High-resolution molecular phenotyping of human IPSC organoids using CLARITY and 2-photon microscopy

High-resolution molecular phenotyping of human IPSC organoids using CLARITY and 2-photon microscopy Simone Tomasi, Soraya Scuderi, Anahita Amiri, Giovanna G. Altobelli, Jessica Mariani, Cheryl Dambrot, Gianfilippo Coppola, Flora M. Vaccarino To understand the role of gene regulation in human brain development and neuropsychiatric disorders, it is essential to develop cellular models of the human brain. Induced pluripotent stem cells (iPSC)-derived brain organoids can be used to investigate the role of gene regulatory elements, noncoding RNA, and in general, noncoding disease associated gene variants in brain development and function. Organoids enable gradients of morphogenes and other extracellular cues to build up in the intercellular milieu and to interact with the genetic and epigenetic background of a given progenitor cell during the course of brain development. We have developed an iPSC-derived organoid model of the early human forebrain, where differentiation of cortical excitatory and inhibitory neurons can be studied in a reproducible fashion, enabling a more precise identification of molecular events crucially involved in the specification of distinct neuronal subtypes. However, a precise assessment of protein and RNA expression in intact organoids is hampered by the limited penetration of molecular probes, therefore requiring the preparation of thin sections and greatly limiting the capacity of exploring molecular and cellular features in a 3D environment. Here, we labeled telencephalic excitatory and inhibitory lineages in using pLenti-CAMKII-GFP and pLenti-DlxI12b-BG-DsRed vectors, then used two-photon microscopy to image the genetically-encoded fluorescence at higher resolution in live forebrain organoids. Next, we used CLARITY to clear the organoids and perform immunostainings on the intact cell aggregates. Our current protocol enables a 3D reconstruction of GFP/TdTomato filled cells allowing the analysis of axonal and dendritic arborization, dendrite length, synapse and spine distribution, as well as stereological counts of structures labeled by specific markers. Using these combined approaches, we aim at comparing intra-organoid layer cytoarchitecture and its emerging connectivity with parallel data from RNA-seq and ChIP-seq experiments, and develop new tools for linking the molecular and cellular features of organoids derived from different individuals.