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

Endocrinology ◽  
2018 ◽  
Vol 159 (12) ◽  
pp. 3910-3924 ◽  
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.

STEM-27. A PERIVASCULAR STEM CELL UNDERLIES VERTEBRATE MENINGEAL TUMORIGENESIS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi26-vi27
Author(s):  
Abrar Choudhury ◽  
Martha Cady ◽  
Calixto Lucas ◽  
Brisa Palikuqi ◽  
Ophir Klein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Tumor Cells ◽  
Rna Sequencing ◽  
Single Cells ◽  
Cell Types ◽  
Human Meningioma ◽  
Single Cell Rna Sequencing ◽  
Human Meningiomas

Abstract BACKGROUND Meningiomas are the most common primary intracranial tumors in humans and dogs, but biologic drivers and cell types underlying meningeal tumorigenesis are incompletely understood. Here we integrate meningioma single-cell RNA sequencing with stem cell approaches to define a perivascular stem cell underlying vertebrate meningeal tumorigenesis. METHODS Single-cell RNA sequencing was performed on 57,114 cells from 8 human meningiomas, 54,607 cells from 3 dog meningiomas, and human meningioma xenografts in mice. Results were validated using immunofluorescence (IF), immunohistochemistry (IHC), and deconvolution of bulk RNA sequencing of 200 human meningiomas. Mechanistic and functional studies were performed using clonogenic and limiting dilution assays, xenografts, and genetically engineered mouse models. RESULTS Copy number variant identification from human meningioma single cells distinguished tumor cells with loss of chr22q from non-tumor cells with intact chr22q. A single cluster distinguished by expression of Notch3 and other cancer stem cell genes had an intermediate level of loss of chr22q, suggesting this cluster may represent meningioma stem cells. In support of this hypothesis, pseudotime trajectory analysis demonstrated transcriptomic progression starting from Notch3+ cells and encompassing all other meningioma cell types. Notch3+ meningioma cells had transcriptomic concordance to mural pericytes, and IF/IHC of prenatal and adult human meninges, as well as lineage tracing using a Notch3-CreERT2 allele in mice, confirmed Notch3+ cells were restricted to the perivascular stem cell niche in mammalian meningeal development and homeostasis. Integrating human and dog meningioma single cells revealed Notch3+ cells in tumor and non-tumor clusters in dog meningiomas. Notch3 IF/IHC and cell-type deconvolution of bulk RNA sequencing showed Notch3+ cells were enriched in high-grade human meningiomas. Notch3 overexpression in human meningioma cells increased clonogenic growth in vitro, and increased tumorigenesis and tumor growth in vivo, decreasing overall survival. CONCLUSIONS Notch3+ stem cells in the perivascular niche underlie vertebrate meningeal tumorigenesis.

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

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.

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

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1751 ◽  
Author(s):  
Rishikesh Kumar Gupta ◽  
Jacek Kuznicki
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Future Directions ◽  
Strong Basis ◽  
Bodily Fluids ◽  
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.

scholarly journals Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

2021 ◽  
Author(s):  
Suraj Kannan ◽  
Matthew Miyamoto ◽  
Brian L. Lin ◽  
Chulan Kwon
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Single Cell ◽  
Pluripotent Stem Cell ◽  
Directed Differentiation ◽  
Trajectory Reconstruction ◽  
Single Cell Rna Sequencing

ABSTRACTA primary limitation in the clinical application of pluripotent stem cell derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, perinatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Here, we used our previously developed large particle fluorescence-activated cell sorting approach to generate an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. We subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through trajectory reconstruction methods, we identified a perinatal maturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of our trajectories with previously published human datasets, we identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, we demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level, and can be leveraged towards improving the clinical viability of PSC-CMs.Significance StatementThere is a significant clinical need to generate mature cardiomyocytes from pluripotent stem cells. However, to date, most differentiation protocols yield phenotypically immature cardiomyocytes. The mechanisms underlying this poor maturation state are unknown. Here, we used single cell RNA-sequencing to compare cardiomyocyte maturation pathways in endogenous and pluripotent stem cell-derived cardiomyocytes. We found that in vitro, cardiomyocytes fail to undergo critical perinatal gene expression changes necessary for complete maturation. We found that key transcription factors regulating these changes are poorly expressed in vitro. Our study provides a better understanding of cardiomyocyte maturation both in vivo and in vitro, and may lead to improved approaches for engineering mature cardiomyocytes from stem cells.

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

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.

scholarly journals A cell atlas of the adult Drosophila midgut

2020 ◽  
Vol 117 (3) ◽  
pp. 1514-1523 ◽  
Author(s):  
Ruei-Jiun Hung ◽  
Yanhui Hu ◽  
Rory Kirchner ◽  
Yifang Liu ◽  
Chiwei Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Biology ◽  
Single Cells ◽  
Intestinal Stem Cells ◽  
Cell Type ◽  
Molecular Features ◽  
Single Cell Rna Sequencing

Studies of the adult Drosophila midgut have led to many insights in our understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. We used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing functions of genes in the midgut epithelium.

scholarly journals Identification of ASCL1 as a determinant for human iPSC-derived dopaminergic neurons

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Aaron M. Earley ◽  
Lena F. Burbulla ◽  
Dimitri Krainc ◽  
Rajeshwar Awatramani
Keyword(s):  
Stem Cells ◽  
Gene Regulation ◽  
Transcription Factor ◽  
Transcription Factors ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Types ◽  
Fine Tuning ◽  
Transcriptional Networks ◽  
Single Cell Rna Sequencing

AbstractDuring cellular specification, transcription factors orchestrate cellular decisions through gene regulation. By hijacking these transcriptional networks, human pluripotent stem cells (hPSCs) can be specialized into neurons with different molecular identities for the purposes of regenerative medicine and disease modeling. However, molecular fine tuning cell types to match their in vivo counterparts remains a challenge. Directing cell fates often result in blended or incomplete neuron identities. A better understanding of hPSC to neuron gene regulation is needed. Here, we used single cell RNA sequencing to resolve some of these graded molecular identities during human neurogenesis from hPSCs. Differentiation platforms were established to model neural induction from stem cells, and we characterized these differentiated cell types by 10x single cell RNA sequencing. Using single cell trajectory and co-expression analyses, we identified a co-regulated transcription factor module expressing achaete-scute family basic helix-loop-helix transcription factor 1 (ASCL1) and neuronal differentiation 1 (NEUROD1). We then tested the function of these transcription factors in neuron subtype differentiation by gene knockout in a novel human system that reports the expression of tyrosine hydroxylase (TH), the rate limiting enzyme in dopamine synthesis. ASCL1 was identified as a necessary transcription factor for regulating dopaminergic neurotransmitter selection.

scholarly journals High resolution mouse subventricular zone stem cell niche transcriptome reveals features of lineage, anatomy, and aging

2020 ◽  
Author(s):  
Xuanhua P. Xie ◽  
Dan R. Laks ◽  
Daochun Sun ◽  
Asaf Poran ◽  
Ashley M. Laughney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Neural Stem Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Subventricular Zone ◽  
Stem Cell Niche ◽  
Advanced Age ◽  
Single Cell Rna Sequencing ◽  
Cell Niche

AbstractAdult neural stem cells (NSC) serve as a reservoir for brain plasticity and origin for certain gliomas. Lineage tracing and genomic approaches have portrayed complex underlying heterogeneity within the major anatomical location for NSC, the subventricular zone (SVZ). To gain a comprehensive profile of NSC heterogeneity, we utilized a well validated stem/progenitor specific reporter transgene in concert with single cell RNA sequencing to achieve unbiased analysis of SVZ cells from infancy to advanced age. The magnitude and high specificity of the resulting transcriptional data sets allow precise identification of the varied cell types embedded in the SVZ including specialized parenchymal cells (neurons, glia, microglia), and non-central nervous system cells (endothelial, immune). Initial mining of the data delineates four quiescent NSC and three progenitor cell subpopulations formed in a linear progression. Further evidence indicates that distinct stem and progenitor populations reside in different regions of the SVZ. As stem/progenitor populations progress from neonatal to advanced age, they acquire a deficiency in transition from quiescence to proliferation. Further data mining identifies stage specific biological processes, transcription factor networks, and cell surface markers for investigation of cellular identities, lineage relationships, and key regulatory pathways in adult NSC maintenance and neurogenesis.Significance StatementAdult neural stem cells (NSC) are closely related to multiple neurological disorders and brain tumors. Comprehensive investigation of their composition, lineage, and aging will provide new insights that may lead to enhanced patient treatment. This study applies a novel transgene to label and manipulate neural stem/progenitor cells, and monitor their evolution during aging. Together with high-throughput single cell RNA sequencing, we are able to analyze the subventricular zone (SVZ) cells from infancy to advanced age with unprecedented granularity. Diverse new cell states are identified in the stem cell niche, and an aging related NSC deficiency in transition from quiescence to proliferation is identified. The related biological features provide rich resources to inspect adult NSC maintenance and neurogenesis.

scholarly journals Transcriptomic entropy benchmarks stem cell-derived cardiomyocyte maturation against endogenous tissue at single cell level

2020 ◽  
Author(s):  
Suraj Kannan ◽  
Michael Farid ◽  
Brian L. Lin ◽  
Matthew Miyamoto ◽  
Chulan Kwon
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Pluripotent Stem Cell ◽  
Developmental Trajectory ◽  
Single Cell Level ◽  
Batch Effects ◽  
Cell Level ◽  
Single Cell Rna Sequencing

The immaturity of pluripotent stem cell (PSC)-derived tissues has emerged as a universal problem for their biomedical applications. While efforts have been made to generate adult-like cells from PSCs, direct benchmarking of PSC-derived tissues against in vivo development has not been established. Thus, maturation status is often assessed on an ad-hoc basis. Single cell RNA-sequencing (scRNA-seq) offers a promising solution, though cross-study comparison is limited by dataset-specific batch effects. Here, we developed a novel approach to quantify PSC-derived cardiomyocyte (CM) maturation through transcriptomic entropy. Transcriptomic entropy is robust across datasets regardless of differences in isolation protocols, library preparation, and other potential batch effects. With this new model, we analyzed over 45 scRNA-seq datasets and over 52,000 CMs, and established a cross-study, cross-species CM maturation reference. This reference enabled us to directly compare PSC-CMs with the in vivo developmental trajectory and thereby to quantify PSC-CM maturation status. We further found that our entropy-based approach can be used for other cell types, including pancreatic beta cells and hepatocytes. Our study presents a biologically relevant and interpretable metric for quantifying PSC-derived tissue maturation, and is extensible to numerous tissue engineering contexts.Significance StatementThere is significant interest in generating mature cardiomyocytes from pluripotent stem cells. However, there are currently few effective metrics to quantify the maturation status of a single cardiomyocyte. We developed a new metric for measuring cardiomyocyte maturation using single cell RNA-sequencing data. This metric, called entropy score, uses the gene distribution to estimate maturation at the single cell level. Entropy score enables comparing pluripotent stem cell-derived cardiomyocytes directly against endogenously-isolated cardiomyocytes. Thus, entropy score can better assist in development of approaches to improve the maturation of pluripotent stem cell-derived cardiomyocytes.

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