Changes in axonal myelination are an important hallmark of aging and a number of neurological diseases. Demyelinated axons are impaired in their function and degenerate over time. Oligodendrocytes, the cells responsible for myelination of axons, are sensitive to mechanical properties of their environment. Growing evidence indicates that mechanical properties of demyelinating lesions are different from the healthy state and thus have the potential to affect myelinating potential of oligodendrocytes. We performed a high-resolution spatial mapping of the mechanical heterogeneity of demyelinating lesions using atomic force microscope-enabled indentation. Our results indicate that the stiffness of specific regions of mouse brain tissue is influenced by age and degree of myelination. Here we specifically demonstrate that acquired acute but not genetic demyelination leads to decreased tissue stiffness, which could influence the remyelination potential of oligodendrocytes. We also demonstrate that specific brain regions have unique ranges of stiffness in white and grey matter. Our ex vivo findings may help the design of future in vitro models to mimic the mechanical environment of the brain in healthy and diseased states. The mechanical properties of demyelinating lesions reported here may facilitate novel approaches in treating demyelinating diseases such as multiple sclerosis. Mechanical characteristics of a cell's environment can have a profound influence on its biological properties. Neuronal and glial cells are sensitive to mechanical input during development, in disease and regeneration. Sustained tensile strain can promote differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes, which are responsible for the myelination of axons. Changing myelination is an important hallmark in human aging and disease, such as multiple sclerosis. Our hypothesis is that these diseases might be characterized by altered tissue stiffness and that this has an influence on remyelination potential. Here we investigate tissue stiffness profiles of healthy, aged and disease model mice. Manipulating the tissue stiffness might be another promising approach for new treatments.
Three-dimensional organoid constructs serve as increasingly widespread in vitro models for development and disease modeling. Current approaches to recreate morphogenetic processes in vitro rely on poorly controllable and ill-defined matrices, thereby largely overlooking the contribution of biochemical and biophysical extracellular matrix (ECM) factors in promoting multicellular growth and reorganization. Here, we show how defined synthetic matrices can be used to explore the role of the ECM in the development of complex 3D neuroepithelial cysts that recapitulate key steps in early neurogenesis. We demonstrate how key ECM parameters are involved in specifying cytoskeleton-mediated symmetry-breaking events that ultimately lead to neural tube-like patterning along the dorsal-ventral (DV) axis. Such synthetic materials serve as valuable tools for studying the discrete action of extrinsic factors in organogenesis, and allow for the discovery of relationships between cytoskeletal mechanobiology and morphogenesis.
Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.
Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.
Abstract The alteration or decrease of axonal myelination is an important hallmark of aging and disease. Demyelinated axons are impaired in their function and degenerate over time. Oligodendrocytes, the cells responsible for myelination of axons, are sensitive to mechanical properties of their environment. Growing evidence indicates that mechanical properties of demyelinating lesions are different from the healthy state and thus have the potential to affect myelinating potential of oligodendrocytes. We performed a high-resolution spatial mapping of the mechanical heterogeneity of demyelinating lesions using Atomic Force Microscope enabled indentation. Our results indicate that the stiffness of specific regions of mouse brain tissue is influenced by age and degree of myelination. Here we specifically demonstrate that acute but not inherited demyelination leads to decreased tissue stiffness, which could lower remyelination potential of oligodendrocytes. We also demonstrate that specific brain regions have unique ranges of stiffness in white and grey matter. Our ex vivo findings may help the design of future in vitro models to mimic mechanical environment of the brain in healthy and disease state. Reported here, mechanical properties of demyelinating lesions may facilitate novel approaches in treating demyelinating diseases such as multiple sclerosis.
Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.
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