The enteric nervous system of the human and mouse colon at a single-cell resolution

Drokhlyansky, Eugene; Smillie, Christopher S.; Wittenberghe, Nicholas Van; Ericsson, Maria; Griffin, Gabriel K.; Cuoco, Michael S.; Goder-Reiser, Max N.; Sharova, Tatyana; Aguirre, Andrew J.; Boland, Genevieve M.; Graham, Daniel B.; Rozenblatt-Rosen, Orit; Xavier, Ramnik J.; Regev, Aviv

doi:10.1101/746743

Cited by 19 publications

(29 citation statements)

References 118 publications

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“…3B ), in this and all other tumors tested ( Fig. 3H ), since the nuclear membrane, ER, and ribosomes remain attached to the nucleus when using this method (Drokhlyansky et al, 2019). The same trends were preserved when down-sampling by the total number of sequencing reads ( Supp.…”

Section: Resultsmentioning

confidence: 61%

“…1 ). We initially divided larger samples or used multiple biopsies to compare four isolation methods (EZPrep(Habib et al, 2017), Nonidet™ P40 with salts and Tris (NST) [modified from Gao, R., et al (Gao et al, 2017)], CHAPS, with salts and Tris (CST) (Drokhlyansky et al, 2019), and Tween with salts and Tris (TST) (Drokhlyansky et al, 2019), which differ primarily in the mechanical force ( e.g. , chopping or douncing), buffer, and/or detergent composition ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…On dry ice, tissue was split and subjected to one of three salt-Tris (ST)-based nucleus isolation protocols (Drokhlyansky et al, 2019) and the EZ nuclei isolation buffer (Habib et al, 2017), as detailed below.…”

Section: Methodsmentioning

confidence: 99%

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A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors

Slyper

Porter

Ashenberg

et al. 2019

Preprint

Self Cite

114

176

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AbstractSingle cell genomics is essential to chart the complex tumor ecosystem. While single cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumor tissues, single nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each strategy requires modifications to fit the unique characteristics of different tissue and tumor types, posing a barrier to adoption. Here, we developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We tested eight tumor types of varying tissue and sample characteristics (resection, biopsy, ascites, and orthotopic patient-derived xenograft): lung cancer, metastatic breast cancer, ovarian cancer, melanoma, neuroblastoma, pediatric sarcoma, glioblastoma, pediatric high-grade glioma, and chronic lymphocytic leukemia. Analyzing 212,498 cells and nuclei from 39 clinical samples, we evaluated protocols by cell quality, recovery rate, and cellular composition. We optimized protocols for fresh tissue dissociation for different tumor types using a decision tree to account for the technical and biological variation between clinical samples. We established methods for nucleus isolation from OCT embedded and fresh-frozen tissues, with an optimization matrix varying mechanical force, buffer, and detergent. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types and intrinsic expression profiles, but at different proportions. Our work provides direct guidance across a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

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Section: Resultsmentioning

confidence: 61%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors

Slyper

Porter

Ashenberg

et al. 2019

Preprint

Self Cite

114

176

View full text Add to dashboard Cite

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“…Prior scRNAseq analysis of postnatal ENS did not detect and characterize the true identity of the MEN lineage. This is because previous studies were performed either exclusively on neural crest-derived ENS cells (Zeisel et al, 2018), or when done in a more agnostic manner, only applied known canonical neural markers to identify the ENS population (Drokhlyansky et al, 2019;Morarach et al, 2020). In either case, the discovery of the distinct identity and derivation of MENs would not have been possible.…”

Section: A Shift In the Balance Between The Two Lineages In The Adultmentioning

confidence: 99%

Age-associated changes in lineage composition of the enteric nervous system regulate gut health and disease

Kulkarni

Saha

Becker

et al. 2020

Preprint

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The enteric nervous system (ENS), a collection of neurons contained in the wall of the gut, is of fundamental importance to gastrointestinal and systemic health. According to the prevailing paradigm, the ENS arises from progenitor cells migrating from the embryonic neural crest and remains largely unchanged thereafter. Here, we show that the composition of maturing ENS changes with time, with a decline in neural-crest derived neurons and their replacement by mesoderm-derived neurons. Single cell transcriptomics and immunochemical approaches establish a distinct expression profile of mesoderm-derived neurons. The dynamic balance between the proportions of neurons from these two different lineages in the post-natal gut is dependent on the availability of their respective trophic signals, GDNF-RET and HGF-MET. With increasing age, the mesoderm-derived neurons become the dominant form of neurons in the ENS, a change associated with significant functional effects on intestinal motility. Normal intestinal function in the adult gastrointestinal tract therefore appears to require an optimal balance between these two distinct lineages within the ENS.

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“…Analysis of ligand-receptor gene expression patterns in single-cell datasets has been instrumental in deconstructing the complex cellular communication network in the context of tissue function, development and cancer biology (Camp et al, 2017;Cohen et al, 2018;Kumar et al, 2018;Smillie et al, 2019;Vento-Tormo et al, 2018). The possibility of applying this powerful approach to predict neuron-non-neuronal cell interactions has only just begun to be explored (Drokhlyansky et al, 2019). Here, we demonstrate the utility of this strategy in uncovering novel neuroimmune crosstalk by systematically testing predictions from our in silico cellular interaction analysis using optogenetic experiments.…”

mentioning

confidence: 99%

Sensory Neurons Innervate Peripheral Lymph Nodes and Locally Regulate Gene Expression in Postsynaptic Endothelium, Stromal Cells, and Innate Leukocytes

Huang

Ziegler

Austin

et al. 2019

Preprint

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Immune responses within barrier tissues are regulated, in part, by nociceptors, specialized peripheral sensory neurons that detect noxious stimuli. Previous work has shown that nociceptor ablation not only alters local responses to immune challenge at peripheral sites, but also within draining lymph nodes (LNs). The mechanisms and significance of nociceptor-dependent modulation of LN function are unknown. Indeed, although sympathetic innervation of LNs is well documented, it has been unclear whether the LN parenchyma itself is innervated by sensory neurons. Here, using a combination of high-resolution imaging, retrograde viral tracing, single-cell 30 transcriptomics (scRNA-seq), and optogenetics, we identified and functionally tested a sensory neuro-immune circuit that is preferentially located in the outermost cortex of skin-draining LNs. Transcriptomic profiling revealed that there are at least four discrete subsets of sensory neurons that innervate LNs with a predominance of peptidergic nociceptors, and an innervation pattern that is distinct from that in the surrounding skin. To uncover potential LN-resident communication partners for LN-innervating sensory neurons, we employed scRNA-seq to generate a draft atlas of all murine LN cells and, based on receptor-ligand expression patterns, nominated candidate target populations among stromal and immune cells. Using selective optogenetic stimulation of LN-innervating sensory axons, we directly experimentally tested our inferred connections. Acute neuronal activation triggered rapid transcriptional changes preferentially within our top-ranked putative interacting partners, principally endothelium and other nodal stroma cells, as well as several innate leukocyte populations. Thus, LNs are monitored by a unique 40 population of sensory neurons that possesses immunomodulatory potential. 840 and Harvard. J.O

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The enteric nervous system of the human and mouse colon at a single-cell resolution

Cited by 19 publications

References 118 publications

A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors

A single-cell and single-nucleus RNA-seq toolbox for fresh and frozen human tumors

Age-associated changes in lineage composition of the enteric nervous system regulate gut health and disease

Sensory Neurons Innervate Peripheral Lymph Nodes and Locally Regulate Gene Expression in Postsynaptic Endothelium, Stromal Cells, and Innate Leukocytes

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