Biological and Medical Importance of Cellular Heterogeneity Deciphered by Single-Cell RNA Sequencing

The present review discusses recent progress in single-cell RNA sequencing (scRNA-seq), which can describe cellular heterogeneity in various organs, bodily fluids, and pathologies (e.g., cancer and Alzheimer’s disease). We outline scRNA-seq techniques that are suitable for investigating cellular heterogeneity that is present in cell populations with very high resolution of the transcriptomic landscape. We summarize scRNA-seq findings and applications of this technology to identify cell types, activity, and other features that are important for the function of different bodily organs. We discuss future directions for scRNA-seq techniques that can link gene expression, protein expression, cellular function, and their roles in pathology. We speculate on how the field could develop beyond its present limitations (e.g., performing scRNA-seq in situ and in vivo). Finally, we discuss the integration of machine learning and artificial intelligence with cutting-edge scRNA-seq technology, which could provide a strong basis for designing precision medicine and targeted therapy in the future.

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Single-cell RNA sequencing of cultured human endometrial CD140b+CD146+ perivascular cells highlights the importance of in vivo microenvironment

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02354-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Dandan Cao ◽

Rachel W. S. Chan ◽

Ernest H. Y. Ng ◽

Kristina Gemzell-Danielsson ◽

William S. B. Yeung

Keyword(s):

Stem Cells ◽

Single Cell ◽

Rna Sequencing ◽

Marker Gene ◽

Cellular Heterogeneity ◽

Stromal Fibroblast ◽

Perivascular Cells ◽

Endometrial Cells ◽

Single Cell Rna Sequencing

Abstract Background Endometrial mesenchymal-like stromal/stem cells (eMSCs) have been proposed as adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b&CD146) has been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity, relationship to primary counterpart, remains largely unclear. Methods In this study, we applied 10X genomics single-cell RNA sequencing (scRNA-seq) to cultured human CD140b+CD146+ endometrial perivascular cells (ePCs) from menstrual and secretory endometrium. We also analyzed publicly available scRNA-seq data of primary endometrium and performed transcriptome comparison between cultured ePCs and primary ePCs at single-cell level. Results Transcriptomic expression-based clustering revealed limited heterogeneity within cultured menstrual and secretory ePCs. A main subpopulation and a small stress-induced subpopulation were identified in secretory and menstrual ePCs. Cell identity analysis demonstrated the similar cellular composition in secretory and menstrual ePCs. Marker gene expression analysis showed that the main subpopulations identified from cultured secretory and menstrual ePCs simultaneously expressed genes marking mesenchymal stem cell (MSC), perivascular cell, smooth muscle cell, and stromal fibroblast. GO enrichment analysis revealed that genes upregulated in the main subpopulation enriched in actin filament organization, cellular division, etc., while genes upregulated in the small subpopulation enriched in extracellular matrix disassembly, stress response, etc. By comparing subpopulations of cultured ePCs to the publicly available primary endometrial cells, it was found that the main subpopulation identified from cultured ePCs was culture-unique which was unlike primary ePCs or primary endometrial stromal fibroblast cells. Conclusion In summary, these data for the first time provides a single-cell atlas of the cultured human CD140b+CD146+ ePCs. The identification of culture-unique relatively homogenous cell population of CD140b+CD146+ ePCs underscores the importance of in vivo microenvironment in maintaining cellular identity.

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RNA-seq library preparation from single pancreatic acinar cells

10.1101/085696 ◽

2016 ◽

Author(s):

Damian Wollny ◽

Sheng Zhao ◽

Ana Martin-Villalba

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Acinar Cells ◽

Cell Types ◽

Cellular Heterogeneity ◽

Pancreatic Acinar Cells ◽

Library Preparation ◽

Cell Type ◽

Promising Tool ◽

Single Cell Rna Sequencing

Single cell RNA sequencing technology has emerged as a promising tool to uncover previously neglected cellular heterogeneity. Multiple methods and protocols have been developed to apply single cell sequencing to different cell types from various organs. However, library preparation for RNA sequencing remains challenging for cell types with high RNAse content due to rapid degradation of endogenous RNA molecules upon cell lysis. To this end, we developed a protocol based on the SMART-seq2 technology for single cell RNA sequencing of pancreatic acinar cells, the cell type with one of the highest ribonuclease concentration measured to date. This protocol reliably produces high quality libraries from single acinar cells reaching a total of 5x106 reads / cell and ∼ 80% transcript mapping rate with no detectable 3´end bias. Thus, our protocol makes single cell transcriptomics accessible to cell type with very high RNAse content.

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Single-cell RNA sequencing deconvolutes the in vivo heterogeneity of human bone marrow-derived mesenchymal stem cells

10.1101/2020.04.06.027904 ◽

2020 ◽

Author(s):

Zun Wang ◽

Xiaohua Li ◽

Junxiao Yang ◽

Yun Gong ◽

Huixi Zhang ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mesenchymal Stem Cells ◽

Single Cell ◽

Rna Sequencing ◽

Critical Role ◽

Cellular Heterogeneity ◽

Bone Homeostasis ◽

Single Cell Rna Sequencing

AbstractBone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stromal cells, which have a critical role in the maintenance of skeletal tissues such as bone, cartilage, and the fat found in bone marrow. In addition to providing microenvironmental support for hematopoietic processes, BM-MSCs can differentiate into various mesodermal lineages including osteoblast/osteocyte, chondrocyte, and adipocyte cells that are crucial for bone metabolism. While BM-MSCs have high cell-to-cell heterogeneity in gene expression, the cell subtypes that contribute to this heterogeneity in vivo in humans have not been characterized. To investigate the transcriptional diversity of BM-MSCs, we applied single-cell RNA sequencing (scRNA-seq) on freshly isolated CD271+ BM-derived mononuclear cells (BM-MNCs) from two human subjects. We successfully identified LEPRhiCD45low BM-MSCs within the CD271+ BM-MNC population, and further codified the BM-MSCs into distinct subpopulations corresponding to the osteogenic, chondrogenic, and adipogenic differentiation trajectories, as well as terminal-stage quiescent cells. Biological functional annotations of transcriptomes suggest that osteoblast precursors may induce angiogenesis coupled with osteogenesis, and chondrocyte precursors may have the potential to differentiate into myocytes. We discovered transcripts for several cluster of differentiation (CD) markers that were highly expressed (e.g., CD167b, CD91, CD130 and CD118) or absent (e.g., CD74, CD217, CD148 and CD68) in BM-MSCs and could be novel markers for human BM-MSC purification. This study is the first systematic in vivo dissection of human BM-MSCs cell subtypes at the single-cell resolution, revealing insight into the extent of their cellular heterogeneity and bone homeostasis.

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Application of single-cell RNA sequencing methodologies in understanding haematopoiesis and immunology

Essays in Biochemistry ◽

10.1042/ebc20180072 ◽

2019 ◽

Vol 63 (2) ◽

pp. 217-225 ◽

Cited By ~ 2

Author(s):

Anna M. Ranzoni ◽

Paulina M. Strzelecka ◽

Ana Cvejic

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Immune Cell ◽

Cell Types ◽

Tree Model ◽

Future Directions ◽

Single Cell Rna Sequencing ◽

Lineage Tree ◽

Cell Variation ◽

Cellular Components

Abstract The blood and immune system are characterised by utmost diversity in its cellular components. This heterogeneity can solely be resolved with the application of single-cell technologies that enable precise examination of cell-to-cell variation. Single-cell transcriptomics is continuously pushing forward our understanding of processes driving haematopoiesis and immune responses in physiological settings as well as in disease. Remarkably, in the last five years, a number of studies involving single-cell RNA sequencing (scRNA-seq) allowed the discovery of new immune cell types and revealed that haematopoiesis is a continuous rather than a stepwise process, thus challenging the classical haematopoietic lineage tree model. This review summarises the most recent studies which applied scRNA-seq to answer outstanding questions in the fields of haematology and immunology and discusses the present challenges and future directions.

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Single-cell molecular and cellular architecture of the mouse neurohypophysis

10.1101/744466 ◽

2019 ◽

Author(s):

Qiyu Chen ◽

Dena Leshkowitz ◽

Janna Blechman ◽

Gil Levkowitz

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Adult Male ◽

Spatial Organization ◽

Cell Types ◽

Intermediate Lobe ◽

Posterior Lobe ◽

Single Cell Rna Sequencing ◽

The Brain

AbstractThe neurohypophysis (NH), located at the posterior lobe of the pituitary, is a major neuroendocrine tissue, which mediates osmotic balance, blood pressure, reproduction, and lactation by means of releasing the neurohormones oxytocin and arginine-vasopressin from the brain into the peripheral blood circulation. The major cellular components of the NH are hypothalamic axonal termini, fenestrated endothelia and pituicytes, the resident astroglia. However, despite the physiological importance of the NH, the exact molecular signature defining neurohypophyseal cell types and in particular the pituicytes, remains unclear. Using single cell RNA sequencing, we captured seven distinct cell types in the NH and intermediate lobe (IL) of adult male mouse. We revealed novel pituicyte markers showing higher specificity than previously reported. Single molecule in situ hybridization revealed spatial organization of the major cell types implying intercellular communications. We present a comprehensive molecular and cellular characterization of neurohypophyseal cell-types serving as a valuable resource for further functional research.Significance StatementThe neurohypophysis (NH) is a major neuroendocrine interface, which allows the brain to regulate the function of peripheral organs in response to specific physiological demands. Despite its importance, a comprehensive molecular description of cell identities in the NH is still lacking. Utilizing single cell RNA sequencing technology, we identified the transcriptomes of five major neurohypophyseal cell types in the adult male mice and mapped the spatial distribution of selected cell types in situ. We revealed an unexpected cellular heterogeneity of the neurohypophysis and provide novel molecular markers for neurohypophyseal cell types with higher specificity than previously reported.

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Single-Cell RNA Sequencing Reveals Novel Markers of Male Pituitary Stem Cells and Hormone-Producing Cell Types

Endocrinology ◽

10.1210/en.2018-00750 ◽

2018 ◽

Vol 159 (12) ◽

pp. 3910-3924 ◽

Cited By ~ 21

Author(s):

Leonard Y M Cheung ◽

Akima S George ◽

Stacey R McGee ◽

Alexandre Z Daly ◽

Michelle L Brinkmeier ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pituitary Gland ◽

Single Cell ◽

Rna Sequencing ◽

Clustering Algorithms ◽

White Blood Cells ◽

Cell Types ◽

Single Cell Rna Sequencing

Abstract Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3′ transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

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Molecular and Cellular Dynamics of Aortic Aneurysms Revealed by Single-Cell Transcriptomics

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.121.315852 ◽

2021 ◽

Author(s):

Yanming Li ◽

Scott A. LeMaire ◽

Ying H. Shen

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Cell Types ◽

Cellular Level ◽

Aortic Aneurysms ◽

Cellular Heterogeneity ◽

Cell Populations ◽

Sequencing Analysis ◽

Molecular Features ◽

Single Cell Rna Sequencing

The aorta is highly heterogeneous, containing many different types of cells that perform sophisticated functions to maintain aortic homeostasis. Recently, single-cell RNA sequencing studies have provided substantial new insight into the heterogeneity of vascular cell types, the comprehensive molecular features of each cell type, and the phenotypic interrelationship between these cell populations. This new information has significantly improved our understanding of aortic biology and aneurysms at the molecular and cellular level. Here, we summarize these findings, with a focus on what single-cell RNA sequencing analysis has revealed about cellular heterogeneity, cellular transitions, communications among cell populations, and critical transcription factors in the vascular wall. We also review the information learned from single-cell RNA sequencing that has contributed to our understanding of the pathogenesis of vascular disease, such as the identification of cell types in which aneurysm-related genes and genetic variants function. Finally, we discuss the challenges and future directions of single-cell RNA sequencing applications in studies of aortic biology and diseases.

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Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

Nature Communications ◽

10.1038/s41467-021-24730-4 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

David S. Fischer ◽

Meshal Ansari ◽

Karolin I. Wagner ◽

Sebastian Jarosch ◽

Yiqi Huang ◽

...

Keyword(s):

T Cells ◽

Single Cell ◽

Rna Sequencing ◽

Ex Vivo ◽

Severe Disease ◽

Human Leukocyte ◽

Cell Receptor ◽

Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

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Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

Genome Medicine ◽

10.1186/s13073-021-00885-z ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Sunny Z. Wu ◽

Daniel L. Roden ◽

Ghamdan Al-Eryani ◽

Nenad Bartonicek ◽

Kate Harvey ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

High Throughput ◽

Single Cells ◽

Cellular Heterogeneity ◽

Tumour Heterogeneity ◽

Fresh Tissue ◽

Human Cancers ◽

Cryopreserved Cell ◽

Single Cell Rna Sequencing

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

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MBRS-46. CHARTING NEOPLASTIC AND IMMUNE CELL HETEROGENEITY IN HUMAN AND GEM MODELS OF MEDULLOBLASTOMA USING scRNAseq

Neuro-Oncology ◽

10.1093/neuonc/noaa222.555 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii406-iii406

Author(s):

Andrew Donson ◽

Kent Riemondy ◽

Sujatha Venkataraman ◽

Ahmed Gilani ◽

Bridget Sanford ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Immune Cell ◽

Genetically Engineered ◽

Cellular Heterogeneity ◽

Cell Heterogeneity ◽

Transcriptomic Data ◽

Single Cell Rna Sequencing ◽

Transcript Profiles

Abstract We explored cellular heterogeneity in medulloblastoma using single-cell RNA sequencing (scRNAseq), immunohistochemistry and deconvolution of bulk transcriptomic data. Over 45,000 cells from 31 patients from all main subgroups of medulloblastoma (2 WNT, 10 SHH, 9 GP3, 11 GP4 and 1 GP3/4) were clustered using Harmony alignment to identify conserved subpopulations. Each subgroup contained subpopulations exhibiting mitotic, undifferentiated and neuronal differentiated transcript profiles, corroborating other recent medulloblastoma scRNAseq studies. The magnitude of our present study builds on the findings of existing studies, providing further characterization of conserved neoplastic subpopulations, including identification of a photoreceptor-differentiated subpopulation that was predominantly, but not exclusively, found in GP3 medulloblastoma. Deconvolution of MAGIC transcriptomic cohort data showed that neoplastic subpopulations are associated with major and minor subgroup subdivisions, for example, photoreceptor subpopulation cells are more abundant in GP3-alpha. In both GP3 and GP4, higher proportions of undifferentiated subpopulations is associated with shorter survival and conversely, differentiated subpopulation is associated with longer survival. This scRNAseq dataset also afforded unique insights into the immune landscape of medulloblastoma, and revealed an M2-polarized myeloid subpopulation that was restricted to SHH medulloblastoma. Additionally, we performed scRNAseq on 16,000 cells from genetically engineered mouse (GEM) models of GP3 and SHH medulloblastoma. These models showed a level of fidelity with corresponding human subgroup-specific neoplastic and immune subpopulations. Collectively, our findings advance our understanding of the neoplastic and immune landscape of the main medulloblastoma subgroups in both humans and GEM models.

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