Mapping human tissues with highly multiplexed RNA in situ hybridization

In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. There has been a surge of multiplexed RNA in situ mapping techniques but their application to human tissues has been limited due to their large size, general lower tissue quality and high autofluorescence. Here we report DART-FISH, a padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections. We introduce an omni-cell type cytoplasmic stain that substantially improves the segmentation of cell bodies. Our enzyme-free isothermal decoding procedure allows us to image 121 genes in large sections from the human neocortex in \u3c10 h. We successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled \u3e20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts

Mapping human tissues with spatial transcriptomics

The structure and function of tissues are highly intertwined, necessitating an understanding of their architecture to comprehend their normal and pathological states. While traditional histology and immunostaining offer high spatial resolution, they are limited in molecular resolution. Conversely, single-cell sequencing provides molecular resolution but lacks spatial context due to tissue dissociation. Spatial transcriptomics bridges this gap by combining microscopy and DNA technology, enabling high resolution molecular readouts with spatial information.In the first part of this dissertation, we utilize a spatial RNA capture technology to map cell types in the human kidney at a near-cellular resolution, revealing cellular neighborhoods across different anatomical regions of the kidney, identifying niches for novel populations, and uncovering strong mitochondrial gene expression patterns in a specific epithelial subtype.While sequencing-based methods have a fixed spatial resolution, multiplexed in situ detection methods offer cellular resolution but face challenges in sensitivity, throughput, and cost which severely limit their application in human sections that are large and have low quality. In aim 2, I developed a novel multiplexed in situ RNA detection method, DART-FISH, capable of profiling hundreds of genes in large human tissue sections with ease of implementation. I further developed an accompanying suite of computational tools for designing and processing the output of in situ experiments. Finally, in aim 3, the utility of DART-FISH is demonstrated by applying it to multiple tissue types. This includes the human brain where the layered organization of excitatory neurons was recapitulated and a rare subclass of inhibitory neurons uncovered. In the human kidney, I profiled healthy and pathological cell states in the cortex, and identified interactions between disease-altered epithelial cells and myofibroblasts. Finally, I showed how DART-FISH can be utilized for organ-scale measurements by imaging a cross section of a mouse kidney where all segments of the nephron can be visualized and pathological neighborhoods can be systematically analyzed with single-cell resolution. In summary, this dissertation provides several experimental and computational solutions to advance the field of spatial transcriptomics and lower its cost. It also shows the application of spatial transcriptomics to map the architecture of human and mouse tissues

DART-FISH (Kalhor, Chen et al) raw data

<p>Data and code deposit for Kalhor, Chen et al. "Mapping Human Tissues with Highly Multiplexed RNA in situ Hybridization".</p> <p>https://doi.org/10.1101/2023.08.16.553610</p> <p>Each data set has the following directory structure:</p> <p>.<br> ├── 1_Registered/ # registered (aligned) image stacks<br> ├── 2_Projected/ # maximum projected and stitched images<br> ├── 3_Decoded/ # pre- and post-filtering spots tables<br> ├── 4_CellAssignment/ # segmentation mask and cell-assigned spots<br> ├── 5_Analysis/ # various QC plots<br> ├── Codes/ # all the codes used to generate folders above</p&gt

Mapping human tissues with highly multiplexed RNA in situ hybridization.

In situ transcriptomic techniques promise a holistic view of tissue organization and cell-cell interactions. There has been a surge of multiplexed RNA in situ mapping techniques but their application to human tissues has been limited due to their large size, general lower tissue quality and high autofluorescence. Here we report DART-FISH, a padlock probe-based technology capable of profiling hundreds to thousands of genes in centimeter-sized human tissue sections. We introduce an omni-cell type cytoplasmic stain that substantially improves the segmentation of cell bodies. Our enzyme-free isothermal decoding procedure allows us to image 121 genes in large sections from the human neocortex in <10 h. We successfully recapitulated the cytoarchitecture of 20 neuronal and non-neuronal subclasses. We further performed in situ mapping of 300 genes on a diseased human kidney, profiled >20 healthy and pathological cell states, and identified diseased niches enriched in transcriptionally altered epithelial cells and myofibroblasts

An atlas of healthy and injured cell states and niches in the human kidney.

Understanding kidney disease relies on defining the complexity of cell types and states, their associated molecular profiles and interactions within tissue neighbourhoods1. Here we applied multiple single-cell and single-nucleus assays (>400,000 nuclei or cells) and spatial imaging technologies to a broad spectrum of healthy reference kidneys (45 donors) and diseased kidneys (48 patients). This has provided a high-resolution cellular atlas of 51 main cell types, which include rare and previously undescribed cell populations. The multi-omic approach provides detailed transcriptomic profiles, regulatory factors and spatial localizations spanning the entire kidney. We also define 28 cellular states across nephron segments and interstitium that were altered in kidney injury, encompassing cycling, adaptive (successful or maladaptive repair), transitioning and degenerative states. Molecular signatures permitted the localization of these states within injury neighbourhoods using spatial transcriptomics, while large-scale 3D imaging analysis (around 1.2 million neighbourhoods) provided corresponding linkages to active immune responses. These analyses defined biological pathways that are relevant to injury time-course and niches, including signatures underlying epithelial repair that predicted maladaptive states associated with a decline in kidney function. This integrated multimodal spatial cell atlas of healthy and diseased human kidneys represents a comprehensive benchmark of cellular states, neighbourhoods, outcome-associated signatures and publicly available interactive visualizations