyses roles of the RPE and Müller cells for successful retinal tissue regeneration. It is predicted that these results will become biomedically relevant, as stem cell biology will soon open ways to produce large amounts of human retinal precursors.

Cerebellar glia cells induce a correct laminar organization in chicken retinal reaggregates.

We investigated the functional role of glia cells during retinogenesis using the rotation culture system. Reaggregating cells from the embryonic chick retina have the unique capacity to reassemble into laminated cellular spheres. These spheres are composed of several compartments holding the constituents of many retinal layers in a topologically correct, yet inverse orientation. However, when these spheres are cultured in the presence of conditioned media derived from monolayers of cerebellar glia cells, the reassembling retinal cells behave totally differently. The anlage of the originally reversed lamina polarity is progressively transformed within a week into a sphere with a compound and correctly laminated orientation. Conditioned media from fibroblasts, other glia cells (except Müller cells) or a set of already characterized retinogenetic factors are not able to produce this dramatic transformation. Additionally, we were able to show that only retinal cells are able to respond with a reorganization process. Reaggregating cells from the chick cerebellum also form spheroids; however, neither in the presence of cerebellar glia cell-derived conditioned medium nor their control counterparts are they able to reassemble histotypically. This indicates that cerebellar glia cells produce diffusible factors to which retinal cells can respond and that these factors can act as important determinants for the correct establishment of the retinal polarity. Since all types of laminar disorganization are of great clinical significance, the knowledge of factors which determine and sustain the normal retinal architecture are biomedically highly relevant.

Inhibition of alpha(1-6)-linked fucose decreases inner retinal cells and increases photoreceptors in chicken retinal reaggregates.

We investigated the developmental role of alpha(1-6)-linked fucose, applying Aleuria aurantia lectin to a specific retinal regeneration system. Thereby, dissociated retinal cells of chicken embryos reaggregate, proliferate, and differentiate in vitro into histotypical spheres, so-called retinospheroids. Under the influence of A. aurantia lectin, processes of proliferation, differentiation and histogenesis of retinospheroids were disturbed. Extending these in vitro studies, we here show that A. aurantia lectin treatment decreases cells of the inner half retina and their processes into inner plexiform layer areas, as revealed by quantitative enzyme histochemistry for butyryl- and acetylcholinesterase, and immunohistochemistry using antibodies to acetylcholinesterase, Pax-6, calbindin-D, and F11. Concomitantly, the number of rod and red/green photoreceptors dramatically increases, using the antibodies rho4D2 and CERN901 (both specific for rods) and CERN906 (specific for red/green cones). These findings show that glycoproteins exhibiting fucose in alpha(1-6)-linkage are involved in processes determining retinal cell fate, strongly shifting the relative ratio of cells of the inner towards cells of the outer retina.

Müller glia cells reorganize reaggregating chicken retinal cells into correctly laminated in vitro retinae.

Müller cells, that belong to the family of radial glia cells, have central functions during retinogenesis. They form a stabilizing scaffold, they are candidate targets for the mediation of extraneous retinogenetic factors, and they are an important source for retina-borne retinogenetic factors. Reaggregate cultures allow the analysis of retinogenesis from dispersed cells to fully laminated tissues. Reaggregating cells from the embryonic chick retina reassemble to reversed laminated cellular spheres including constituents of all retinal layers, yet the outer nuclear layer is represented by internal rosettes. Using spheroids, we tested whether Müller cells have a decisive function in establishing retinal polarity and in determining the lamination pattern. To this end, we established confluent monolayers of highly enriched Müller cells derived from E6 or E13 chicken retinas, and then let dispersed E5.5 retinal cells reaggregate either in the absence of these monolayers or on top of them. In the presence of Müller cells, the reversed lamina polarity of rosetted spheroids progressively transformed within a week into correctly laminated retinal spheres, whereas all initial rosettes vanished. Moreover, photoreceptors formed a regular outer nuclear layer, as visualized by the rod-specific CERN901 antibody. In correctly laminated spheroids, staining for vimentin and glutamine synthetase was much more pronounced than in rosetted spheroids; in particular, a well-established inner limiting membrane stood out wherever the retinal lamination was complete. Because these effects can be similarly achieved by supernatants derived from Müller cells, direct cell-cell contacts or cellular replenishment from the monolayer do not account for these effects. We conclude that Müller cells are involved in the establishment of a correct retinal lamination and in the arrangement of the cells in the reaggregate cultures. In particular, rosette formation is counteracted and the formation of an inner limiting membrane is induced. Because rosettes are objects of concern in several ophthalmological defects, these results are highly relevant, both biomedically and also for normal retinogenesis.

Fucose in alpha(1-6)-linkage regulates proliferation and histogenesis in reaggregated retinal spheroids of the chick embryo.

We have used the lectin from Aleuria aurantia (AAL) which is highly specific for alpha(1-6)-linked fucose, to examine its effect on chicken retinogenesis in a reaggregation culture system. When dispersed cells of the embryonic chick retina are reaggregated to form histotypic retinospheroids, AAL elicits strong inhibition of spheroid growth. The action of AAL is specific, since its effect is dose-dependent, saturable, and inhibited by an excess of fucose. Fucosidase treatment entirely abolishes reaggregation. In contrast, Anguilla anguilla agglutinin (AAA) binding to fucose in alpha(1-2)-linkage does not show any effects. Incubation with CAB4-a specific monoclonal antibody for fucose in alpha(1-6)-linkage-reduces spheroid size and shape. AAL does not much affect primary aggregation, but rather subsequent processes of cell proliferation and histogenesis. In particular, AAL inhibits uptake of bromo-desoxyuridine (BrdU), most efficiently so during days in vitro 2 (div2) and div3. As a consequence, the histological differentiation is entirely disturbed, as evidenced by vimentin immunostaining; particularly, rosettes are not forming and the radial glia scaffold is disorganized. We conclude that glycoproteins exhibiting fucose in alpha(1-6)-linkage may play major roles in early processes of retinal tissue formation.

Inductive effects of the retinal pigmented epithelium (RPE) on histogenesis of the avian retina as revealed by retinospheroid technology.

During eye formation, inductive phenomena occurring between retinal pigmented epithelium (RPE) and retina are not well understood. After briefly summarizing the normal development of retina and RPE, we present three-dimensional in vitro models of the chick embryonic retina which allows elucidation of RPE-retina interactions. In such retinospheroids, a complete arrangement of layers is achieved, provided that dispersed retinal cells are: (1) young enough; and (2) reaggregated on a monolayer of RPE. Thereby, the RPE extends cell proliferation, while differentiation is much delayed. These findings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.

Müller glia cells and their possible roles during retina differentiation in vivo and in vitro.

Müller cells are astrocyte-like radial glia cells which are formed exclusively in the retina. Here we present evidence that Müller cells are crucially involved in the development of the retina's architecture and circuitry. There is increasing evidence that Müller cells are present from the very early beginning of retinogenesis. We postulate the "gradual maturation hypothesis of Müller cells". According to this hypothesis, Müller cells are continuously generated by a gradual transition of neuroepithelial stem cells into mature Müller cells. This process may be partly reversible. Müller cells, or their immature precursors, are able to subserve different functions. They are primary candidates for stabilizing the complex retinal architecture and for providing an orientation scaffold. Thereby, they introduce a reference system for the migration and correct allocation of neurons. Moreover, they may provide spatial information and microenvironmental cues for differentiating neurons, and may also be important for the segregation of cell and fibre layers. Additionally, they seem to be involved in the guidance of axonal fibres both in radial and in lateral directions, as they are involved in the support and stabilization of synapses.

Pigmented epithelium sustains cell proliferation and decreases expression of opsins and acetylcholinesterase in reaggregated chicken retinospheroids.

We investigated the effect of the retinal pigmented epithelium on cell proliferation and differentiation in rosetted retinospheroids, which are retina-like spheres reaggregated in the complete absence of retinal pigmented epithelium from dissociated retinal cells of 6-day-old chick embryos in a rotation culture system. In spheroids raised in the absence of retinal pigmented epithelium (controls), acetylcholinesterase was expressed in cells of an inner nuclear-like layer and their neuropil matrices. Moreover, the ratio between rods and cones was found to be approximately normal throughout the spheroid. When spheroids were cultured in the presence of retinal pigmented epithelium monolayers, cell proliferation in spheroids as determined by BrdU labelling was significantly increased and extended for 1 week, while acetylcholinesterase protein levels and specific activities in homogenates were decreased to approximately 30%. At the same time, opsin immunoreactivity was completely suppressed within the spheroid and appeared slowly in cells around its periphery; i.e. the proportion of rhodopsin-positive cells decreased from 14 to 3%. This study reveals that the retinal pigmented epithelium in vitro sustains cell proliferation but inhibits the differentiation of acetylcholinesterase-positive cells and of photoreceptors.

Pigmented epithelium induces complete retinal reconstitution from dispersed embryonic chick retinae in reaggregation culture.

Reaggregation of dispersed retinal cells of the chick embryo leads to histotypic retinospheroids in which the laminar organization remains incomplete: photoreceptors form rosettes which are surrounded by constituents of the other retinal layers. Here, for the first time, a complete arrangement of layers is achieved in cellular spheres (stratoids), provided that fully dispersed retinal cells are younger than embryonic day E6, and are reaggregated in the presence of a monolayer of retinal pigmented epithelium (RPE). A remarkable mechanism of stratoid formation from 1 to 15 days in vitro is revealed by the establishment of a radial Müller glia scaffold and of photoreceptors. During the first two days of reaggregation on RPE, rosettes are still observed. At this stage immunostaining with vimentin and F11 antibodies for radial Müller glia reveal a disorganized pattern. Subsequently, radial glia processes organize into long parallel fibre bundles which are arranged like spokes to stabilize the surface and centre of the stratoid. The opsin-specific antibody CERN 901 detects photoreceptors as they gradually build up an outer nuclear layer at the surface. These findings assign to the RPE a decisive role for the genesis and regeneration of a vertebrate retina.

The neural cell recognition molecule F11 is expressed on Müller cells and Schwann cells in vitro.

The neural cell recognition molecule F11 is a member of the immunoglobulin superfamily. During development it is expressed on axons in neuropils, and it is implicated in neurite outgrowth and in fasciculation. F11 is a multifunctional protein that interacts with L1/Ng-CAM, Nr-CAM, tenascin-C, tenascin-R (restrictin) and receptor protein tyrosine phosphatase beta. Here we show that F11 is not only expressed on neurons, but also on Müller cells and Schwann cells in vitro. Using two independent cell culture techniques, stratospheroids which represent an in vitro regeneration system of the chicken retina, and purified Schwann cells from chicken sciatic nerve, we show that Müller cells and Schwann cells express F11. On semithin cryosections of stratospheroids we were able to colocalize F11 with the Müller cell markers vimentin and 3A7. Expression of F11 by purified Schwann cells in demonstrated histologically and by immunoblot analysis. These in vitro data suggest the involvement of F11 in glia-neuron or glia-neurite interactions.

On the role of Müller glia cells in histogenesis: only retinal spheroids, but not tectal, telencephalic and cerebellar spheroids develop histotypical patterns.

The establishment of cell and fibre layers and the specification of different cell types are crucial processes during development of the central nervous system. Here we investigated the developmental architecture of radial glia cells in these processes using so-called spheroids that arise from dissociated chicken embryonic neural cells in rotation culture. We were able to produce retinal, tectal, and telencephalic spheroids from E6 embryos and cerebellar spheroids from E10 embryos. Cell and fibre differentiation can be observed in all types of spheroids, however, it is most abundant in retinal spheroids. Moreover, only in retinal spheroids a histotypic organization can be detected. Using immunohistochemistry and electron microscopy, we assign this -at least partially- to the capacity of Müller cells to form radial scaffolds, since we observe a congruency between these radial scaffolds and the presence of rosettes formed by photoreceptor precursors and Müller cells. Tectal, telencephalic and cerebellar spheroids do not show organized radial glia scaffolds, instead, the radial glia cells are randomly arranged and the spheroids do not show histotypical organization. The application of the specific gliotoxin 6-aminonicotinamide to growing retinal spheroids leads to a significant decrease in the number and size of the rosettes. Concomitantly, the degree of histotypical organization is also drastically reduced. This organizing capacity of Müller cells in vitro now strongly suggests the presence of a comparable function also in vivo. Moreover, since non-retinal radial glia cells are not able to re-organize an histotypic organization in vitro, Müller cells seem to be qualitatively different from other radial glia cells. In future studies we want to untangle these differences.

Lateral and radial growth uncoupled in reaggregated retinospheroids of embryonic avian retina.

According to an earlier resented model (Layer and Willbold, Int. Rev. Cytol. 146: 1-47, 1993), growth of the retina can be conceived of as an areal increase of an epithelial tissue sheet ("lateralization") plus a concomitant establishment of the layered retina ("radialization"). To provide further support for this model, here we have reaggregated dissociated retinal plus pigmented cells from chick or quail embryos and observed their development into histotypic three-dimensional spheres in rotation culture. These so-called stratospheroids consist of a continuous fully laminated retinal part with a coiled-up pigmented epithelial core. Using BrdU-labeling, we show that radial growth, i.e. the sequential production of cell types in spheroids, is comparable to normal vitreal-scleral retinogenesis. The region next to the pigmented epithelial core represents a "lateral growth zone" (equivalent to an ora serrata in vivo), where mitotic cell numbers are highest, even when in the laminated part proliferation has already ceased. Gradients of lateral differentiation emanate from this growth zone into the retinal tissue, as revealed by immunostaining of the photoreceptor protein opsin and the cell recognition molecule F11. Moreover, we found that stratospheroids derived from older embryos consist only of a hollow monolayered neuroepithelium which develops in the absence of any radial growth. This indicates that cell production is sustained longer in lateral than in radial direction. These differently staged stratospheroids will be excellent models to characterize genes involved in the regulation of lateral and radial growth processes.