Toward Retinal Organoids in High-Throughput.

Human retinal organoids recapitulate the cellular diversity, arrangement, gene expression, and functional aspects of the human retina. Protocols to generate human retinal organoids from pluripotent stem cells are typically labor intensive, include many manual handling steps, and the organoids need to be maintained for several months until they mature. To generate large numbers of human retinal organoids for therapy development and screening purposes, scaling up retinal organoid production, maintenance, and analysis is of utmost importance. In this review, we discuss strategies to increase the number of high-quality retinal organoids while reducing manual handling steps. We further review different approaches to analyze thousands of retinal organoids with currently available technologies and point to challenges that still await to be overcome both in culture and analysis of retinal organoids.

Multimodal spatiotemporal phenotyping of human retinal organoid development.

Organoids generated from human pluripotent stem cells provide experimental systems to study development and disease, but quantitative measurements across different spatial scales and molecular modalities are lacking. In this study, we generated multiplexed protein maps over a retinal organoid time course and primary adult human retinal tissue. We developed a toolkit to visualize progenitor and neuron location, the spatial arrangements of extracellular and subcellular components and global patterning in each organoid and primary tissue. In addition, we generated a single-cell transcriptome and chromatin accessibility timecourse dataset and inferred a gene regulatory network underlying organoid development. We integrated genomic data with spatially segmented nuclei into a multimodal atlas to explore organoid patterning and retinal ganglion cell (RGC) spatial neighborhoods, highlighting pathways involved in RGC cell death and showing that mosaic genetic perturbations in retinal organoids provide insight into cell fate regulation.

Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq.

We present FLASH-seq (FS), a full-length single-cell RNA sequencing (scRNA-seq) method with increased sensitivity and reduced hands-on time compared to Smart-seq3. The entire FS protocol can be performed in ~4.5 hours, is simple to automate and can be easily miniaturized to decrease resource consumption. The FS protocol can also use unique molecular identifiers (UMIs) for molecule counting while displaying reduced strand-invasion artifacts. FS will be especially useful for characterizing gene expression at high resolution across multiple samples.

Cell Types of the Human Retina and Its Organoids at Single-Cell Resolution.

Human organoids recapitulating the cell-type diversity and function of their target organ are valuable for basic and translational research. We developed light-sensitive human retinal organoids with multiple nuclear and synaptic layers and functional synapses. We sequenced the RNA of 285,441 single cells from these organoids at seven developmental time points and from the periphery, fovea, pigment epithelium and choroid of light-responsive adult human retinas, and performed histochemistry. Cell types in organoids matured in vitro to a stable "developed" state at a rate similar to human retina development in vivo. Transcriptomes of organoid cell types converged toward the transcriptomes of adult peripheral retinal cell types. Expression of disease-associated genes was cell-type-specific in adult retina, and cell-type specificity was retained in organoids. We implicate unexpected cell types in diseases such as macular degeneration. This resource identifies cellular targets for studying disease mechanisms in organoids and for targeted repair in human retinas.

Self-organized developmental patterning and differentiation in cerebral organoids.

Cerebral organoids recapitulate human brain development at a considerable level of detail, even in the absence of externally added signaling factors. The patterning events driving this self-organization are currently unknown. Here, we examine the developmental and differentiative capacity of cerebral organoids. Focusing on forebrain regions, we demonstrate the presence of a variety of discrete ventral and dorsal regions. Clearing and subsequent 3D reconstruction of entire organoids reveal that many of these regions are interconnected, suggesting that the entire range of dorso-ventral identities can be generated within continuous neuroepithelia. Consistent with this, we demonstrate the presence of forebrain organizing centers that express secreted growth factors, which may be involved in dorso-ventral patterning within organoids. Furthermore, we demonstrate the timed generation of neurons with mature morphologies, as well as the subsequent generation of astrocytes and oligodendrocytes. Our work provides the methodology and quality criteria for phenotypic analysis of brain organoids and shows that the spatial and temporal patterning events governing human brain development can be recapitulated in vitro.

Cerebral organoids model human brain development and microcephaly.

The complexity of the human brain has made it difficult to study many brain disorders in model organisms, highlighting the need for an in vitro model of human brain development. Here we have developed a human pluripotent stem cell-derived three-dimensional organoid culture system, termed cerebral organoids, that develop various discrete, although interdependent, brain regions. These include a cerebral cortex containing progenitor populations that organize and produce mature cortical neuron subtypes. Furthermore, cerebral organoids are shown to recapitulate features of human cortical development, namely characteristic progenitor zone organization with abundant outer radial glial stem cells. Finally, we use RNA interference and patient-specific induced pluripotent stem cells to model microcephaly, a disorder that has been difficult to recapitulate in mice. We demonstrate premature neuronal differentiation in patient organoids, a defect that could help to explain the disease phenotype. Together, these data show that three-dimensional organoids can recapitulate development and disease even in this most complex human tissue.