A close spatiotemporal relation between end of mitosis, of butyryl- (BChE) and then acetylcholinesterase (AChE) expressions in early neural tissues has been established, yet the underlying mechanisms are not understood. With this observation in mind, cell culture work analyzing cholinergic functions in the developing vertebrate retina was revealing. For proper cell stratification in avian and rodent retinal 3D-reaggregates (organoids) and explants, Müller glial cells (MCs) and the retinal pigmented epithelium (RPE) are essential actors. Network formation depends on a ChAT+ amacrine cell and BChE+ MCs. Further, cholinergic effects at the photoreceptor (PR) to RPE interface remain unclear. Acetylcholine (ACh) reportedly is produced in, and released from PRs, which is received by alpha-7 nicotinic acetylcholine receptors (α7-nAChRs) on adjacent RPE cells. If properly activated, RPE cells in turn signal ACh back to MCs. Notably, MCs under certain conditions act as stem cells in retinal regeneration. Hence, we postulate that a cholinergic signaling loop between MCs, PRs and RPE, which probably already constituted an ancient proto eye, can regulate stem cells. Since a similar cholinergic triad regulates skin regeneration, comparable cholinergic triads could appear at the core of stem cell biology, deserving more research (e.g., cancer biology, tissue regeneration).

Reaching back more than a century, suspension cultures have provided major insights into processes of histogenesis; e.g., cell communication, distinction of self/nonself, cell sorting and cell adhesion. Besides studies on lower animals, the vertebrate retina served as excellent reaggregate model to analyze 3D reconstruction of a complex neural laminar tissue. Methodologically, keeping cells under suspension is essential to achieve tissue organisation in vitro; thereby, the environmental conditions direct the emergent histotypic particulars. Recent progress in regenerative medicine is based to a large extent on human induced pluripotent stem cells (hiPSCs), which are cultured under suspension. Following their genetically directed differentiation into various histologic 3D structures, organoids provide excellent multipurpose in vitro assay models, as well as tissues for repair transplantations. Historically, a nearly fully laminated retinal spheroid from avian embryos was achieved already in 1984, foreshadowing the potential of culturing stem cells under suspension for tissue reconstruction purposes.

Abstract The post-genomic era raises questions about neo-Darwinian genetic determinism. Instead, open aspects of macroevolution become intelligible by Evo-Devo research. At all developmental levels, self-organization acts robustly towards “wholeness”, as exemplified by organoid technologies. In retinal reaggregates histotypical features are reached along different formative routes. Thus, tissue formation is not merely gene-directed, but channeled by unpredictable external conditions. These insights restrict conceptions of onto- and phylogenesis. Neither is characterized by unlimited randomness nor by finite genocentrism. A re-examination of Driesch’s drive to wholeness, i.e., robustness and intentionality, appears timely, while his teleologic postulates remain undecidable by reductionist reasoning.

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.

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.

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.

ZusammenfassungSowohl die frühe phylogenetische und ontogenetische Existenz von cholinergen Systemen, als auch das Vorkommen in nicht‐neuronalen Geweben legen cholinerge und nicht‐cholinerge Funktionalitäten nahe, die weit über deren klassische Funktion an Synapsen hinausgehen. Die Fähigkeit von Cholinesterasen zur Bildung riesiger Proteinkomplexe eröffneten ihnen vielseitige Funktionsfelder. Schon in Stammzellen vertreten, begünstigen Cholinesterasen im Verbund mit Komponenten der Zellmatrix die Zelldifferenzierung; dabei erscheint ihre Enzymaktivität (teilweise) als nicht notwendig. Dies wurde durch Effekte inaktiver AChE in nicht‐neuronalen Zellen einerseits, und davon unabhängig durch die Entdeckung der CLAM‐Proteinfamilie beeindruckend untermauert. Vieles spricht somit dafür, dass die ursprünglichen Funktionsfelder der Cholinesterasen, wie auch von cholinergen Systemen insgesamt, in allgemeinen Zell‐Zell‐Wechselwirkungen zu suchen sind. Diese Einsichten wurden hier an einigen Zellkulturstudien und ausgewählten Beispielen der Normalentwicklung dargestellt. In der Wirbeltierretina beeinflussen die als erste differenzierenden cholinergen Amakrinzellen die Netzwerkbildung. Nicht weniger bedeutend ist das cholinerge System bei der Bildung von Röhrenknochen. Acetylcholin beschleunigt die Knochenbildung, und die Cholinesterasen regulieren dabei nicht nur dessen Konzentration, sondern spielen beide zudem strukturelle Rollen. Ebenso überzeugend ist eine Studie an Froschlarven, die zeigt, dass bei der Darmbildung von Xenopus laevis sehr wohl das AChE‐Protein, aber nicht dessen Enzymaktivität beteiligt ist. Die volle Aufklärung der Wirkungsweise der Cholinesterasen ist notwendig, denn eine Vielzahl von spezifischen Anticholinesterasen finden breite Anwendungen in wichtigen gesellschaftlichen Bereichen (Landwirtschaft, Gesundheit, Sicherheit). Die Forschung hat dies erkannt und widmet sich verstärkt nicht nur den Cholinesterasen, sondern insgesamt der Aufklärung nicht‐neuronaler cholinerger Systeme (NNCS).

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.

Molecular developmental biology has expanded our conceptions of gene actions, underpinning that embryonic development is not only governed by a set of specific genes, but as much by space–time conditions of its developing modules (determinate vs. regulative development; or, nature vs. nurture discussion). Typically, formation of cellular spheres, their transformation into planar epithelia, followed by tube formations and laminations are modular steps leading to the development of nervous tissues. Thereby, actions of organising centres, morphogenetic movements (in- and evaginations), inductive events between epithelia, tissue polarity reversal, widening of epithelia, and all these occurring orderly in space and time, are driving forces of emergent laminar neural tissues, e.g. the vertebrate retina. Analyses of self-organisational formation of retina-like 3D structures from dispersed cells (so-called retinal spheroids, also called retinal organoids) under defined cell culture conditions (in vitro) demonstrate that not only particular genetic networks, but—at least as important—the applied culture conditions (in vitro constraints) define phenotypes of emergent tissues. Such in vitro approaches allow assigning emerging tissue formation to ground-laying genetic networks separately from contributions by conditional constraints.

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.

Recently we could establish major cholinergic impact on vertebrate in vivo and in vitro skeletogenesis (1,2). Cholinergic mechanisms are also at the core of formation of the vertebrate retina. Retinal histogenesis of a so-called inner plexiform layer (IPL) was disturbed in an AChE KO mouse (3). Characterized best by their ChAT expression, the only cholinergic cells in all vertebrate retinae are so-called starburst amacrine cells (SACs), which send processes into synaptic IPL sublaminae. We documented that SACs are derived from a larger pool of postmitotic AChE+ cells. A developmental comparison of ChAT+ and AChE+ cells revealed a close spatial localization of both proteins first within individual cells (nuclear ChAT, vs. extranuclear AChE), and later between adjacent cells, e.g., ACh-secreting and -degrading cells have the same cell lineage origin, and later remain in close apposition (4). Using our 3D stem cell organoid approach (retinal spheroids), we could show that ChAT+ cells were first to initiate IPL formation by establishing two synaptic sublaminae. Unexpectedly, the earliest ChAT+ cells co-expressed markers of Müller glial precursors (MCPs), indicating that a direct SAC precursor i) gives rise to neurons and glial cells, and ii) that these premature cholinergic cells drive earliest processes of network formation in vertebrate retinae, e.g. could function as IPL founder cells (5, cf. also 6,7). These findings could have profound relevance for a basic understanding of neuronal network formation.

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.

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.

Besides invention of iPSC technology, recent progress of stem cell-based organoids is founded on long-standing 3D-reaggregate approaches from embryonic tissues. In particular, histotypic in vitro reconstruction of avian retinal spheroids was most prolific. For instance, a complete reconstitution of all retinal layers was possible, which was supported by Wnt signalling and factors from the retinal pigmented epithelium (RPE); similar in vitro findings are still missing for mammals. Using an established model of reaggregates from dispersed retinal cells of the neonatal Gerbil, we show here that in contrast to supernatant from RPE (RPECM), supplementation with Wnt3a induced a correct inside-out polarity of retinal layers. XAP1+ precursors of photoreceptors (PRs) were correctly found on the external face of the sphere, but general cell differentiation remained limited.

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 24 h 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.

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.

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.

ZusammenfassungDie Entwicklung von medizinisch hoch relevanten Organoiden aus Stammzellen beruht auf zwei zellbiologischen Forschungslinien: Erstens auf der mehr als ein Jahrhundert zurückliegenden Erkenntnis, dass sich vereinzelte Zellen aus einem embryonalen Gewebeverband durch selbstorganisatorische Prozesse in der Zellkulturschale wieder zu histotypischem Gewebe entwickeln. Am Beispiel der Retina von Wirbeltieren (Huhn, Maus) führte dies unter geeigneten Bedingungen zu kugelförmigen Gewebestrukturen (retinale Sphäroide), die der dreifach geschichteten normalen Retina sehr ähnlich waren und gleichzeitig das erste Beispiel von einem neuronalen Organoid darstellten. Damit war die prinzipielle Möglichkeit der künstlichen Herstellung von Gewebe aus geeigneten Stammzellen aufgezeigt, was nun – zweitens – mit der Erfindung von induzierten pluripotenten Stammzellen (iPSCs) aus ausdifferenziertem menschlichen Gewebe tatsächlich verwirklicht wurde.