Spatially defined single-cell transcriptional profiling characterizes diverse chondrocyte subtypes and nucleus pulposus progenitors in human intervertebral discs

AbstractA comprehensive understanding of the cellular heterogeneity and molecular mechanisms underlying the development, homeostasis, and disease of human intervertebral disks (IVDs) remains challenging. Here, the transcriptomic landscape of 108 108 IVD cells was mapped using single-cell RNA sequencing of three main compartments from young and adult healthy IVDs, including the nucleus pulposus (NP), annulus fibrosus, and cartilage endplate (CEP). The chondrocyte subclusters were classified based on their potential regulatory, homeostatic, and effector functions in extracellular matrix (ECM) homeostasis. Notably, in the NP, a PROCR+ resident progenitor population showed enriched colony-forming unit-fibroblast (CFU-F) activity and trilineage differentiation capacity. Finally, intercellular crosstalk based on signaling network analysis uncovered that the PDGF and TGF-β cascades are important cues in the NP microenvironment. In conclusion, a single-cell transcriptomic atlas that resolves spatially regulated cellular heterogeneity together with the critical signaling that underlies homeostasis will help to establish new therapeutic strategies for IVD degeneration in the clinic.

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Single-cell Atlas Unveils Cellular Heterogeneity and Novel Markers in Human Neonatal and Adult Intervertebral Discs

10.1101/2021.10.20.465219 ◽

2021 ◽

Author(s):

Wensen Jiang ◽

Juliane Dagmar Glaeser ◽

Khosrowdad Salehi ◽

Giselle Kaneda ◽

Pranav Mathkar ◽

...

Keyword(s):

Single Cell ◽

Cell Biology ◽

Annulus Fibrosus ◽

Developmental Trajectories ◽

Intervertebral Discs ◽

Cellular Heterogeneity ◽

Gradual Transition ◽

Nucleus Pulposus Cells ◽

And Function ◽

Ivd Degeneration

The origin, composition, distribution, and function of cells in the human intervertebral disc (IVD) has not been fully understood. Here, cell atlases of both human neonatal and adult IVDs have been generated and further assessed by gene ontology pathway enrichment, pseudo-time trajectory, histology, and immunofluorescence. Comparison of cell atlases revealed the presence of several sub-populations of notochordal cells (NC) in the neonatal IVD and a small quantity of NCs and associated markers in the adult IVD. Developmental trajectories predicted that most neonatal NCs develop into adult nucleus pulposus cells (NPCs) while some keep their identity throughout adulthood. A high heterogeneity and gradual transition of annulus fibrosus cells (AFCs) in the neonatal IVD was detected and their potential relevance in IVD development was assessed. Collectively, comparing single-cell atlases between neonatal and adult IVDs delineates the landscape of IVD cell biology and may help discover novel therapeutic targets for IVD degeneration.

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ETMR-05. SINGLE-CELL RNA-SEQ OF ETMR REVEALS CELL PROGRAMS OF DEVELOPMENTAL HIERARCHY AND CELLULAR DIVERSITY IN THE TUMOR MICROENVIRONMENT

Neuro-Oncology ◽

10.1093/neuonc/noaa222.209 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii323-iii323

Author(s):

Flavia W de Faria ◽

Marta Interlandi ◽

Natalia Moreno ◽

Monika Graf ◽

Viktoria Melcher ◽

...

Keyword(s):

Stem Cell ◽

Single Cell ◽

Molecular Mechanisms ◽

Interaction Analysis ◽

Cellular Heterogeneity ◽

Receptor Interaction ◽

Acid Oxidation ◽

Bmp Receptors ◽

New Findings ◽

Developmental Hierarchy

Abstract Embryonal tumors with multilayered rosettes (ETMR) are deadly brain malignancies affecting young children. No standard treatment is available and the median survival is less than 12 months. Molecularly, the disease is characterized by the miRNA C19MC cluster amplification, with the expression of multiples miRNAs related to a stem cell program. The discoveries on the purely molecular mechanisms of the disease did not help to create a bridge for new treatment strategies so far and the cellular diversity of ETMR remains poorly understood. In this study, we used single-cell RNA sequencing of murine and human tumors to describe ETMR cellular heterogeneity. Our findings support that intra-tumoral heterogeneity is mainly characterized by 4 cellular programs defining a developmental hierarchy related to different metabolic states: 1) Early quiescent NSC-like cells supported by fatty-acid oxidation 2) Late NSC and NP-like proliferative cells fueled by glycolytic metabolism; 3) Post-mitotic neuroblast-like cells, relying on oxidative-phosphorylation; 4) NSC-like proliferative cells, with metabolic plasticity and capable of performing the three types of metabolism. Tumor-specific ligand-receptor interaction analysis revealed that ETMR exchange with microglia and vascular mural cells (MC) signals related to extracellular matrix (ECM) organization (Cxcl12-CxCr4), stem cell signaling (BMPs-BMP receptors), anti-apoptosis and survival (Ntf3-Ntrk), not seen in the control brain. In addition, the vascular MC showed a cancer-associated fibroblast (CAF) phenotype, with potential prognostic implications, as previously demonstrated for other tumors. This study provides new findings to build up a more robust understanding of ETMR biology and opens space for further studies in the field.

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Single-Cell Atlas Unveils Cellular Heterogeneity and Novel Markers in Human Neonatal and Adult Intervertebral Discs

SSRN Electronic Journal ◽

10.2139/ssrn.3977258 ◽

2021 ◽

Author(s):

Wensen Jiang ◽

Juliane Dagmar Glaeser ◽

Khosrowdad Salehi ◽

Giselle Kaneda ◽

Pranav Mathkar ◽

...

Keyword(s):

Single Cell ◽

Intervertebral Discs ◽

Cellular Heterogeneity

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Single-Cell RNA-Sequencing Identifies Infrapatellar Fat Pad Macrophage Polarization in Acute Synovitis/Fat Pad Fibrosis and Cell Therapy

Bioengineering ◽

10.3390/bioengineering8110166 ◽

2021 ◽

Vol 8 (11) ◽

pp. 166

Author(s):

Dimitrios Kouroupis ◽

Thomas M. Best ◽

Lee D. Kaplan ◽

Diego Correa ◽

Anthony J. Griswold

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Transcriptional Profiling ◽

Macrophage Polarization ◽

Myeloid Cells ◽

Cellular Heterogeneity ◽

Joint Disease ◽

Infrapatellar Fat Pad ◽

Fat Pad ◽

Single Cell Rna Sequencing

The pathogenesis and progression of knee inflammatory pathologies is modulated partly by residing macrophages in the infrapatellar fat pad (IFP), thus, macrophage polarization towards pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes is important in joint disease pathologies. Alteration of M1/M2 balance contributes to the initiation and progression of joint inflammation and can be potentially altered with mesenchymal stem cell (MSC) therapy. In an acute synovial/IFP inflammation rat model a single intra-articular injection of IFP-MSC was performed, having as controls (1) diseased rats not receiving IFP-MSC and (2) non-diseased rats. After 4 days, cell specific transcriptional profiling via single-cell RNA-sequencing was performed on isolated IFP tissue from each group. Eight transcriptomically distinct cell populations were identified within the IFP across all three treatment groups with a noted difference in the proportion of myeloid cells across the groups. Largely myeloid cells consisted of macrophages (>90%); one M1 sub-cluster highly expressing pro-inflammatory markers and two M2 sub-clusters with one of them expressing higher levels of canonical M2 markers. Notably, the diseased samples (11.9%) had the lowest proportion of cells expressing M2 markers relative to healthy (14.8%) and MSC treated (19.4%) samples. These results suggest a phenotypic polarization of IFP macrophages towards the pro-inflammatory M1 phenotype in an acute model of inflammation, which are alleviated by IFP-MSC therapy inducing a switch towards an alternate M2 status. Understanding the IFP cellular heterogeneity and associated transcriptional programs may offer insights into novel therapeutic strategies for disabling joint disease pathologies.

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Quantitative Single-Cell Transcript Assessment of Biomarkers Supports Cellular Heterogeneity in the Bovine IVD

Veterinary Sciences ◽

10.3390/vetsci6020042 ◽

2019 ◽

Vol 6 (2) ◽

pp. 42 ◽

Cited By ~ 1

Author(s):

Kangning Li ◽

Devin Kapper ◽

Sumona Mondal ◽

Thomas Lufkin ◽

Petra Kraus

Keyword(s):

In Situ Hybridization ◽

Single Cell ◽

Gaussian Mixture ◽

Cellular Heterogeneity ◽

Rna In Situ Hybridization ◽

Ivd Degeneration

Severe and chronic low back pain is often associated with intervertebral disc (IVD) degeneration. While imposing a considerable socio-economic burden worldwide, IVD degeneration is also severely impacting on the quality of life of affected individuals. Cell-based regenerative medicine approaches have moved into clinical trials, yet IVD cell identities in the mature disc remain to be fully elucidated and tissue heterogeneity exists, requiring a better characterization of IVD cells. The bovine coccygeal IVD is an accepted research model to study IVD mechano-biology and disc homeostasis. Recently, we identified novel IVD biomarkers in the outer annulus fibrosus (AF) and nucleus pulposus (NP) of the mature bovine coccygeal IVD through RNA in situ hybridization (AP-RISH) and z-proportion test. Here we follow up on Lam1, Thy1, Gli1, Gli3, Noto, Ptprc, Scx, Sox2 and Zscan10 with fluorescent RNA in situ hybridization (FL-RISH) and confocal microscopy. This permits sub-cellular transcript localization and the addition of quantitative single-cell derived values of mRNA expression levels to our previous analysis. Lastly, we used a Gaussian mixture modeling approach for the exploratory analysis of IVD cells. This work complements our earlier cell population proportion-based study, confirms the previously proposed biomarkers and indicates even further heterogeneity of cells in the outer AF and NP of a mature IVD.

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A Single-Cell Transcriptome Atlas of the Mouse Glomerulus

Journal of the American Society of Nephrology ◽

10.1681/asn.2018030238 ◽

2018 ◽

Vol 29 (8) ◽

pp. 2060-2068 ◽

Cited By ~ 53

Author(s):

Nikos Karaiskos ◽

Mahdieh Rahmatollahi ◽

Anastasiya Boltengagen ◽

Haiyue Liu ◽

Martin Hoehne ◽

...

Keyword(s):

Gene Expression ◽

Endothelial Cells ◽

Single Cell ◽

Mesangial Cells ◽

Transcriptional Profiling ◽

Wild Type Mouse ◽

Cell Types ◽

Cellular Heterogeneity ◽

Marker Genes ◽

Glomerular Cell

Background Three different cell types constitute the glomerular filter: mesangial cells, endothelial cells, and podocytes. However, to what extent cellular heterogeneity exists within healthy glomerular cell populations remains unknown.Methods We used nanodroplet-based highly parallel transcriptional profiling to characterize the cellular content of purified wild-type mouse glomeruli.Results Unsupervised clustering of nearly 13,000 single-cell transcriptomes identified the three known glomerular cell types. We provide a comprehensive online atlas of gene expression in glomerular cells that can be queried and visualized using an interactive and freely available database. Novel marker genes for all glomerular cell types were identified and supported by immunohistochemistry images obtained from the Human Protein Atlas. Subclustering of endothelial cells revealed a subset of endothelium that expressed marker genes related to endothelial proliferation. By comparison, the podocyte population appeared more homogeneous but contained three smaller, previously unknown subpopulations.Conclusions Our study comprehensively characterized gene expression in individual glomerular cells and sets the stage for the dissection of glomerular function at the single-cell level in health and disease.

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Single-Cell Genomics: Catalyst for Cell Fate Engineering

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.748942 ◽

2021 ◽

Vol 9 ◽

Author(s):

Boxun Li ◽

Gary C. Hon

Keyword(s):

Single Cell ◽

Cell Fate ◽

Molecular Mechanisms ◽

Cell Types ◽

Cellular Heterogeneity ◽

State Transitions ◽

Small Scale ◽

Specific Cell ◽

Direct Reprogramming ◽

Cell State

As we near a complete catalog of mammalian cell types, the capability to engineer specific cell types on demand would transform biomedical research and regenerative medicine. However, the current pace of discovering new cell types far outstrips our ability to engineer them. One attractive strategy for cellular engineering is direct reprogramming, where induction of specific transcription factor (TF) cocktails orchestrates cell state transitions. Here, we review the foundational studies of TF-mediated reprogramming in the context of a general framework for cell fate engineering, which consists of: discovering new reprogramming cocktails, assessing engineered cells, and revealing molecular mechanisms. Traditional bulk reprogramming methods established a strong foundation for TF-mediated reprogramming, but were limited by their small scale and difficulty resolving cellular heterogeneity. Recently, single-cell technologies have overcome these challenges to rapidly accelerate progress in cell fate engineering. In the next decade, we anticipate that these tools will enable unprecedented control of cell state.

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Single cell and single nucleus RNA-Seq reveal cellular heterogeneity and homeostatic regulatory networks in adult mouse stria vascularis

10.1101/756635 ◽

2019 ◽

Author(s):

Soumya Korrapati ◽

Ian Taukulis ◽

Rafal Olszewski ◽

Madeline Pyle ◽

Shoujun Gu ◽

...

Keyword(s):

Single Cell ◽

Gene Regulatory Networks ◽

Regulatory Networks ◽

Molecular Mechanisms ◽

Stria Vascularis ◽

Cell Types ◽

Cellular Heterogeneity ◽

Genetic Mechanisms ◽

Single Nucleus ◽

Gene Regulatory

AbstractThe stria vascularis (SV) generates the endocochlear potential (EP) in the inner ear and is necessary for proper hair cell mechanotransduction and hearing. While channels belonging to SV cell types are known to play crucial roles in EP generation, relatively little is known about gene regulatory networks that underlie the ability of the SV to generate and maintain the EP. Using single cell and single nucleus RNA-sequencing, we identify and validate known and rare cell populations in the SV. Furthermore, we establish a basis for understanding molecular mechanisms underlying SV function by identifying potential gene regulatory networks as well as druggable gene targets. Finally, we associate known deafness genes with adult SV cell types. This work establishes a basis for dissecting the genetic mechanisms underlying the role of the SV in hearing and will serve as a basis for designing therapeutic approaches to hearing loss related to SV dysfunction.

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Propionibacterium acnes Accelerates Intervertebral Disc Degeneration by Inducing Pyroptosis of Nucleus Pulposus Cells via the ROS-NLRP3 Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2021/4657014 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Guoqing Tang ◽

Xiaoguang Han ◽

Zhijie Lin ◽

Hongbin Qian ◽

Bing Chen ◽

...

Keyword(s):

Nucleus Pulposus ◽

Propionibacterium Acnes ◽

Strong Association ◽

Intervertebral Discs ◽

Inflammatory Factors ◽

Death Process ◽

Dependent Manner ◽

Nucleus Pulposus Cells ◽

Alternative Treatment Strategy ◽

Ivd Degeneration

Our previous study verified the occurrence of Propionibacterium acnes (P. acnes), a low-virulence anaerobic bacterium, latently residing in degenerated intervertebral discs (IVDs), and the infection had a strong association with IVD degeneration. We explored whether P. acnes induces nucleus pulposus cell (NPC) pyroptosis, a more dangerous cell death process than apoptosis, and accelerates IVD degeneration via the pyroptotic products interleukin- (IL-) 1β and IL-18. After coculturing with P. acnes, human NPCs showed significant upregulation of NOD-like receptor 3 (NLRP3), cleaved IL-1β, cleaved caspase-1, and cleaved gasdermin D in response to the overexpression of IL-1β and IL-18 in a time- and dose-dependent manner. In addition, the gene expression of inflammatory factors and catabolic enzymes significantly increased in normal NPCs when cocultured with pyroptotic NPCs in a transwell system, and the adverse effects were inhibited when NPC pyroptosis was suppressed. Furthermore, inoculation of P. acnes into the IVDs of rats caused significant pyroptosis of NPCs and remarkable IVD degeneration. Finally, coculture of NPCs with P. acnes induced the overexpression of reactive oxygen species (ROS) and NLRP3, while inhibition of both factors reduced NPC pyroptosis. Therefore, P. acnes induces NPC pyroptosis via the ROS-NLRP3 signaling pathway, and the pyroptotic NPCs cause an IVD degeneration cascade. Targeting the P. acnes-induced pyroptosis of NPCs may become an alternative treatment strategy for IVD degeneration in the future.

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Single-Cell RNA Sequencing Defines the Regulation of Spermatogenesis by Sertoli-Cell Androgen Signaling

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.763267 ◽

2021 ◽

Vol 9 ◽

Author(s):

Congcong Cao ◽

Qian Ma ◽

Shaomei Mo ◽

Ge Shu ◽

Qunlong Liu ◽

...

Keyword(s):

Single Cell ◽

Sertoli Cells ◽

Molecular Mechanisms ◽

Cell Types ◽

Cellular Heterogeneity ◽

Cell Populations ◽

Wild Type ◽

Cellular Processes ◽

Transcriptomic Sequencing ◽

Transcriptional Changes

Androgen receptor (AR) signaling is essential for maintaining spermatogenesis and male fertility. However, the molecular mechanisms by which AR acts between male germ cells and somatic cells during spermatogenesis have not begun to be revealed until recently. With the advances obtained from the use of transgenic mice lacking AR in Sertoli cells (SCARKO) and single-cell transcriptomic sequencing (scRNA-seq), the cell specific targets of AR action as well as the genes and signaling pathways that are regulated by AR are being identified. In this study, we collected scRNA-seq data from wild-type (WT) and SCARKO mice testes at p20 and identified four somatic cell populations and two male germ cell populations. Further analysis identified that the distribution of Sertoli cells was completely different and uncovered the cellular heterogeneity and transcriptional changes between WT and SCARKO Sertoli cells. In addition, several differentially expressed genes (DEGs) in SCARKO Sertoli cells, many of which have been previously implicated in cell cycle, apoptosis and male infertility, have also been identified. Together, our research explores a novel perspective on the changes in the transcription level of various cell types between WT and SCARKO mice testes, providing new insights for the investigations of the molecular and cellular processes regulated by AR signaling in Sertoli cells.

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