BLab-seq, a non-toxic, transgene-based method for determining birth dates and transcriptomic profiles of neuronal subtypes in human organoids

About This Grant

The human nervous system is complex, comprising thousands of functionally distinct neuronal subtypes, each defined by unique gene expression profiles. The timing of cell cycle exit and terminal differentiation plays a critical role in determining neuronal fate. Existing chemical-based birth dating strategies are limited by cytotoxicity, the need for tissue fixation, and incompatibility with transcriptomic profiling. Conversely, single-cell transcriptomic approaches can infer developmental trajectories but do not directly link birth timing to maturation and terminal fate. To address this gap, we propose to develop Birth Labeled sequencing (BLab-seq), a novel transgene-based strategy that integrates non-toxic, fluorescent birth dating with single-cell RNA- sequencing to directly link neuronal birth timing and fate specification in human organoids. Unlike traditional cell lineage reporters, BLab-seq will offer a temporally precise, nontoxic, and multiomics-compatible strategy for studying neurogenesis across all cell types in human organoids—capabilities not currently available with existing tools. To validate BLab-seq and generate new mechanistic insights, we will test BLab-seq using human retinal organoids. Human vision is dependent on the retina, a multilayered neural tissue composed of a diverse array of neuronal classes and subtypes that detect, process, and relay light information. Despite significant progress, critical conceptual gaps in our understanding of human retinal development remain. While studies in model organisms have shown that the seven retinal cell classes are born in broad, temporally ordered windows, these developmental timelines have not been established in humans. Furthermore, the birth timing of the ~130 retinal cell subtypes has not been characterized in any species, and the roles of extrinsic signaling in subtype specification remain largely unknown. Human stem cell-derived retinal organoids offer an experimentally tractable model that recapitulates the developmental timing and cellular diversity of the human retina. Our preliminary studies demonstrate that birth dating in organoids is a valuable strategy to understand developmental mechanisms. Moreover, our findings suggest that retinoic acid and thyroid hormone signaling regulate the developmental timing of photoreceptors and possibly other retinal cell types. The second main goal of this study is to use BLab-seq to determine the birth timing of retinal cell classes and subtypes and to assess how extrinsic signaling influences these processes in human organoids (Aim 2). This aim will validate BLab-seq as a birth dating method, determine the birth order of human retinal cell classes and subtypes, and describe how signaling influences retinal cell birth timing.