Identification of a basal stem cell subpopulation in the prostate via functional, lineage tracing and single-cell RNA-seq analyses

AbstractThe basal cell compartment in many epithelial tissues such as the prostate, bladder, and mammary gland are generally believed to serve as an important pool of stem cells. However, basal cells are heterogenous and the stem cell subpopulation within basal cells is not well elucidated. Here we uncover that the core epithelial-to-mesenchymal transition (EMT) inducer Zeb is exclusively expressed in a prostate basal cell subpopulation based on both immunocytochemical and cell lineage tracing analysis. The Zeb1+prostate epithelial cells are multipotent prostate basal stem cells (PBSCs) that can self-renew and generate functional prostatic glandular structures with all three epithelial cell types at the single-cell level. Genetic ablation studies reveal an indispensable role for Zeb1 in prostate basal cell development. Utilizing unbiased single cell transcriptomic analysis of over 9000 mouse prostate basal cells, we find that Zeb1+basal cell subset shares gene expression signatures with both epithelial and mesenchymal cells and stands out uniquely among all the basal cell clusters. Moreover, Zeb1+epithelial cells can be detected in mouse and clinical samples of prostate tumors. Identification of the PBSC and its transcriptome profile is crucial to advance our understanding of prostate development and tumorigenesis.

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Altered Epithelial-mesenchymal Plasticity as a Result of Ovol2 Deletion Minimally Impacts the Self-renewal of Adult Mammary Basal Epithelial Cells

Journal of Mammary Gland Biology and Neoplasia ◽

10.1007/s10911-021-09508-0 ◽

2022 ◽

Author(s):

Peng Sun ◽

Yingying Han ◽

Maksim Plikus ◽

Xing Dai

Keyword(s):

Epithelial Cells ◽

Basal Cell ◽

Epithelial To Mesenchymal Transition ◽

Basal Cells ◽

Self Renewal ◽

Mesenchymal Transition ◽

Transcriptional State ◽

Specific Deletion ◽

Basal Epithelial Cells

AbstractStem-cell containing mammary basal epithelial cells exist in a quasi-mesenchymal transcriptional state characterized by simultaneous expression of typical epithelial genes and typical mesenchymal genes. Whether robust maintenance of such a transcriptional state is required for adult basal stem cells to fuel self-renewal and regeneration remains unclear. In this work, we utilized SMA-CreER to direct efficient basal cell-specific deletion of Ovol2, which encodes a transcription factor that inhibits epithelial-to-mesenchymal transition (EMT), in adult mammary gland. We identified a basal cell-intrinsic role of Ovol2 in promoting epithelial, and suppressing mesenchymal, molecular traits. Interestingly, Ovol2-deficient basal cells display minimal perturbations in their ability to support tissue homeostasis, colony formation, and transplant outgrowth. These findings underscore the ability of adult mammary basal cells to tolerate molecular perturbations associated with altered epithelia-mesenchymal plasticity without drastically compromising their self-renewal potential.

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Cadm1 regulates airway stem cell growth and differentiation via modulation of Stat3 activity

10.1101/090761 ◽

2016 ◽

Author(s):

Pooja Seedhar ◽

Elizabeth K Sage ◽

Sabari Vallath ◽

Gabrielle Sturges ◽

Adam Giangreco

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Differentiation ◽

Basal Cell ◽

Ciliated Cell ◽

Basal Cells ◽

Airway Injury ◽

Cell Growth And Differentiation ◽

Stat3 Signalling ◽

Cell Adhesion Molecule 1

AbstractAirway homeostasis, repair, and regeneration are imperfectly understood processes involving the proliferation and differentiation of endogenous lung stem cells. Here, we establish that epithelial Cell adhesion molecule 1 (Cadm1) regulates the growth and differentiation of airway basal cells, previously identified as lung stem cells. Immunohistochemistry and gene expression analysis reveals that Cadm1 is broadly expressed throughout the murine tracheobronchial epithelium, exhibits transient downregulation concomitant with airway injury, and is subsequently restored during basal cell differentiation. Using Cadm1 null (KO) and keratin 14 (K14)-specific Cadm1 overexpressing transgenic mice, we demonstrate that maintaining Cadm1 expression reduces basal stem cell proliferation after tracheal polidocanol injury, whereas Cadm1 deletion causes increased ciliated cell differentiation and sustained downstream Stat3 signalling. Altogether, this study defines a previously uncharacterised role for Cadm1 in directing airway basal cell homeostasis and repair via modulation of Stat3 activity.

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Stem Cells and Cellular Origins of Mammary Gland: Updates in Rationale, Controversies, and Cancer Relevance

Stem Cells International ◽

10.1155/2019/4247168 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Jiaojiao Zhou ◽

Qishan Chen ◽

Yiheng Zou ◽

Shu Zheng ◽

Yiding Chen

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Mammary Gland ◽

Cell Biology ◽

Mammary Epithelium ◽

Lineage Tracing ◽

Basal Cells ◽

Mammary Stem Cells ◽

Mammary Stem ◽

Cell Niche

Evidences have supported the pivotal roles of stem cells in mammary gland development. Many molecular markers have been identified to characterize mammary stem cells. Cellular fate mapping of mammary stem cells by lineage tracing has put unprecedented insights into the mammary stem cell biology, which identified two subtypes of mammary stem cells, including unipotent and multipotent, which specifically differentiate to luminal or basal cells. The emerging single-cell sequencing profiles have given a more comprehensive understanding on the cellular hierarchy and lineage signatures of mammary epithelium. Besides, the stem cell niche worked as an essential regulator in sustaining the functions of mammary stem cells. In this review, we provide an overview of the characteristics of mammary stem cells. The cellular origins of mammary gland are discussed to understand the stem cell heterogeneity and their diverse differentiations. Importantly, current studies suggested that the breast cancer stem cells may originate from the mammary stem cells after specific mutations, indicating their close relationships. Here, we also outline the recent advances and controversies in the cancer relevance of mammary stem cells.

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EPEN-22. SINGLE-CELL RNA SEQUENCING IDENTIFIES UPREGULATION OF IKZF1 IN PFA2 MYELOID SUBPOPULATION DRIVING AN ANTI-TUMOR PHENOTYPE

Neuro-Oncology ◽

10.1093/neuonc/noaa222.159 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii312-iii312

Author(s):

Andrea Griesinger ◽

Eric Prince ◽

Andrew Donson ◽

Kent Riemondy ◽

Timothy Ritzman ◽

...

Keyword(s):

T Cell ◽

Single Cell ◽

T Cell Activation ◽

Expression Profiles ◽

Myeloid Cells ◽

Cell Subset ◽

Lineage Tracing ◽

Machine Learning Techniques ◽

Cell Subpopulation ◽

Immune Gene

Abstract We have previously shown immune gene phenotype variations between posterior fossa ependymoma subgroups. PFA1 tumors chronically secrete IL-6, which pushes the infiltrating myeloid cells to an immune suppressive function. In contrast, PFA2 tumors have a more immune activated phenotype and have a better prognosis. The objective of this study was to use single-cell(sc) RNAseq to descriptively characterize the infiltrating myeloid cells. We analyzed approximately 8500 cells from 21 PFA patient samples and used advanced machine learning techniques to identify distinct myeloid and lymphoid subpopulations. The myeloid compartment was difficult to interrupt as the data shows a continuum of gene expression profiles exist within PFA1 and PFA2. Through lineage tracing, we were able to tease out that PFA2 myeloid cells expressed more genes associated with an anti-viral response (MHC II, TNF-a, interferon-gamma signaling); while PFA1 myeloid cells had genes associated with an immune suppressive phenotype (angiogenesis, wound healing, IL-10). Specifically, we found expression of IKZF1 was upregulated in PFA2 myeloid cells. IKZF1 regulates differentiation of myeloid cells toward M1 or M2 phenotype through upregulation of either IRF5 or IRF4 respectively. IRF5 expression correlated with IKZF1, being predominately expressed in the PFA2 myeloid cell subset. IKZF1 is also involved in T-cell activation. While we have not completed our characterization of the T-cell subpopulation, we did find significantly more T-cell infiltration in PFA2 than PFA1. Moving forward these studies will provide us with valuable information regarding the molecular switches involved in the tumor-immune microenvironment and to better develop immunotherapy for PFA ependymoma.

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Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics

Nature Communications ◽

10.1038/s41467-021-21650-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Lars Velten ◽

Benjamin A. Story ◽

Pablo Hernández-Malmierca ◽

Simon Raffel ◽

Daniel R. Leonce ◽

...

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Cancer Stem Cells ◽

Single Cell ◽

Lineage Tracing ◽

Specific Gene ◽

Leukemic Stem Cells ◽

Hematopoietic Stem ◽

Mutational Status

AbstractCancer stem cells drive disease progression and relapse in many types of cancer. Despite this, a thorough characterization of these cells remains elusive and with it the ability to eradicate cancer at its source. In acute myeloid leukemia (AML), leukemic stem cells (LSCs) underlie mortality but are difficult to isolate due to their low abundance and high similarity to healthy hematopoietic stem cells (HSCs). Here, we demonstrate that LSCs, HSCs, and pre-leukemic stem cells can be identified and molecularly profiled by combining single-cell transcriptomics with lineage tracing using both nuclear and mitochondrial somatic variants. While mutational status discriminates between healthy and cancerous cells, gene expression distinguishes stem cells and progenitor cell populations. Our approach enables the identification of LSC-specific gene expression programs and the characterization of differentiation blocks induced by leukemic mutations. Taken together, we demonstrate the power of single-cell multi-omic approaches in characterizing cancer stem cells.

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Heterogeneous disease-propagating stem cells in juvenile myelomonocytic leukemia

Journal of Experimental Medicine ◽

10.1084/jem.20180853 ◽

2021 ◽

Vol 218 (2) ◽

Cited By ~ 1

Author(s):

Eleni Louka ◽

Benjamin Povinelli ◽

Alba Rodriguez-Meira ◽

Gemma Buck ◽

Wei Xiong Wen ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Single Cell ◽

Rna Sequencing ◽

Childhood Leukemia ◽

Clonal Evolution ◽

Juvenile Myelomonocytic Leukemia ◽

Hematopoietic Stem ◽

Ras Pathway ◽

Myelomonocytic Leukemia

Juvenile myelomonocytic leukemia (JMML) is a poor-prognosis childhood leukemia usually caused by RAS-pathway mutations. The cellular hierarchy in JMML is poorly characterized, including the identity of leukemia stem cells (LSCs). FACS and single-cell RNA sequencing reveal marked heterogeneity of JMML hematopoietic stem/progenitor cells (HSPCs), including an aberrant Lin−CD34+CD38−CD90+CD45RA+ population. Single-cell HSPC index-sorting and clonogenic assays show that (1) all somatic mutations can be backtracked to the phenotypic HSC compartment, with RAS-pathway mutations as a “first hit,” (2) mutations are acquired with both linear and branching patterns of clonal evolution, and (3) mutant HSPCs are present after allogeneic HSC transplant before molecular/clinical evidence of relapse. Stem cell assays reveal interpatient heterogeneity of JMML LSCs, which are present in, but not confined to, the phenotypic HSC compartment. RNA sequencing of JMML LSC reveals up-regulation of stem cell and fetal genes (HLF, MEIS1, CNN3, VNN2, and HMGA2) and candidate therapeutic targets/biomarkers (MTOR, SLC2A1, and CD96), paving the way for LSC-directed disease monitoring and therapy in this disease.

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HERV-K HML-2 transcription in diverse cancers is related with cancer stem cell and epithelial-mesenchymal transition markers

10.1101/451997 ◽

2018 ◽

Cited By ~ 1

Author(s):

Audrey T. Lin ◽

Cindy G. Santander ◽

Fabricia F. Nascimento ◽

Emanuele Marchi ◽

Timokratis Karamitros ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cancer Stem Cell ◽

Human Genome ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Embryonic Stem ◽

Endogenous Retroviruses ◽

Human Endogenous Retrovirus ◽

Mesenchymal Transition

AbstractEndogenous retroviruses (ERVs) are remnants of ancient retroviral infections that make up 8% of the human genome. Although these elements are mostly fragmented and inactive, many proviruses belonging to the HERV-K (HML-2) family, the youngest lineage in the human genome, have intact open reading frames, some encoding for accessory genes called np9 and rec that interact with oncogenic pathways. Many studies have established that ERVs are transiently expressed in both stem cells and cancer, resulting in aberrant self-renewal and uncontrolled proliferation. np9 and rec expression are significantly correlated with a range of cancer stem cell (CSC) and epithelial to mesenchymal transition (EMT) biomarkers, including cellular receptors, transcription factors, and histone modifiers. Surprisingly, these ERV genes are negatively correlated with genes known to promote pluripotency in embryonic stem cell lines, such as Oct4. These results indicate that HERV-K (HML-2) is part of the transcriptional landscape responsible for cancer cells undergoing the phenotypic switch that characterises EMT. The discovery of np9 and rec’s correlation with CSC and EMT biomarkers suggest a yet undescribed role affecting the transitional CSC-like state in EMT and the shift towards cancer malignancy.ImportanceIn this study, we find that human endogenous retrovirus HERV-K (HML-2)-encoded genes np9 and rec are correlated with the expression of many biomarkers associated with cancer stem cells (CSC) and epithelial-mesenchymal transition (EMT). There has been a significant effort to develop novel treatments targeting CSC and EMT-specific signalling pathways and cell surface markers. This research describes HERV-K (HML-2) as interacting or being part of the regulatory network that make up reversible cell state switching in EMT. Our findings suggest these specific HERVs may be good candidate biomarkers in identifying the transitional CSC-like states that are present during the progression of EMT and cancer metastasis.

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Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

10.1101/2020.07.27.212076 ◽

2020 ◽

Cited By ~ 4

Author(s):

Ameen A. Salahudeen ◽

Shannon S. Choi ◽

Arjun Rustagi ◽

Junjie Zhu ◽

Sean M. de la O ◽

...

Keyword(s):

Lung Disease ◽

Single Cell ◽

Basal Cell ◽

Human Lung ◽

Basal Cells ◽

Ciliated Cells ◽

Club Cells ◽

Distal Lung

ABSTRACTThe distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 “apical-out” organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

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Basal stem cell fate specification is mediated by SMAD signaling in the developing human lung

10.1101/461103 ◽

2018 ◽

Cited By ~ 3

Author(s):

Alyssa J. Miller ◽

Qianhui Yu ◽

Michael Czerwinski ◽

Yu-Hwai Tsai ◽

Renee F. Conway ◽

...

Keyword(s):

Single Cell ◽

Progenitor Cells ◽

Cell Fate ◽

Basal Cell ◽

Human Lung ◽

Branching Morphogenesis ◽

Basal Cells ◽

Smad Signaling ◽

Cell Fate Decisions ◽

Cell Transition

AbstractBasal stem cells (basal cells), located in the bronchi and trachea of the human lung epithelium, play a critical role in normal airway homeostasis and repair, and have been implicated in the development of diseases such as cancer1-4. Additionally, basal-like cells contribute to alveolar regeneration and fibrosis following severe injury5-8. However, the developmental origin of basal cells in humans is unclear. Previous work has shown that specialized progenitor cells exist at the tips of epithelial tubes during lung branching morphogenesis, and in mice, give rise to all alveolar and airway lineages9,10. These ‘bud tip progenitor cells’ have also been described in the developing human lung11-13, but the mechanisms controlling bud tip differentiation into specific cell lineages, including basal cells, are unknown. Here, we interrogated the bud tip-to-basal cell transition using human tissue specimens, bud tip progenitor organoid cultures11, and single-cell transcriptomics. We used single-cell mRNA sequencing (scRNAseq) of developing human lung specimens from 15-21 weeks gestation to identify molecular signatures and cell states in the developing human airway epithelium. We then inferred differentiation trajectories during bud tip-to-airway differentiation, which revealed a previously undescribed transitional cell state (‘hub progenitors’) and implicated SMAD signaling as a regulator of the bud tip-to-basal cell transition. We used bud tip progenitor organoids to show that TGFT1 and BMP4 mediated SMAD signaling robustly induced the transition into functional basal-like cells, and these in vitro-derived basal cells exhibited clonal expansion, self-renewal and multilineage differentiation. This work provides a framework for deducing and validating key regulators of cell fate decisions using single cell transcriptomics and human organoid models. Further, the identification of SMAD signaling as a critical regulator of newly born basal cells in the lung may have implications for regenerative medicine, basal cell development in other organs, and understanding basal cell misregulation in disease.

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Airway tissue stem cells reutilize the embryonic proliferation regulator, Tgfß-Id2 axis, for tissue regeneration

10.1101/2020.11.23.394908 ◽

2020 ◽

Author(s):

Hirofumi Kiyokawa ◽

Akira Yamaoka ◽

Chisa Matsuoka ◽

Tomoko Tokuhara ◽

Takaya Abe ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Tissue Regeneration ◽

Basal Cell ◽

Molecular Mechanisms ◽

Fine Tuning ◽

Basal Cells ◽

Adult Tissue ◽

Epithelial Regeneration ◽

Slow Cycling

SummaryDuring development, quiescent basal stem cells are derived from proliferative primordial progenitors through the cell cycle slowdown. In contrast, quiescent basal cells contribute to tissue repair during adult tissue regeneration by shifting from slow-cycling to proliferating and subsequently back to slow-cycling. Although sustained basal cell proliferation results in tumorigenesis, the molecular mechanisms regulating these transitions remain unknown. Using temporal single-cell transcriptomics of developing murine airway progenitors and in vivo genetic validation experiments, we found that Tgfß signaling slowed down cell cycle by inhibiting Id2 expression in airway progenitors and contributed to the specification of slow-cycling basal cell population during development. In adult tissue regeneration, reduced Tgfß signaling restored Id2 expression and initiated epithelial regeneration. Id2 overexpression and Tgfbr2 knockout enhanced epithelial proliferation; however, persistent Id2 expression in basal cells drove hyperplasia at a rate that resembled a precancerous state. Together, the Tgfß-Id2 axis commonly regulates the proliferation transitions in airway basal cells during development and regeneration, and its fine-tuning is critical for normal regeneration while avoiding basal cell hyperplasia.

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