scholarly journals scATAC-Seq reveals epigenetic heterogeneity associated with an EMT-like process in male germline stem cells and its regulation by G9a

2020 ◽  
Author(s):  
Jinyue Liao ◽  
Hoi Ching Suen ◽  
Alfred Chun Shui Luk ◽  
Annie Wing Tung Lee ◽  
Judy Kin Wing Ng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Regulatory Networks ◽  
Epithelial Mesenchymal Transition ◽  
Stem Cell Differentiation ◽  
Germline Stem Cells ◽  
Gene Markers ◽  
Mesenchymal Transition

AbstractBackgroundEpithelial-mesenchymal transition (EMT) is a phenomenon in which epithelial cells acquire mesenchymal traits. It contributes to organogenesis and tissue homeostasis, as well as stem cell differentiation. Emerging evidence indicates that heterogeneous expression of EMT gene markers presents in sub-populations of germline stem cells (GSCs). However, the functional implications of such heterogeneity are largely elusive.ResultsWe unravelled an EMT-like process in GSCs by in vitro extracellular matrix (ECM) model and single-cell genomics approaches. We found that histone methyltransferase G9a regulated an EMT-like program in GSC in vitro and contributed to neonatal germ cell migration in vivo. Through modulating ECM, we demonstrated that GSCs exist in interconvertible epithelial-like and mesenchymal-like cell states. GSCs gained higher migratory ability after transition to a mesenchymal-like cell state, which was largely mediated by the TGF-β signaling pathway. Dynamics of epigenetic regulation at the single-cell level was also found to align with the EMT-like process. Chromatin accessibility profiles generated by single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) clustered GSCs into epithelial-like and mesenchymal-like states, which were associated with differentiation status. The high-resolution data revealed regulators in the EMT-like process, including transcription factors Zeb1. We further identified putative enhancer-promoter interactions and cis-co-accessibility networks at loci such as Tgfb1, Notch1 and Lin28a. Lastly, we identified HES1 as the putative target underlying G9a’s regulation.ConclusionOur work provides the foundation for understanding the EMT-like process and a comprehensive resource for future investigation of epigenetic regulatory networks in GSCs.

scholarly journals Insights into Differentiation of Melanocytes from Human Stem Cells and Their Relevance for Melanoma Treatment

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2508
Author(s):  
Madalina Mirea ◽  
Stefan Eckensperger ◽  
Markus Hengstschläger ◽  
Mario Mikula
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Developmental Process ◽  
Epithelial Mesenchymal Transition ◽  
Stem Cell Differentiation ◽  
Human Stem Cells ◽  
Mesenchymal Transition ◽  
Melanocytic Differentiation

Malignant melanoma represents a highly aggressive form of skin cancer. The metastatic process itself is mostly governed by the so-called epithelial mesenchymal transition (EMT), which confers cancer cells migrative, invasive and resistance abilities. Since EMT represents a conserved developmental process, it is worthwhile further examining the nature of early developmental steps fundamental for melanocyte differentiation. This can be done either in vivo by analyzing the physiologic embryo development in different species or by in vitro studies of melanocytic differentiation originating from embryonic human stem cells. Most importantly, external cues drive progenitor cell differentiation, which can be divided in stages favoring neural crest specification or melanocytic differentiation and proliferation. In this review, we describe ectopic factors which drive human pluripotent stem cell differentiation to melanocytes in 2D, as well as in organoid models. Furthermore, we compare developmental mechanisms with processes described to occur during melanoma development. Finally, we suggest differentiation factors as potential co-treatment options for metastatic melanoma patients.

scholarly journals Studying human reproductive biology through single-cell analysis and in vitro differentiation of stem cells into germ cell-like cells

2020 ◽  
Vol 26 (5) ◽  
pp. 670-688 ◽  
Author(s):  
Lin Li ◽  
Risako Yang ◽  
Chenghong Yin ◽  
Kehkooi Kee
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Germ Cells ◽  
Regulatory Networks ◽  
Single Cell Analysis ◽  
Reproductive Development ◽  
In Vitro Differentiation ◽  
Gene Markers ◽  
Human Reproductive

Abstract BACKGROUND Understanding the molecular and cellular mechanisms of human reproductive development has been limited by the scarcity of human samples and ethical constraints. Recently, in vitro differentiation of human pluripotent stem cells into germ cells and single-cell analyses have opened new avenues to directly study human germ cells and identify unique mechanisms in human reproductive development. OBJECTIVE AND RATIONALE The goal of this review is to collate novel findings and insightful discoveries with these new methodologies, aiming at introducing researchers and clinicians to the use of these tools to study human reproductive biology and develop treatments for infertility. SEARCH METHODS PubMed was used to search articles and reviews with the following main keywords: in vitro differentiation, human stem cells, single-cell analysis, spermatogenesis, oogenesis, germ cells and other key terms related to these subjects. The search period included all publications from 2000 until now. OUTCOMES Single-cell analyses of human gonads have identified many important gene markers at different developmental stages and in subpopulations of cells. To validate the functional roles of these gene markers, researchers have used the in vitro differentiation of human pluripotent cells into germ cells and confirmed that some genetic requirements are unique in human germ cells and are not conserved in mouse models. Moreover, transcriptional regulatory networks and the interaction of germ and somatic cells in gonads were elucidated in these studies. WIDER IMPLICATIONS Single-cell analyses allow researchers to identify gene markers and potential regulatory networks using limited clinical samples. On the other hand, in vitro differentiation methods provide clinical researchers with tools to examine these newly identify gene markers and study the causative effects of mutations previously associated with infertility. Combining these two methodologies, researchers can identify gene markers and networks which are essential and unique in human reproductive development, thereby producing more accurate diagnostic tools for assessing reproductive disorders and developing treatments for infertility.

scholarly journals Lgr5+CD44+EpCAM+ Strictly Defines Cancer Stem Cells in Human Colorectal Cancer

2018 ◽  
Vol 46 (2) ◽  
pp. 860-872 ◽  
Author(s):  
Zhengwei Leng ◽  
Qinghua Xia ◽  
Jinhuang Chen ◽  
Yong Li ◽  
Jiqian Xu ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Colony Formation ◽  
Epithelial Mesenchymal Transition ◽  
Tumor Xenograft ◽  
Self Renewal ◽  
Mesenchymal Transition

Background/Aims: Although EpCAM+CD44+ cells exhibit more stem-like properties than did EpCAM-CD44- cells, the specificity of EpCAM combined with CD44 in defining CSCs needs further improvement. Lgr5 is used as a biomarker to isolate cancer stem cells (CSCs) in colorectal cancer. However, it remains unclear whether Lgr5, along with EpCAM and CD44, can further identify and define CSCs in colorectal cancer. Methods: Lgr5+CD44+EpCAM+, Lgr5+CD44+EpCAM-, Lgr5+CD44-EpCAM+, Lgr5-CD44+EpCAM+, and Lgr5-CD44-EpCAM-cells were separately isolated using fluorescence-activated cell sorting (FACS). Colony formation, self-renewal, differentiation, and tumorigenic properties of these cells were investigated through in vitro experiments and in vivo tumor xenograft models. The expression of stemness genes and CSC- and epithelial-mesenchymal transition (EMT)-related genes, such as KLF4, Oct4, Sox2, Nanog, CD133, CD44, CD166, ALDH1, Lgr5, E-cadherin, ZO-1, Vimentin, Snail, Slug, and Twist, was examined using real-time PCR. Results: Lgr5-positive subpopulations exhibited higher capacities for colony formation, self-renewal, differentiation, and tumorigenicity as well as higher expression of stemness genes and mesenchymal genes and lower expression of epithelial genes than did Lgr5-negative subpopulations. Conclusion: Our data revealed that tumorigenic cells were highly restricted to Lgr5-positive subpopulations. Most importantly, Lgr5+CD44+EpCAM+ cells exhibited more pronounced CSC-like traits than did any other subpopulation, indicating that Lgr5 combined with CD44 and EpCAM can further improve the stem-like traits of CSCs in colorectal cancer.

scholarly journals Robust temporal map of human in vitro myelopoiesis using single-cell genomics

2021 ◽  
Author(s):  
Clara Alsinet ◽  
Maria Primo ◽  
Valentina Lorenzi ◽  
Andrew J Knights ◽  
Carmen Sancho-Serra ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Regulatory Networks ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Myeloid Differentiation ◽  
Hematopoietic Stem ◽  
Wide Range

Myeloid cells have a central role in homeostasis and tissue defence. Characterising the current in vitro protocols of myelopoiesis is imperative for their use in research and immunotherapy as well as for understanding the early stages of myeloid differentiation in humans. Here, we profiled the transcriptome of more than 400k cells and generated a robust molecular map of the differentiation of human induced pluripotent stem cells (iPSC) into macrophages. By integrating our in vitro datasets with in vivo single-cell developmental atlases, we found that in vitro macrophage differentiation recapitulates features of in vivo yolk sac hematopoiesis, which happens prior to the appearance of definitive hematopoietic stem cells (HSC). During in vitro myelopoiesis, a wide range of myeloid cells are generated, including erythrocytes, mast cells and monocytes, suggesting that, during early human development, the HSC-independent immune wave gives rise to multiple myeloid cell lineages. We leveraged this model to characterize the transition of hemogenic endothelium into myeloid cells, uncovering poorly described myeloid progenitors and regulatory programs. Taking advantage of the variety of myeloid cells produced, we developed a new protocol to produce type 2 conventional dendritic cells (cDC2) in vitro. We found that the underlying regulatory networks coding for myeloid identity are conserved in vivo and in vitro. Using genetic engineering techniques, we validated the effects of key transcription factors important for cDC2 and macrophage identity and ontogeny. This roadmap of early myeloid differentiation will serve as an important resource for investigating the initial stages of hematopoiesis, which are largely unexplored in humans, and will open up new therapeutic opportunities.

scholarly journals Human Primary Breast Cancer Stem Cells Are Characterized by Epithelial-Mesenchymal Plasticity

2021 ◽  
Vol 22 (4) ◽  
pp. 1808 ◽  
Author(s):  
Juliane Strietz ◽  
Stella S. Stepputtis ◽  
Marie Follo ◽  
Peter Bronsert ◽  
Elmar Stickeler ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Cancer Stem Cells ◽  
Primary Breast Cancer ◽  
Epithelial Mesenchymal Transition ◽  
Treatment Options ◽  
Culture Conditions ◽  
Mesenchymal Transition

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with only limited treatment options available. Recently, cancer stem cells (CSCs) have emerged as the potential drivers of tumor progression due to their ability to both self-renew and give rise to differentiated progeny. The CSC state has been linked to the process of epithelial-mesenchymal transition (EMT) and to the highly flexible state of epithelial-mesenchymal plasticity (EMP). We aimed to establish primary breast cancer stem cell (BCSC) cultures isolated from TNBC specimens. These cells grow as tumor spheres under anchorage-independent culture conditions in vitro and reliably form tumors in mice when transplanted in limiting dilutions in vivo. The BCSC xenograft tumors phenocopy the original patient tumor in architecture and gene expression. Analysis of an EMT-related marker profile revealed the concomitant expression of epithelial and mesenchymal markers suggesting an EMP state for BCSCs of TNBC. Furthermore, BCSCs were susceptible to stimulation with the EMT inducer TGF-β1, resulting in upregulation of mesenchymal genes and enhanced migratory abilities. Overall, primary BCSC cultures are a promising model close to the patient that can be used both in vitro and in vivo to address questions of BCSC biology and evaluate new treatment options for TNBC.

scholarly journals Overexpressed P75CUX1 promotes EMT in glioma infiltration by activating β-catenin

2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Anqi Xu ◽  
Xizhao Wang ◽  
Jie Luo ◽  
Mingfeng Zhou ◽  
Renhui Yi ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
Tumor Grade ◽  
Prognostic Indicator ◽  
Migration And Invasion ◽  
Poor Overall Survival ◽  
Mesenchymal Transition ◽  
Kaplan Meier ◽  
Homeobox Protein

AbstractThe homeobox protein cut-like 1 (CUX1) comprises three isoforms and has been shown to be involved in the development of various types of malignancies. However, the expression and role of the CUX1 isoforms in glioma remain unclear. Herein, we first identified that P75CUX1 isoform exhibited consistent expression among three isoforms in glioma with specifically designed antibodies to identify all CUX1 isoforms. Moreover, a significantly higher expression of P75CUX1 was found in glioma compared with non-tumor brain (NB) tissues, analyzed with western blot and immunohistochemistry, and the expression level of P75CUX1 was positively associated with tumor grade. In addition, Kaplan–Meier survival analysis indicated that P75CUX1 could serve as an independent prognostic indicator to identify glioma patients with poor overall survival. Furthermore, CUX1 knockdown suppressed migration and invasion of glioma cells both in vitro and in vivo. Mechanistically, this study found that P75CUX1 regulated epithelial–mesenchymal transition (EMT) process mediated via β-catenin, and CUX1/β-catenin/EMT is a novel signaling cascade mediating the infiltration of glioma. Besides, CUX1 was verified to promote the progression of glioma via multiple other signaling pathways, such as Hippo and PI3K/AKT. In conclusion, we suggested that P75CUX1 could serve as a potential prognostic indicator as well as a novel treatment target in malignant glioma.

scholarly journals Long noncoding RNA LINC00941 promotes pancreatic cancer progression by competitively binding miR-335-5p to regulate ROCK1-mediated LIMK1/Cofilin-1 signaling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jie Wang ◽  
Zhiwei He ◽  
Jian Xu ◽  
Peng Chen ◽  
Jianxin Jiang
Keyword(s):  
Pancreatic Cancer ◽  
Cell Growth ◽  
Cell Lines ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Epithelial Mesenchymal Transition ◽  
Loss Of Function ◽  
Mesenchymal Transition

AbstractAn accumulation of evidence indicates that long noncoding RNAs are involved in the tumorigenesis and progression of pancreatic cancer (PC). In this study, we investigated the functions and molecular mechanism of action of LINC00941 in PC. Quantitative PCR was used to examine the expression of LINC00941 and miR-335-5p in PC tissues and cell lines, and to investigate the correlation between LINC00941 expression and clinicopathological features. Plasmid vectors or lentiviruses were used to manipulate the expression of LINC00941, miR-335-5p, and ROCK1 in PC cell lines. Gain or loss-of-function assays and mechanistic assays were employed to verify the roles of LINC00941, miR-335-5p, and ROCK1 in PC cell growth and metastasis, both in vivo and in vitro. LINC00941 and ROCK1 were found to be highly expressed in PC, while miR-335-5p exhibited low expression. High LINC00941 expression was strongly associated with larger tumor size, lymph node metastasis, and poor prognosis. Functional experiments revealed that LINC00941 silencing significantly suppressed PC cell growth, metastasis and epithelial–mesenchymal transition. LINC00941 functioned as a molecular sponge for miR-335-5p, and a competitive endogenous RNA (ceRNA) for ROCK1, promoting ROCK1 upregulation, and LIMK1/Cofilin-1 pathway activation. Our observations lead us to conclude that LINC00941 functions as an oncogene in PC progression, behaving as a ceRNA for miR-335-5p binding. LINC00941 may therefore have potential utility as a diagnostic and treatment target in this disease.

scholarly journals EXTH-51. ANTI-INVASIVE EFFICACY AND SURVIVAL BENEFIT OF THE YAP-TEAD INHIBITOR VERTEPORFIN IN PRECLINICAL GLIOBLASTOMA MODELS

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii98-ii98
Author(s):  
Anne Marie Barrette ◽  
Alexandros Bouras ◽  
German Nudelman ◽  
Zarmeen Mussa ◽  
Elena Zaslavsky ◽  
...  
Keyword(s):  
Survival Benefit ◽  
De Novo ◽  
Epithelial Mesenchymal Transition ◽  
Intraperitoneal Administration ◽  
Standard Of Care ◽  
Tumor Burden ◽  
Mesenchymal Transition ◽  
Protein Levels

Abstract Glioblastoma (GBM) remains an incurable disease, in large part due to its malignant infiltrative spread, and current clinical therapy fails to target the invasive nature of tumor cells in disease progression and recurrence. Here, we use the YAP-TEAD inhibitor Verteporfin to target a convergence point for regulating tumor invasion/metastasis and establish the robust anti-invasive therapeutic efficacy of this FDA-approved drug and its survival benefit across several preclinical glioma models. Using patient-derived GBM cells and orthotopic xenograft models (PDX), we show that Verteporfin treatment disrupts YAP/TAZ-TEAD activity and processes related to cell adhesion, migration and epithelial-mesenchymal transition. In-vitro, Verteporfin impairs tumor migration, invasion and motility dynamics. In-vivo, intraperitoneal administration of Verteporfin in mice with orthotopic PDX tumors shows consistent drug accumulation within the brain and decreased infiltrative tumor burden, across three independent experiments. Interestingly, PDX tumors with impaired invasion after Verteporfin treatment downregulate CDH2 and ITGB1 adhesion protein levels within the tumor microenvironment. Finally, Verteporfin treatment confers survival benefit in two independent PDX models: as monotherapy in de-novo GBM and in combination with standard-of-care chemoradiation in recurrent GBM. These findings indicate potential therapeutic value of this FDA-approved drug if repurposed for GBM patients.

scholarly journals Phosphorylation of Serine 11 and Serine 92 as New Positive Regulators of Human Snail1 Function: Potential Involvement of Casein Kinase-2 and the cAMP-activated Kinase Protein Kinase A

2010 ◽  
Vol 21 (2) ◽  
pp. 244-253 ◽  
Author(s):  
Matthew Reid MacPherson ◽  
Patricia Molina ◽  
Serhiy Souchelnytskyi ◽  
Christer Wernstedt ◽  
Jorge Martin-Pérez ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Tumor Metastasis ◽  
Epithelial Mesenchymal Transition ◽  
Cop9 Signalosome ◽  
Mesenchymal Transition ◽  
Depth Analysis ◽  
Positive Regulators

Snail1 is a major factor for epithelial-mesenchymal transition (EMT), an important event in tumor metastasis and in other pathologies. Snail1 is tightly regulated at transcriptional and posttranscriptional levels. Control of Snail1 protein stability and nuclear export by GSK3β phosphorylation is important for Snail1 functionality. Stabilization mechanisms independent of GSK3β have also been reported, including interaction with LOXL2 or regulation of the COP9 signalosome by inflammatory signals. To get further insights into the role of Snail1 phosphorylation, we have performed an in-depth analysis of in vivo human Snail1 phosphorylation combined with mutational studies. We identify new phosphorylation sites at serines 11, 82, and 92 and confirmed previously suggested phosphorylations at serine 104 and 107. Serines 11 and 92 participate in the control of Snail1 stability and positively regulate Snail1 repressive function and its interaction with mSin3A corepressor. Furthermore, serines 11 and 92 are required for Snail1-mediated EMT and cell viability, respectively. PKA and CK2 have been characterized as the main kinases responsible for in vitro Snail1 phosphorylation at serine 11 and 92, respectively. These results highlight serines 11 and 92 as new players in Snail1 regulation and suggest the participation of CK2 and PKA in the modulation of Snail1 functionality.

scholarly journals CD73 facilitates EMT progression and promotes lung metastases in triple-negative breast cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nataliia Petruk ◽  
Sanni Tuominen ◽  
Malin Åkerfelt ◽  
Jesse Mattsson ◽  
Jouko Sandholm ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Epithelial Mesenchymal Transition ◽  
Lung Metastases ◽  
Vimentin Expression ◽  
Mesenchymal Transition ◽  
Cell Protrusion

AbstractCD73 is a cell surface ecto-5′-nucleotidase, which converts extracellular adenosine monophosphate to adenosine. High tumor CD73 expression is associated with poor outcome among triple-negative breast cancer (TNBC) patients. Here we investigated the mechanisms by which CD73 might contribute to TNBC progression. This was done by inhibiting CD73 with adenosine 5′-(α, β-methylene) diphosphate (APCP) in MDA-MB-231 or 4T1 TNBC cells or through shRNA-silencing (sh-CD73). Effects of such inhibition on cell behavior was then studied in normoxia and hypoxia in vitro and in an orthotopic mouse model in vivo. CD73 inhibition, through shRNA or APCP significantly decreased cellular viability and migration in normoxia. Inhibition of CD73 also resulted in suppression of hypoxia-induced increase in viability and prevented cell protrusion elongation in both normoxia and hypoxia in cancer cells. Sh-CD73 4T1 cells formed significantly smaller and less invasive 3D organoids in vitro, and significantly smaller orthotopic tumors and less lung metastases than control shRNA cells in vivo. CD73 suppression increased E-cadherin and decreased vimentin expression in vitro and in vivo, proposing maintenance of a more epithelial phenotype. In conclusion, our results suggest that CD73 may promote early steps of tumor progression, possibly through facilitating epithelial–mesenchymal transition.

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