Glucose metabolism regulates human cortical cell diversification and maturation

Defining metabolic regulation of progenitor expansion and neurogenic differentiation is essential to understand and treat neurodevelopmental disorders driven by metabolic alterations that have life-long impact on neurological health. In vitro cultures are the only method to temporally study and molecularly manipulate living human brain cells. However, all current cell culture conditions use supraphysiological levels of glucose and oxygen, compared to the developing brain. In our studies, we probed how environmental exposure to endogenous-like concentrations of glucose and oxygen affect metabolic state and developmental progression of human cortical cell types by using pluripotent stem cell (PSC)-derived organoids. In addition to functional assays and immunolabeling, we conducted single cell RNA sequencing of cortical organoids exposed to varying glucose and oxygen conditions using a 10x single cell genomics pipeline. Overall design: Cortical organoids were exposed to standard culture conditions (18 mM glucose media incubated in 20% oxygen) or experimental culture conditions which consisted of a mix of glucose and oxygen conditions (18 mM glucose at 8% oxygen, 5 mM glucose at 8% oxygen, and 5 mM glucose at 20% oxygen). The 5 mM medias are DMEM/F12 (shortend to DMEM), Plasmax, and RPMI, while the control media is DMEM/F12 (shortened to DMEM) with standard glucose concentrations (18 mM). The DMEM and RPMI with 5 mM media was created by adding 5 mM of glucose to a special ordered basal medium containing no glucose. The Plasmax basal media is manufactured with 5 mM glucose at baseline. Organoids across three PSC lines were exposed to the media and oxygen conditions for 6 or 10 weeks in culture and then captured for single cell RNA analysis, resulting in 36 experimental samples collected.