Bone morphogenic protein (BMP) signalling contributes towards maintenance of pluripotency and favours mesodermal over neural fates upon differentiation, but the mechanisms by which BMP controls differentiation are not well understood. We report that BMP regulates differentiation by blocking downregulation of Cdh1, an event that accompanies the earliest stages of neural and mesodermal differentiation. We find that loss of Cdh1 is a limiting requirement for differentiation of pluripotent cells, and that experimental suppression of Cdh1 activity rescues the BMP-imposed block to differentiation. We further show that BMP acts prior to and independently of Cdh1 to prime pluripotent cells for mesoderm differentiation, thus helping to reinforce the block to neural differentiation. We conclude that differentiation depends not only on exposure to appropriate extrinsic cues but also on morphogenetic events that control receptivity to those differentiation cues, and we explain how a key pluripotency signal, BMP, feeds into this control mechanism.DOI: http://dx.doi.org/10.7554/eLife.01197.001
Progress made in the last two decades in chemical vapour deposition technology has enabled the production of inexpensive, high-quality coatings made from diamond to become a scientific and commercial reality. Two properties of diamond make it a highly desirable candidate material for biomedical applications: first, it is bioinert, meaning that there is minimal immune response when diamond is implanted into the body, and second, its electrical conductivity can be altered in a controlled manner, from insulating to near-metallic. In vitro, diamond can be used as a substrate upon which a range of biological cells can be cultured. In vivo, diamond thin films have been proposed as coatings for implants and prostheses. Here, we review a large body of data regarding the use of diamond substrates for in vitro cell culture. We also detail more recent work exploring diamond-coated implants with the main targets being bone and neural tissue. We conclude that diamond emerges as one of the major new biomaterials of the twenty-first century that could shape the way medical treatment will be performed, especially when invasive procedures are required.
Brain Computer Interfaces (BCI) currently represent a field of intense research aimed both at understanding neural circuit physiology and at providing functional therapy for traumatic or degenerative neurological conditions. Due to its chemical inertness, biocompatibility and stability, diamond is currently being actively investigated as a potential substrate material for culturing cells and for use as the electrically active component of a neural sensor. Here we provide a protocol for the differentiation of mature, electrically active neurons on microcrystalline synthetic thin-film diamond substrates starting from undifferentiated pluripotent stem cells. Furthermore, we investigate the optimal characteristics of the diamond microstructure for long-term neuronal sustainability. We also analyze the effect of boron as a dopant for such a culture. We found that the diamond crystalline structure has a significant influence on the neuronal culture unlike the boron doping. Specifically, small diamond microcrystals promote higher neurite density formation. We find that boron incorporated into the diamond does not influence the neurite density and has no deleterious effect on cell survival.
Macroautophagy/autophagy cytoplasmic quality control pathways are required during neural development and are critical for the maintenance of functional neuronal populations in the adult brain. Robust evidence now exists that declining neuronal autophagy pathways contribute to human neurodegenerative diseases, including Parkinson disease (PD). Reliable and relevant human neuronal model systems are therefore needed to understand the biology of disease-vulnerable neural populations, to decipher the underlying causes of neurodegenerative disease, and to develop assays to test therapeutic interventions in vitro. Human induced pluripotent stem cell (hiPSC) neural model systems can meet this demand: they provide a renewable source of material for differentiation into regional neuronal subtypes for functional assays; they can be expanded to provide a platform for screening, and they can potentially be optimized for transplantation/neurorestorative therapy. So far, however, hiPSC differentiation protocols for the generation of ventral midbrain dopaminergic neurons (mDANs)the predominant neuronal sub-type afflicted in PDhave been somewhat restricted by poor efficiency and/or suitability for functional and/or imaging-based in vitro assays. Here, we describe a reliable, monolayer differentiation protocol for the rapid and reproducible production of high numbers of mDANs from hiPSC in a format that is amenable for autophagy/mitophagy research. We characterize these cells with respect to neuronal differentiation and macroautophagy capability and describe qualitative and quantitative assays for the study of autophagy and mitophagy in these important cells.
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