Change in the developmental fate of the chick optic vesicle from the neural retina to the telencephalon.

The forebrain develops into the telencephalon, diencephalon, and optic vesicle (OV). The OV further develops into the optic cup, the inner and outer layers of which develop into the neural retina and retinal pigmented epithelium (RPE), respectively. We studied the change in fate of the OV by using embryonic transplantation and explant culture methods. OVs excised from 10-somite stage chick embryos were freed from surrounding tissues (the surface ectoderm and mesenchyme) and were transplanted back to their original position in host embryos. Expression of neural retina-specific genes, such as Rax and Vsx2 (Chx10), was downregulated in the transplants. Instead, expression of the telencephalon-specific gene Emx1 emerged in the proximal region of the transplants, and in the distal part of the transplants close to the epidermis, expression of an RPE-specific gene Mitf was observed. Explant culture studies showed that when OVs were cultured alone, Rax was continuously expressed regardless of surrounding tissues (mesenchyme and epidermis). When OVs without surrounding tissues were cultured in close contact with the anterior forebrain, Rax expression became downregulated in the explants, and Emx1 expression became upregulated. These findings indicate that chick OVs at stage 10 are bi-potential with respect to their developmental fates, either for the neural retina or for the telencephalon, and that the surrounding tissues have a pivotal role in their actual fates. An in vitro tissue culture model suggests that under the influence of the anterior forebrain and/or its surrounding tissues, the OV changes its fate from the retina to the telencephalon.

RPE specification in the chick is mediated by surface ectoderm-derived BMP and Wnt signalling.

The retinal pigment epithelium (RPE) is indispensable for vertebrate eye development and vision. In the classical model of optic vesicle patterning, the surface ectoderm produces fibroblast growth factors (FGFs) that specify the neural retina (NR) distally, whereas TGFß family members released from the proximal mesenchyme are involved in RPE specification. However, we previously proposed that bone morphogenetic proteins (BMPs) released from the surface ectoderm are essential for RPE specification in chick. We now show that the BMP- and Wnt-expressing surface ectoderm is required for RPE specification. We reveal that Wnt signalling from the overlying surface ectoderm is involved in restricting BMP-mediated RPE specification to the dorsal optic vesicle. Wnt2b is expressed in the dorsal surface ectoderm and subsequently in dorsal optic vesicle cells. Activation of Wnt signalling by implanting Wnt3a-soaked beads or inhibiting GSK3ß at optic vesicle stages inhibits NR development and converts the entire optic vesicle into RPE. Surface ectoderm removal at early optic vesicle stages or inhibition of Wnt, but not Wnt/ß-catenin, signalling prevents pigmentation and downregulates the RPE regulatory gene Mitf. Activation of BMP or Wnt signalling can replace the surface ectoderm to rescue MITF expression and optic cup formation. We provide evidence that BMPs and Wnts cooperate via a GSK3ß-dependent but ß-catenin-independent pathway at the level of pSmad to ensure RPE specification in dorsal optic vesicle cells. We propose a new dorsoventral model of optic vesicle patterning, whereby initially surface ectoderm-derived Wnt signalling directs dorsal optic vesicle cells to develop into RPE through a stabilising effect of BMP signalling.

PEDF counteracts DL-α-aminoadipate toxicity and rescues gliotoxic damages in RPE-free chicken retinal explants.

Gliotoxic responses complicate human eye diseases, the causes of which often remain obscure. Here, we activated Müller cells (MCs) by the gliotoxin DL-α-aminoadipate (AAA) and assayed possible protective effects by pigment epithelium-derived factor (PEDF) in RPE-free retinal explants of the E6 chick embryo. These models are suited to analyze gliotoxic reactions in vitro, since the avian retina contains only Müller cells (MCs) as glial components, and the RPE-free explants are devoid of a major PEDF source. ChAT- and AChE-immunohistochemistry (IHC) revealed that AAA treatment disrupted the differentiation of cholinergic amacrine cells in the inner plexiform layer. At the applied concentration of 1 mM AAA, apoptosis of MCs was slightly increased, as shown by TUNEL and caspase-3 activity assays. Concomitantly, cell-free gaps emerged in the middle of the retina, where MCs were swollen and amassed glutamine synthetase (shown by GS and Vimentin IHC). AAA treatment strongly activated MCs, as shown by GFAP IHC, and by an increase of stress-related catalase activity. Remarkably, nearly all effects of AAA on MCs were effectively counter-balanced by 50 ng/ml PEDF co-treatment, as also shown by RT-PCR. These findings suggest that supplementation with PEDF can protect the retina against gliotoxic attacks. Further studies should establish whether PEDF similarly protects a gliotoxic human retina.

Nuclei of chicken neurons in tissues and three-dimensional cell cultures are organized into distinct radial zones.

We used chicken retinospheroids (RS) to study the nuclear architecture of vertebrate cells in a three-dimensional (3D) cell culture system. The results showed that the different neuronal cell types of RS displayed an extreme form of radial nuclear organization. Chromatin was arranged into distinct radial zones which became already visible after DAPI staining. The distinct zones were enriched in different chromatin modifications and in different types of chromosomes. Active isoforms of RNA polymerase II were depleted in the outermost zone. Also chromocenters and nucleoli were radially aligned in the nuclear interior. The splicing factor SC35 was enriched at the central zone and did not show the typical speckled pattern of distribution. Evaluation of neuronal and non-neuronal chicken tissues showed that the highly ordered form of radial nuclear organization was also present in neuronal chicken tissues. Furthermore, the data revealed that the neuron-specific nuclear organization was remodeled when cells spread on a flat substrate. Monolayer cultures of a chicken cell line did not show this extreme form of radial organization. Rather, such monolayer cultures displayed features of nuclear organization which have been described before for many different types of monolayer cells. The finding that an extreme form radial nuclear organization, which has not been described before, is present in RS and tissues, but not in cells spread on a flat substrate, suggests that it would be important to complement studies on nuclear architecture performed with monolayer cells by studies on 3D cell culture systems and tissues.

Simple explant culture of the embryonic chicken retina with long-term preservation of photoreceptors.

Structurally stable in vitro-model systems are indispensible to analyse neural development during embryogenesis, follow cellular differentiation and evaluate neurotoxicological or growth factor effects. Here we describe a three-dimensional, long-term in vitro-culture system of the embryonic chick retina which supports photoreceptor development. Retinal tissue was isolated from E6 chick eye, and cultured as explants by continuous orbital rotation to allow free floatation without any supporting materials. Young stage (E6) immature retinas were cultured for various time periods in order to follow the differentiation of cell types and plexiform layers by immunocytochemical methods. These explants could be cultured for at least 2-3 weeks with remarkable retention of retinal architecture. Interestingly, photoreceptors developed in the absence of pigment epithelium. Electron microscopic studies revealed formation of structures resembling photoreceptor outer segments, a feature not reported previously. Thus, the verification of photoreceptors, Müller cells, inner retinal cells and the inner plexiform layer described in our study establishes this explant culture as a valuable in vivo-like model system.

Electromagnetic exposure of scaffold-free three-dimensional cell culture systems.

In recent years, a number of in vitro studies have reported on the possible athermal effects of electromagnetic exposure on biological tissue. Typically, this kind of study is performed on monolayers of primary cells or cell lines. However, two-dimensional cell layer systems lack physiological relevance since cells in vivo are organized in a three-dimensional (3D) architecture. In monolayer studies, cell-cell and cell-ECM interactions obviously differ from live tissue and scale-ups of experimental results to in vivo systems should be considered carefully. To overcome this problem, we used a scaffold-free 3D cell culture system, suitable for the exploration of electrophysiological effects due to electromagnetic fields (EMF) at 900 MHz. Dissociated cardiac myocytes were reaggregated into cellular spheres by constant rotation, and non-invasive extracellular recordings of these so-called spheroids were performed with microelectrode arrays (MEA). In this study, 3D cell culture systems were exposed to pulsed EMFs in a stripline setup. We found that inhomogeneities in the EMF due to electrodes and conducting lines of the MEA chip had only a minor influence on the field distribution in the spheroid if the exposure parameters were chosen carefully.

Non-neuronal Role of Acetylcholinesterase in Bone Development and Degeneration.

Acetylcholinesterase (AChE), an enzyme catalyzing the degradation of acetylcholine, plays an important suppressive role in the cholinergic regulation by terminating the action of acetylcholine. The expression of acetylcholinesterase and other cholinergic components is not restricted to only brain and nerve tissues but can also be found in non-neuronal tissues like the immune system and bone tissue. Primary identification of these components has been achieved. However, the information about their specific functions and underlying molecular mechanisms in bone remains scattered. Here, the physiological process of bone development, homeostasis, and degeneration are introduced. Next, the cholinergic system and its expression in bone tissue is documented. Among them, special attention goes to AChE, as the structure of this enzyme suggests diverse binding affinities, enabled by a peripheral site and a catalytic site. The peripheral site supports the non-enzymatic function of AChE in non-neuronal systems. Based on recent studies, the non-neuronal roles of acetylcholinesterase, both enzymatically and non-enzymatically, in bone development, homeostasis and degeneration are summarized briefly together with potential mechanisms to support these functions. We conclude that AChE may be a potential therapeutic target for bone diseases like osteoporosis.

Differences in the Response to DNA Double-Strand Breaks between Rod Photoreceptors of Rodents, Pigs, and Humans.

Genome editing (GE) represents a powerful approach to fight inherited blinding diseases in which the underlying mutations cause the degeneration of the light sensing photoreceptor cells of the retina. Successful GE requires the efficient repair of DNA double-stranded breaks (DSBs) generated during the treatment. Rod photoreceptors of adult mice have a highly specialized chromatin organization, do not efficiently express a variety of DSB response genes and repair DSBs very inefficiently. The DSB repair efficiency in rods of other species including humans is unknown. Here, we used ionizing radiation to analyze the DSB response in rods of various nocturnal and diurnal species, including genetically modified mice, pigs, and humans. We show that the inefficient repair of DSBs in adult mouse rods does not result from their specialized chromatin organization. Instead, the DSB repair efficiency in rods correlates with the level of Kruppel-associated protein-1 (KAP1) expression and its ataxia-telangiectasia mutated (ATM)-dependent phosphorylation. Strikingly, we detected robust KAP1 expression and phosphorylation only in human rods but not in rods of other diurnal species including pigs. Hence, our study provides important information about the uniqueness of the DSB response in human rods which needs to be considered when choosing model systems for the development of GE strategies.

Cholinergic control of bone development and beyond.

There is ample evidence that cholinergic actions affect the health status of bones in vertebrates including man. Nicotine smoking, but also exposure to pesticides or medical drugs point to the significance of cholinergic effects on bone status, as reviewed here in Introduction. Then, we outline processes of endochondral ossification, and review respective cholinergic actions. In Results, we briefly summarize our in vivo and in vitro studies on bone development of chick and mouse [1,2], including (i) expressions of cholinergic components (AChE, BChE, ChAT) in chick embryo, (ii) characterisation of defects during skeletogenesis in prenatal ChE knockout mice, (iii) loss-of-function experiments with beads soaked in cholinergic components and implanted into chicken limb buds, and finally (iv) we use an in vitro mesenchymal 3D-micromass model that mimics cartilage and bone formation, which also had revealed complex crosstalks between cholinergic, radiation and inflammatory mechanisms [3]. In Discussion, we evaluate non-cholinergic actions of cholinesterases during bone formation by considering: (i) how cholinesterases could function in adhesive mechanisms; (ii) whether and how cholinesterases can form bone-regulatory complexes with alkaline phosphatase (ALP) and/or ECM components, which could regulate cell division, migration and adhesion. We conclude that cholinergic actions in bone development are driven mainly by classic cholinergic, but non-neural cycles (e.g., by acetylcholine); in addition, both cholinesterases can exert distinct ACh-independent roles. Considering their tremendous medical impact, these results bring forward novel research directions that deserve to be pursued.

Basic fibroblast growth factor increases the precursor pool of photoreceptors, but inhibits their differentiation and apoptosis in chicken retinal reaggregates.

The role(s) of basic fibroblast growth factor (bFGF, FGF-2) in the differentiation and survival of photoreceptor (PR) cells was investigated in three-dimensional reaggregated histotypic spheres, derived from dispersed cells of the embryonic day 6 chicken embryo retina. Novel data processing methods are introduced to reliably quantify sphere sizes and spatial distributions of immunochemical signals in spheroids. Supplementation with 25 ng/mL FGF-2 increased cell proliferation, detected by bromodeoxyuridine uptake, and growth of spheroids. Immunochemical studies showed that FGF-2 decreased the number of visinin-positive and XAP-1-positive cells, including the total PR pool from early precursor until mature states, whereas the number of Pax6-positive amacrine cells was strongly increased. Notably, the relative number of PR precursors as detected by an Islet2 antibody was increased. The further differentiation of both red/green cones and then rods, as detected by CERN-906 and CERN-901 antibody binding, was much delayed. In contrast, blocking system-inherent FGF-2 by suramin showed opposite effects. Addition of both FGF-2 plus suramin resulted in nearly normal levels of PR differentiation. Terminal deoxynucleotidyl transferase dUTP nick end labelling histochemistry showed that PR apoptosis, which generally progresses with the age of spheres, was strongly increased by suramin treatment. These results suggest that in a three-dimensional retinal tissue context, FGF-2 restricts the pool of PRs in favour of cells of the inner retina, increases and maintains their precursor pool, delays their differentiation, and also protects them from apoptosis.

IPL Sublamination in Chicken Retinal Spheroids Is Initiated via Müller Cells and Cholinergic Differentiation, and Is Disrupted by NMDA Signaling.

Purpose: Reaggregates from E6 embryonic chicken retina exhibit areas corresponding to an inner plexiform layer (IPL), which presents an ideal in vitro model to test conditions and constraints of cholinergic and glutamatergic network formation, providing a basis for retinal tissue engineering. Here, we show that ipl formation is regulated by cholinergic starburst amacrine cells (SACs), a glial scaffold and by L-glutamate. Methods: Rosetted spheroids were cultured in absence or presence of 0.2 to 0.4 mM L-glutamate and analyzed by immuno- and enzyme histochemistry, proliferation, and apoptosis assays. Results: After 2 days in vitro (div), ipl formation was announced by acetylcholinesterase+ (AChE) and choline acetyltransferase+ (ChAT) cells. Individual vimentin+ or transitin+ Müller glial cell precursors (MCPs) in ipl centers coexpressed ChAT. Comparable to in vivo, pairwise arranged ChAT+ SACs formed two laminar subbands. Projections of calretinin+ amacrine cells (ACs) into ipl associated with MCP processes. In L-glutamate-, or NMDA-treated spheroids ipls were disrupted, including loss of SACs and MCs; coincubation with NMDA receptor inhibitor MK-801 prevented these effects. Also, many Pax6+ cells, comprising most ACs, were lost, while rho4D2+ rod photoreceptors were increased. Cell proliferation was slightly increased, while apoptosis remained unaffected. Conclusions: This demonstrated: (1) a far-advanced differentiation of an IPL in retinal spheroids, as never described before; (2) ipl sublamination was initiated by cholinergic precursor cells, which-functioning as "ipl founder cells"-(3) gave rise to neurons and glial cells; (4) these SACs and MCPs together organized ipl formation; and (5) this process was counteracted by NMDA-dependent glutamate actions.

Acetylcholinesterase in cell adhesion, neurite growth and network formation.

The expression of acetylcholinesterase is not restricted to cholinergically innervated tissues and relates to both neurotransmission and multiple biological aspects, including neural development, stress response and neurodegenerative diseases. Therefore, the classical function of acetylcholinesterase has to be distinguished from its non-classical, e.g. enzymatic from non-enzymatic, functions. Here, the roles of acetylcholinesterase in cell adhesion, promoting neurite outgrowth and neural network formation are reviewed briefly, together with potential mechanisms to support these functions. Part of these functions may depend on the structural properties of acetylcholinesterase, for example, protein-protein interactions. Recent findings have revealed that laminin-1 is an interaction partner for acetylcholinesterase. The binding of acetylcholinesterase to this extracellular matrix component may allow cell-to-cell recognition, and also cell signalling via membrane receptors. Studies using monolayer and 3D spheroid retinal cultures, as well as the acetylcholinesterase-knockout mouse, have been instrumental in elaborating the non-classical functions of acetylcholinesterase.

High LET radiation shows no major cellular and functional effects on primary cardiomyocytes in vitro.

It is well known that ionizing radiation causes adverse effects on various mammalian tissues. However, there is little information on the biological effects of heavy ion radiation on the heart. In order to fill this gap, we systematically examined DNA-damage induction and repair, as well as proliferation and apoptosis in avian cardiomyocyte cultures irradiated with heavy ions such as titanium and iron, relevant for manned space-flight, and carbon ions, as used for radiotherapy. Further, and to our knowledge for the first time, we analyzed the effect of heavy ion radiation on the electrophysiology of primary cardiomyocytes derived from chicken embryos using the non-invasive microelectrode array (MEA) technology. As electrophysiological endpoints beat rate and field action potential duration were analyzed. The cultures clearly exhibited the capacity to repair induced DNA damage almost completely within 24 h, even at doses of 7 Gy, and almost completely recovered from radiation-induced changes in proliferative behavior. Interestingly, no significant effects on apoptosis could be detected. Especially the functionality of primary cardiac cells exhibited a surprisingly high robustness against heavy ion radiation, even at doses of up to 7 Gy. In contrast to our previous study with X-rays the beat rate remained more or less unaffected after heavy ion radiation, independently of beam quality. The only change we could observe was an increase of the field action potential duration of up to 30% after titanium irradiation, diminishing within the following three days. This potentially pathological observation may be an indication that heavy ion irradiation at high doses could bear a long-term risk for cardiovascular disease induction.

Non-thermal near-infrared exposure photobiomodulates cellular responses to ionizing radiation in human full thickness skin models.

Ionizing and near-infrared radiation are both part of the therapeutic spectrum in cancer treatment. During cancer therapy ionizing radiation is typically used for non-invasive reduction of malignant tissue, while near-infrared photobiomodulation is utilized in palliative medical approaches, e.g. for pain reduction or impairment of wound healing. Furthermore, near-infrared is part of the solar wavelength spectrum. A combined exposure of these two irradiation qualities - either intentionally during medical treatment or unintentionally due to solar exposure - is therefore presumable for cancer patients. Several studies in different model organisms and cell cultures show a strong impact of near-infrared pretreatment on ionizing radiation-induced stress response. To investigate the risks of non-thermal near-infrared (NIR) pretreatment in patients, a human in vitro full thickness skin models (FTSM) was evaluated for radiation research. FTSM were pretreated with therapy-relevant doses of NIR followed by X-radiation, and then examined for DNA-double-strand break (DSB) repair, cell proliferation and apoptosis. Double-treated FTSM revealed a clear influence of NIR on X-radiation-induced stress responses in cells in their typical tissue environment. Furthermore, over a 24h time period, double-treated FTSM presented a significant persistence of DSBs, as compared to samples exclusively irradiated by X-rays. In addition, NIR pretreatment inhibited apoptosis induction of integrated fibroblasts, and counteracted the radiation-induced proliferation inhibition of basal keratinocytes. Our work suggests that cancer patients treated with X-rays should be prevented from uncontrolled NIR irradiation. On the other hand, controlled double-treatment could provide an alternative therapy approach, exposing the patient to less radiation.

GDNF stimulates rod photoreceptors and dopaminergic amacrine cells in chicken retinal reaggregates.

PURPOSE: To investigate the role(s) of glial cell line-derived neurotrophic factor (GDNF) on expression of rod photoreceptor and dopaminergic amacrine cell-specific genes in an in vitro reaggregate model of the chick retina. METHODS: Retinal reaggregates derived from embryonic day (E)6 chicks (rosetted spheroids) were supplemented with 50 ng/mL GDNF, or, alternatively, endogenous GDNF expression was downregulated by transient transfection of spheroids with a pCMS-EGFP[GDNF] antisense vector. Using mainly semiquantitative RT-PCR analyses, expression of rhodopsin, four separate opsins, and tyrosine hydroxylase (THase) was analyzed after either treatment. RESULTS: Supplementation with GDNF accelerated rhodopsin mRNA expression and sustained it at an increased level, in contrast to untreated control subjects, where rhodopsin mRNA levels were lower and unmaintained. Expression of red, green, blue, and violet opsins were unaffected. Under these conditions, GDNF also massively increased the expression of tyrosine hydroxylase, the rate-limiting enzyme in the synthesis of dopamine. The expression of endogenous GDNF was blocked in spheroids by using antisense transfections, which resulted in both a significant decrease in rhodopsin mRNA expression and a complete suppression of THase expression, as determined by RT-PCR, Western blot analysis, and immunocytochemistry. CONCLUSIONS: GDNF supports expression of both rhodopsin and THase in vitro, two critical molecules involved in the production of rod photoreceptors and dopaminergic amacrine cells, respectively; however, the presence of GDNF does not affect cone production and survival.

Human serum cholinesterase from liver pathological samples exhibit highly elevated aryl acylamidase activity.

BACKGROUND: Although aspartate aminotransferase (AST) and gamma-glutamyltransferase (gamma GT) enzymes are widely used as markers for liver disorders, the ubiquitous enzyme butyrylcholinesterase (BChE), synthesized in liver is also used as marker in the assessment of liver pathophysiology. This BChE enzyme in addition to its esterase activity has yet another enzymatic function designated as aryl acylamidase (AAA) activity. It is determined in in vitro based on the hydrolysis of the synthetic substrate o-nitroacetanilide. In the present study, human serum cholinesterase (BChE) activity was studied with respect to its AAA activity on the BChE protein (AAA(BChE)) in patients with liver disorders. AST and gamma GT values were taken into account in this study as known markers for liver disorders. METHODS: Blood samples were grouped into 3 based on esterase activity associated with BChE protein. They are normal, low, and very low BChE activity but with markedly increased AST and gamma GT levels. These samples were tested for their respective AAA function. Association of AAA with BChE from samples was proved using BChE monoclonal antibody precipitation experiment. RESULTS: The absolute levels of AAA were increased as BChE activity decreased while deviating from normal samples and such deviation was directly proportional to the severity of the liver disorder. Differences between these groups became prominent after determining the ratios of AAA(BChE) to BChE activities. Samples showing very high AAA(BChE) to BChE ratio were also showing high to very high gamma GT values. CONCLUSIONS: These findings establish AAA(BChE) as an independently regulated enzymatic activity on BChE especially in liver disorders. Moreover, since neither the low esterase activity of BChE by itself nor increased levels of AST/gamma GT are sufficient pathological indicators, this pilot study merits replication with large sample numbers.

Expression and possible functions of the cholinergic system in a murine embryonic stem cell line.

The expression of a cholinergic system during embryonic development is a widespread phenomenon. However, no precise function could be assigned to it during early pre-neural stages and there are only few studies that document when it precisely starts to be expressed. Here, we examined the expression of cholinergic components in a murine embryonic stem cell line by RT-PCR, histochemistry, and enzyme activity measurements; the acetylcholine (ACh) content was measured by HPLC. We have demonstrated that embryonic stem cells express ACh, acetylcholine receptors, choline acetyltransferase (ChAT), acetyl- and butyryl-cholinesterase (AChE and BChE). Butyryl-cholinesterase (BChE) expression was higher than AChE. The cholinesterase activity was down-regulated by adding specific inhibitors to culture medium. Inhibition of BChE led to a reduction of proliferation. This is the first demonstration that mouse embryonic stem cells express the full molecular equipment of a cholinergic system. Locally produced ACh might function as an intercellular signal, modulating the proliferation of stem cells.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures.

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation.

BMP-induced reprogramming of the neural retina into retinal pigment epithelium requires Wnt signalling.

In vertebrates, the retinal pigment epithelium (RPE) and photoreceptors of the neural retina (NR) comprise a functional unit required for vision. During vertebrate eye development, a conversion of the RPE into NR can be induced by growth factors in vivo at optic cup stages, but the reverse process, the conversion of NR tissue into RPE, has not been reported. Here, we show that bone morphogenetic protein (BMP) signalling can reprogram the NR into RPE at optic cup stages in chick. Shortly after BMP application, expression of Microphthalmia-associated transcription factor (Mitf) is induced in the NR and selective cell death on the basal side of the NR induces an RPE-like morphology. The newly induced RPE differentiates and expresses Melanosomalmatrix protein 115 (Mmp115) and RPE65. BMP-induced Wnt2b expression is observed in regions of the NR that become pigmented. Loss of function studies show that conversion of the NR into RPE requires both BMP and Wnt signalling. Simultaneous to the appearance of ectopic RPE tissue, BMP application reprogrammed the proximal RPE into multi-layered retinal tissue. The newly induced NR expresses visual segment homeobox-containing gene (Vsx2), and the ganglion and photoreceptor cell markers Brn3α and Visinin are detected. Our results show that high BMP concentrations are required to induce the conversion of NR into RPE, while low BMP concentrations can still induce transdifferentiation of the RPE into NR. This knowledge may contribute to the development of efficient standardized protocols for RPE and NR generation for cell replacement therapies.

A deep conical agarose microwell array for adhesion independent three-dimensional cell culture and dynamic volume measurement.

Multicellular spheroids represent a well-established 3D model to study healthy and diseased cells in vitro. The use of conventional 3D cell culture platforms for the generation of multicellular spheroids is limited to cell types that easily self-assemble into spheroids because less adhesive cells fail to form stable aggregates. A high-precision micromoulding technique developed in our laboratory produces deep conical agarose microwell arrays that allow the cultivation of uniform multicellular aggregates, irrespective of the spheroid formation capacity of the cells. Such hydrogel arrays warrant a steady nutrient supply for several weeks, permit live volumetric measurements to monitor cell growth, enable immunohistochemical staining, fluorescence-based microscopy, and facilitate immediate harvesting of cell aggregates. This system also allows co-cultures of two distinct cell types either in direct cell-cell contact or at a distance as the hydrogel permits diffusion of soluble compounds. Notably, we show that co-culture of a breast cancer cell line with bone marrow stromal cells enhances 3D growth of the cancer cells in this system.