03002nas a2200421   4500000000100000000000100001008004100002260001200043653003800055653003500093653003500128653003000163653002300193100001900216700002500235700002200260700001800282700002500300700002200325700002000347700002200367700002400389700001900413700002000432700002100452700002600473700002100499700001800520700001700538700001700555700002400572245012900596856005500725300001400780490000700794520176500801022001402566       2023                            d  c2023-1210aDevelopment of the nervous system10aDiseases of the nervous system10aExperimental models of disease10aStem-cell differentiation10aStem-cell research1 aDaniel Reumann1 aChristian Krauditsch1 aMaria Novatchkova1 aEdoardo Sozzi1 aSakurako Nagumo Wong1 aMichael Zabolocki1 aMarthe Priouret1 aBalint Doleschall1 aKaja I. Ritzau-Reid1 aMarielle Piber1 aIlaria Morassut1 aCharles Fieseler1 aAlessandro Fiorenzano1 aMolly M. Stevens1 aManuel Zimmer1 aCedric Bardy1 aMalin Parmar1 aJürgen A. Knoblich00aIn vitro modeling of the human dopaminergic system using spatially arranged ventral midbrain–striatum–cortex assembloids  uhttps://www.nature.com/articles/s41592-023-02080-x  a2034-20470 v203 aVentral midbrain dopaminergic neurons project to the striatum as well as the cortex and are involved in movement control and reward-related cognition. In Parkinson’s disease, nigrostriatal midbrain dopaminergic neurons degenerate and cause typical Parkinson’s disease motor-related impairments, while the dysfunction of mesocorticolimbic midbrain dopaminergic neurons is implicated in addiction and neuropsychiatric disorders. Study of the development and selective neurodegeneration of the human dopaminergic system, however, has been limited due to the lack of an appropriate model and access to human material. Here, we have developed a human in vitro model that recapitulates key aspects of dopaminergic innervation of the striatum and cortex. These spatially arranged ventral midbrain–striatum–cortical organoids (MISCOs) can be used to study dopaminergic neuron maturation, innervation and function with implications for cell therapy and addiction research. We detail protocols for growing ventral midbrain, striatal and cortical organoids and describe how they fuse in a linear manner when placed in custom embedding molds. We report the formation of functional long-range dopaminergic connections to striatal and cortical tissues in MISCOs, and show that injected, ventral midbrain-patterned progenitors can mature and innervate the tissue. Using these assembloids, we examine dopaminergic circuit perturbations and show that chronic cocaine treatment causes long-lasting morphological, functional and transcriptional changes that persist upon drug withdrawal. Thus, our method opens new avenues to investigate human dopaminergic cell transplantation and circuitry reconstruction as well as the effect of drugs on the human dopaminergic system.  a1548-7105