Dopamine signaling in the adult ventral forebrain regulates behavior, stress response, and memory formation and in neurodevelopment regulates neural differentiation and cell migration. Excessive dopamine levels, including those due to cocaine use in utero and in adults, could lead to long-term adverse consequences. The mechanisms underlying both homeostatic and pathological changes remain unclear, in part due to the diverse cellular responses elicited by dopamine and the reliance on animal models that exhibit species-specific differences in dopamine signaling. In this study, we use the human-derived ventral forebrain organoid model of Xiang–Tanaka and characterize their response to cocaine or dopamine. We explore dosing regimens of dopamine or cocaine to simulate acute or chronic exposure. We then use calcium imaging, cAMP imaging, and bulk RNA-sequencing to measure responses to cocaine or dopamine exposure. We observe an upregulation of inflammatory pathways in addition to indicators of oxidative stress following exposure. Using inhibitors of reactive oxygen species (ROS), we then show ROS to be necessary for multiple transcriptional responses of cocaine exposure. These results highlight novel response pathways and validate the potential of cerebral organoids as in vitro human models for studying complex biological processes in the brain.

The mesolimbic pathway connects ventral tegmental area dopaminergic neurons and striatal medium spiny neurons, playing a critical role in reward and stress behaviors. Exposure to substances of abuse during development and adulthood has been linked to adverse outcomes and molecular changes. The rise of human cell repositories and whole-genome sequences enables human functional genomics ‘in a dish’, offering insights into human-specific responses to substances of abuse. Continued development of new models is needed, and the characterization of in vitro models is also necessary to ensure appropriate experimental designs and the accurate interpretation of results. This study introduces new culture conditions for generating medium spiny neurons and dopaminergic neurons with an early common media, allowing for coculture and assembloid generation. It then provides a comprehensive characterization of these and prior models and their responses to substances of abuse. Single-cell analysis reveals cell-type-specific transcriptomic responses to dopamine, cocaine, and morphine, including compound and cell-type-specific transcriptomic signatures related to neuroinflammation and alterations in signaling pathways. These findings offer a resource for future genomics studies leveraging human stem cell-derived models.

Objective: To determine how fibronectin fragment (Fn-f), a matrikine that induces an osteoarthritis (OA)-like phenotype, reshapes chondrocyte states and transcriptional programs at single-cell resolution. Design: Primary human ankle chondrocytes from two cadaveric donors without OA were treated with PBS or purified recombinant Fn-f for 18 hours and profiled by single-cell RNA sequencing. Transcriptional states were characterized by marker genes and functional enrichment, compared with classifications used for OA chondrocytes, and evaluated for Fn-f-dependent changes in abundance and transcriptional programs. Results: Five transcriptionally distinct populations were identified with features that matched previous classifications of effector, inflammatory, pre-hypertrophic/fibrochondrocyte, reparative, and homeostatic chondrocytes. Fn-f markedly redistributed cells among these states, enriching inflammatory chondrocytes while reducing effector and reparative populations. Within transcriptional states, Fn-f differentially altered matrix/cartilage, inflammatory, and stress/immediate-early programs. These responses did not uniformly correspond to assigned subtype identities, demonstrating that transcriptional programs can be dynamically deployed across chondrocyte states. Conclusions: Controlled perturbations of healthy chondrocytes complement observational OA atlases by resolving stimulus-dependent changes in chondrocyte state abundance and transcriptional programs. Discordance between subtype annotations and dynamic functional programs further highlights the need for harmonized definitions of chondrocytes across phenotypic states.