Differentiation, Transcriptomic Profiling, and Calcium Imaging of Human Hypothalamic Neurons

Abstract

AbstractNeurons in the hypothalamus orchestrate homeostatic physiological processes and behaviors essential for life. Human pluripotent stem cells (hPSCs) can be differentiated into many types of hypothalamic neurons, progenitors, and glia. This updated unit includes published studies and protocols with new advances in the differentiation, maturation, and interrogation by transcriptomic profiling and calcium imaging of human hypothalamic cell populations. Specifically, new methods to freeze and thaw hypothalamic progenitors after they have been patterned and before substantial neurogenesis has occurred are provided that will facilitate experimental flexibility and planning. Also included are updated recipes and protocols for neuronal maturation, with details on the equipment and methods for examining their transcriptomic response and cell‐autonomous properties in culture in the presence of synaptic blockers. Together, these protocols facilitate the adoption and use of this model system for fundamental biological discovery and therapeutic translation to human diseases such as obesity, diabetes, sleep disorders, infertility, and chronic stress. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: hPSC maintenanceBasic Protocol 2: Hypothalamic neuron differentiationSupport Protocol 1: Cortical neuron (control) differentiationBasic Protocol 3: Neuronal maturationSupport Protocol 2: Cryopreservation and thawing of neuronal progenitorsSupport Protocol 3: Quality control: Confirmation of hypothalamic patterning and neurogenesisSupport Protocol 4: Bulk RNA sequencing of hypothalamic culturesBasic Protocol 4: Calcium imaging of hypothalamic neurons using Fura‐2 AMAlternate Protocol: Calcium imaging of green fluorescent hypothalamic neurons using Rhod‐3 AM