Deterministic scRNA-seq of individual intestinal organoids reveals new subtypes and coexisting distinct stem cell pools

Abstract

1While Single-cell RNA-sequencing (scRNA-seq) has transformed our ability to resolve cellular 2 properties across systems, current microfluidics-based scRNA-seq technologies are limited to 3 samples with large amounts of cells (> 1,000 cells). This prevents efficient processing of 4 individual, small tissue samples, leading to confounded mosaic cell population read-outs. To 5 overcome these limitations, we developed a deterministic, mRNA-capture bead and cell co-6 encapsulation dropleting system, DisCo, that enables precise particle and cell positioning and 7 droplet sorting control through combined machine-vision and multilayer microfluidics. To 8 underscore the unique capabilities of our approach in processing low-input samples (< 100 cells) 9 at high efficiency (> 70%), we analyzed intestinal organoid development by "DisCo-ing" 31 10 individual organoids at varying developmental stages. This revealed extensive organoid 11 heterogeneity, uncovering a so far uncharacterized "gobloid" subtype consisting predominantly of 12 precursor and mature (Muc2 + ) goblet cells. We also identified spheroids that are comprised of a 13 secondary regenerative fetal-like Ly6a + stem cell population, marked by prolonged YAP1 nuclear 14 translocation and target gene expression, and that persist as symmetrical cysts even under 15 differentiation conditions. These findings illustrate the power of our "no-cell-left-behind" platform 16 in providing high-resolution snapshots of cellular heterogeneity among individual, small tissues. 17 18 19 20 21 22 23 24 25 26

An Lgr5-independent developmental lineage is involved in mouse intestinal regeneration

“…Since adult spheroids were obtained in variable amounts and not from all mice, we initially hypothesized that they originated from regions of the intestine of some animals in which regeneration takes place following minimal "physiological" injuries. However, using a new two-layer sandwich protocol described by the Lutolf group (Bues et al, 2020), we reproducibly obtained adult spheroids from all mice (Fig. S3A-C).…”

Everything You Always Wanted to Know About Organoid-Based Models (and Never Dared to Ask)

“…Standardization of organoid expansion alongside generation of stable organoid lines will form reliable tools for drug screening using high-throughput readouts (eg, single-cell RNA sequencing [RNAseq], Assay for Transposase-Accessible Chromatin using sequencing, ATAC sequencing, bisulfite sequencing, spatially resolved RNAseq, proteomics, and bioimaging). 45 , 188 , 189 , 190 Recently, a multiplex single-cell analysis pipeline was developed on organoids co-cultured with fibroblast and leukocytes to establish post-translational modification signaling networks that can be altered in diseases. 191 For example, growing organoids from patient-derived stem cell aggregates in preformed U-shaped microcavities imprinted in the hydrogel achieves highly homogenous cultures, both in size and maturation level.…”

Organoids in image-based phenotypic chemical screens

“…Furthermore, despite providing a comprehensive picture of gene expression, single-cell sequencing does not deliver information on individual organoid phenotypes and lacks the organoid context of the sequenced cells. Recent protocols try to fill this gap: single organoids can be first imaged to extract phenotypes and then sequenced to profile gene expression changes 60 , 61 . Similarly, in situ sequencing approaches can be used in organoids, enabling the transcriptomic profiling of intact organoid structures 62 .…”