ABSTRACT
3D brain organoids derived from human pluripotent stem cells (hPSCs) possess the remarkable ability to self-organize and differentiate into tissue resembling the early human fetal brain. Brain organoids provide a powerful tool for studying human brain development and disease in an in vitro system. Here we describe a protocol for the differentiation of hPSCs to human cerebral organoids using a commercially available kit (STEMdiff™ Cerebral Organoid Kit) and discuss methods to scale up the protocol in a high-throughput manner.
Subject(s)
Organoids , Pluripotent Stem Cells , Brain , Cell Differentiation , HumansABSTRACT
The capabilities of imaging technologies, fluorescent sensors, and optogenetics tools for cell biology are advancing. In parallel, cellular reprogramming and organoid engineering are expanding the use of human neuronal models in vitro. This creates an increasing need for tissue culture conditions better adapted to live-cell imaging. Here, we identify multiple caveats of traditional media when used for live imaging and functional assays on neuronal cultures (i.e., suboptimal fluorescence signals, phototoxicity, and unphysiological neuronal activity). To overcome these issues, we develop a neuromedium called BrainPhys™ Imaging (BPI) in which we optimize the concentrations of fluorescent and phototoxic compounds. BPI is based on the formulation of the original BrainPhys medium. We benchmark available neuronal media and show that BPI enhances fluorescence signals, reduces phototoxicity and optimally supports the electrical and synaptic activity of neurons in culture. We also show the superior capacity of BPI for optogenetics and calcium imaging of human neurons. Altogether, our study shows that BPI improves the quality of a wide range of fluorescence imaging applications with live neurons in vitro while supporting optimal neuronal viability and function.
Subject(s)
Brain/diagnostic imaging , Brain/physiology , Diagnostic Imaging , Neurons/physiology , Optogenetics , Action Potentials/physiology , Animals , Cell Survival , Cells, Cultured , Cerebrospinal Fluid/metabolism , Culture Media , Fluorescence , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Light , Nerve Net/physiology , Osmolar Concentration , Rats , Signal-To-Noise Ratio , Synapses/physiologyABSTRACT
Expression of the cell surface sialomucin CD34 is common to many adult stem cell types, including muscle satellite cells. However, no clear stem cell or regeneration-related phenotype has ever been reported in mice lacking CD34, and its function on these cells remains poorly understood. Here, we assess the functional role of CD34 on satellite cell-mediated muscle regeneration. We show that Cd34(-/-) mice, which have no obvious developmental phenotype, display a defect in muscle regeneration when challenged with either acute or chronic muscle injury. This regenerative defect is caused by impaired entry into proliferation and delayed myogenic progression. Consistent with the reported antiadhesive function of CD34, knockout satellite cells also show decreased motility along their host myofiber. Altogether, our results identify a role for CD34 in the poorly understood early steps of satellite cell activation and provide the first evidence that beyond being a stem cell marker, CD34 may play an important function in modulating stem cell activity.
Subject(s)
Antigens, CD34/metabolism , Cell Movement , Cell Proliferation , Muscle, Skeletal/physiology , Regeneration , Satellite Cells, Skeletal Muscle/cytology , Animals , Antigens, CD34/genetics , Elapid Venoms/adverse effects , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Muscle, Skeletal/drug effects , Muscle, Skeletal/injuries , Point Mutation , Satellite Cells, Skeletal Muscle/physiology , Time-Lapse ImagingABSTRACT
The posterior bed nuclei of the stria terminalis (BST) are important neural substrate for relaying limbic influences to the paraventricular nucleus (PVN) of the hypothalamus to inhibit hypothalamic-pituitary-adrenal (HPA) axis responses to emotional stress. Androgen receptor-expressing cells within the posterior BST have been identified as projecting to the PVN region. To test a role for androgen receptors in the posterior BST to inhibit PVN motor neurons, we compared the effects of the non-aromatizable androgen dihydrotestosterone (DHT), the androgen receptor antagonist hydroxyflutamide (HF), or a combination of both drugs implanted unilaterally within the posterior BST. Rats bearing unilateral implants were analyzed for PVN Fos induction in response to acute-restraint stress and relative levels of corticotrophin-releasing hormone and arginine vasopressin (AVP) mRNA. Glutamic acid decarboxylase (GAD) 65 and GAD 67 mRNA were analyzed in the posterior BST to test a local involvement of GABA. There were no changes in GAD expression to support a GABA-related mechanism in the BST. For PVN neuropeptide expression and Fos responses, basic effects were lateralized to the sides of the PVN ipsilateral to the implants. However, opposite to our expectations of an inhibitory influence of androgen receptors in the posterior BST, PVN AVP mRNA and stress-induced Fos were augmented in response to DHT and attenuated in response to HF. These results suggest that a subset of androgen receptor-expressing cells within the posterior BST region may be responsible for increasing the biosynthetic capacity and stress-induced drive of PVN motor neurons.
