Poster presented at ISSCR 2023 by Vukasin Jovanovic. The poster is entitled: Scalable Differentiation of Hypothalamic Arcuate Nucleus Neurons from Human Pluripotent Stem Cells.
Authors (underlined, presenting): Vukasin M. Jovanovic, Ravi Tharakan, Chaitali Sen, Jason Inman, Pinar Ormanoglu, Christopher LeClair, Anton Simeonov, Carlos Tristan, Ilyas Singeç
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Abstract
The hypothalamus is the main integration site of endocrine and neuronal systems regulating autonomous and behavioral responses related to sleep,circadian rhythm, thermoregulation, hormonerelease, arousal, reproductive cycling, thirst, hunger, and satiety. Neuroendocrine dysfunction of hypothalamic neurons in the arcuate nucleus is associated with obesity, type 2 diabetes, hyperlipidemia, high blood pressure, and cardiovascular disease. Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus respond to postprandially secreted hormones (e.g., insulin) to inhibit appetite and increase energy expenditure. However, access to the human hypothalamus for translational research and drug development remains very limited. Here we report controlled, efficient, and scalable differentiation of human pluripotent stem cells (hPSCs) into pure populations of POMC neurons with arcuate nucleus identity. Combined use of several small molecules that modulate developmental pathways, specify hPSCs into ventraldiencephalon followed by a hypothalamicidentity. Time-course gene expression analysis (RNA-seq) of independent hPSC lines revealed strong upregulation of typical hypothalamic cell-type specific transcription factors over the course of 21 days (e.g., NKX2.1, RAX, OTP, ARX, ISL1). Importantly, no upregulation of dorsal forebrain markers (e.g., PAX6 and FOXG1) was observed. These findings were further validated by protein expression analysis (immunocytochemistry, Westernblot). Comparison of the top differentially expressed genes of stem cell-derived neurons to human brain tissue confirmed a distinct hypothalamic molecular signature. Additional phenotypic characterization revealed inhibitory properties (GABA, GAD65/67) and a subpopulation of tyrosine hydroxylase positive (TH+) dopamine neurons. Administration of human recombinant insulin to cultures of electrically active neurons increased the firing rate in a dose-dependent manner. Lastly, this differentiation approach was automated using a robotic cell culture system that enables the production of large numbers of hypothalamic neurons for translational applications.
