scholarly journals A53 STUDYING THE ROLE OF ASCL2 IN THE ESOPHAGEAL EPITHELIUM USING ORGANOIDS

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 10-11
Author(s):  
M Hamilton ◽  
D Jean ◽  
V Giroux
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Epithelial Cells ◽  
Notch Pathway ◽  
Stem Cell Population ◽  
Esophageal Epithelium ◽  
Ki 67 ◽  
Self Renewal ◽  
Esophageal Epithelial Cells

Abstract Background The esophagus is lined with a stratified squamous epithelium that assure protection against the austere environment found in the esophageal lumen. The maintenance of this epithelium is ensured by a rare population of cells: stem cells. Those cells have increased capacity of self-renewal and multipotency, which is the capacity to give rise to every cell types of a tissue. The marker Krt15 was used to identify the first stem cell population in the esophagus. Krt15+ cells display an extended lifespan and they are radioresistant, multipotent and capable of self-renewal. Moreover, it was observed by RNA sequencing that the expression of the transcription factor ASCL2 is strongly increased in Krt15+ cells compared to Krt15- cells. Interestingly, ASCL2 is necessary to maintain the stemness of Lgr5+ intestinal stem cells. It is also a target of the Wnt/β-catenin pathway. The overall goal of this project is to determine the role of ACSL2 in the maintenance of esophageal stem cells and to identify its binding partners since ASCL2 needs to dimerize to efficiently bind DNA. Aims Confirm that esophageal organoids are adapted to study ASCL2 in the esophagus. Methods Esophageal organoids were established from esophageal epithelial cells from wildtype mice. Following this, organoids were treated with an inhibitor of the Notch pathway (DAPT) to induce hyperplasia or infected with lentiviruses to invalidate Ascl2 (CRISPR/Cas9 approach). Results To validate that Ascl2 plays an important role in esophageal cell proliferation, Notch pathway was inhibited through DAPT treatment in esophageal organoids to induce hyperplasia, which was confirmed by increased number of proliferative cells (Ki-67+). ASCL2 protein expression was also increased in DAPT-treated organoids supporting its role in proliferation and confirming that organoid is a good model to study ASCL2 role in esophageal epithelial cells. In this optic, organoids lines invalidated for Ascl2 (CRISPR/Cas9 approach) were established. Our preliminary results suggest that Ascl2 loss affects cell proliferation and organoid size under normal conditions. Conclusions The expression of ASCL2 correlates with hyperplasia which supports its role in esophageal epithelium homeostasis. Funding Agencies Canada research chair et NSERC

scholarly journals Wnt/β-catenin signaling promotes self-renewal and inhibits the primed state transition in naïve human embryonic stem cells

2016 ◽  
Vol 113 (42) ◽  
pp. E6382-E6390 ◽  
Author(s):  
Zhuojin Xu ◽  
Aaron M. Robitaille ◽  
Jason D. Berndt ◽  
Kathryn C. Davidson ◽  
Karin A. Fischer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Extrinsic Factors ◽  
Self Renewal ◽  
Wnt Proteins ◽  
Primed State

In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/β-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/β-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of β-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/β-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/β-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.

scholarly journals Glycolipids Recognized by A2B5 Antibody Promote Proliferation, Migration, and Clonogenicity in Glioblastoma Cells

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1267 ◽  
Author(s):  
Baeza-Kallee ◽  
Bergès ◽  
Soubéran ◽  
Colin ◽  
Denicolaï ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Cell Growth ◽  
Self Renewal ◽  
Neuraminidase Treatment ◽  
Attractive Therapeutic Target ◽  
And Migration

A2B5+ cells isolated from human glioblastomas exhibit cancer stem cell properties. The A2B5 epitope belongs to the sialoganglioside family and is synthetized by the ST8 alpha-N-acetyl-neuraminidase α-2,8-sialyltransferase 3 (ST8SIA3) enzyme. Glycolipids represent attractive targets for solid tumors; therefore, the aim of this study was to decipher A2B5 function in glioblastomas. To this end, we developed cell lines expressing various levels of A2B5 either by genetically manipulating ST8SIA3 or by using neuraminidase. The overexpression of ST8SIA3 in low-A2B5-expressing cells resulted in a dramatic increase of A2B5 immunoreactivity. ST8SIA3 overexpression increased cell proliferation, migration, and clonogenicity in vitro and tumor growth when cells were intracranially grafted. Conversely, lentiviral ST8SIA3 inactivation in low-A2B5-expressing cells resulted in reduced proliferation, migration, and clonogenicity in vitro and extended mouse survival. Furthermore, in the shST8SIA3 cells, we found an active apoptotic phenotype. In high-A2B5-expressing cancer stem cells, lentiviral delivery of shST8SIA3 stopped cell growth. Neuraminidase treatment, which modifies the A2B5 epitope, impaired cell survival, proliferation, self-renewal, and migration. Our findings prove the crucial role of the A2B5 epitope in the promotion of proliferation, migration, clonogenicity, and tumorigenesis, pointing at A2B5 as an attractive therapeutic target for glioblastomas.

Loss of Krüppel-Like Factor 4 (KLF4) Leads to Increased Self-Renewal Under Stress Conditions and Improved Survival of Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 861-861 ◽  
Author(s):  
Chun Shik Park ◽  
Takeshi Yamada ◽  
Koramit Suppipat ◽  
Maksim Mamonkin ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Knockout Mice ◽  
Peripheral Blood ◽  
Fold Increase ◽  
Ki 67 ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 861 Hematopoiesis is a highly regulated process in which a small number of hematopoietic stem cells (HSC) generate all mature blood cells. In order to preserve homeostasis of the hematopoietic system throughout lifetime, this pool of HSC must be maintained by the processes of self-renewal and survival. Self-renewal requires a coordination of survival signals and control of proliferation uncoupled from differentiation. Even though extrinsic signals from the microenvironment and cell-intrinsic regulators are required for self-renewal of HSCs, the intricate transcriptional machinery that selectively regulates HSC self-renewal and survival is still poorly understood. Krüppel-like factor 4 (KLF4) is a zinc-finger transcription factor that regulates proliferation, differentiation, apoptosis, and self-renewal. The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with OCT3/4, c-Myc and SOX2 suggests that KLF4 is required for preservation of an undifferentiated state. To investigate the function of KLF4 in HSC maintenance, we used conditional Klf4 knockout mice (Klf4fl/flVav-iCre+) to specifically delete KLF4 gene in hematopoietic cells. We first analyzed the frequency of HSC and progenitor cells in the bone marrow (BM) of Klf4fl/flVav-iCre– (control) and Klf4fl/flVav-iCre+ (knockout) by flow cytometry. We found that KLF4 deficiency leads to a 2.4-fold increase in the number of long-term HSC (Lin–Sca-1+c-Kit+ CD150+ CD48–) and a 2.2-fold increase in short-term HSC compartements (Lin–Sca-1+c-Kit+ CD150+ CD48+) whereas no significant changes were found in myeloid and lymphoid progenitor cells. Consistent with this phenotypic analysis, KLF4 expression in HSC is higher than hematopoietic progenitor cells and mature lineages (n=3; P<0.05). Even though ablation of Klf4 gene does not affect multi-lineage potential of HSC upon transplantation, its deletion leads to a reduction of monocytes and T cell expansion. To assess the effect of Klf4 ablation in self-renewal, we performed serial competitive repopulation assays using a 1:1 mixture of BM cells from control (Klf4fl/flVav-iCre–; CD45.2+) or knockout (Klf4fl/flVav-iCre+; CD45.2+) with B6.SJL (CD45.1+) mice. In primary transplants, the contribution of knockout BM cells in peripheral blood was similar to controls. Interestingly, loss of KLF4 led to enhanced contribution to peripheral blood in secondary transplants (4.5-fold; P<0.005) and tertiary transplants (2.6-fold; P<0.005). Consistent with this result, we found a significant increased number of colony forming units only in the third replating on methylcellulose (P<0.0005). To further characterize the role of KLF4 in HSC proliferation, we determined expression of Ki-67 and DNA content in nuclei of LSK CD150+ cells. The fraction of G0 cells defined as Ki-67– within 2n DNA in Klf4-knockout LSK CD150+ cells was similar to control (control 74.3 ± 0.7% vs 73.2 ± 2.3%). However, Annexin V staining revealed a 2.4-fold increased survival of LSK CD150+ cells in Klf4-knockout mice compared to control mice but not in myeloid progenitor cells (Lin–c-Kit+Sca-1–) suggesting that KLF4 selectively regulates the survival of HSC. These studies indicate that KLF4 controls steady state HSC survival and self-renewal under stress conditions. Disclosures: No relevant conflicts of interest to declare.

Effect of Calcium on the Growth and Differentiation of Cultured Rat Esophageal Epithelial Cells

1982 ◽  
Vol 40 ◽  
pp. 132-133
Author(s):  
W.T. Gunning ◽  
M.R. Marino ◽  
M.S. Babcock ◽  
G.D. Stoner
Keyword(s):  
Epithelial Cells ◽  
Cell Types ◽  
Replication Rate ◽  
Enzymatic Dissociation ◽  
Growth Assay ◽  
Epidermal Growth ◽  
Fetal Bovine ◽  
Esophageal Epithelial Cells ◽  
Cellular Replication

The role of calcium in modulating cellular replication and differentiation has been described for various cell types. In the present study, the effects of Ca++ on the growth and differentiation of cultured rat esophageal epithelial cells was investigated.Epithelial cells were isolated from esophagi taken from 8 week-old male CDF rats by the enzymatic dissociation method of Kaighn. The cells were cultured in PFMR-4 medium supplemented with 0.25 mg/ml dialyzed fetal bovine serum, 5 ng/ml epidermal growth factor, 10-6 M hydrocortisone 10-6 M phosphoethanolamine, 10-6 M ethanolamine, 5 pg/ml insulin, 5 ng/ml transferrin, 10 ng/ml cholera toxin and 50 ng/ml garamycin at 36.5°C in a humidified atmosphere of 3% CO2 in air. At weekly intervals, the cells were subcultured with a solution containing 1% polyvinylpyrrolidone, 0.01% EGTA, and 0.05% trypsin. After various passages, the replication rate of the cells in PFMR-4 medium containing from 10-6 M to 10-3 M Ca++ was determined using a clonal growth assay.

scholarly journals Local anesthetics impair the growth and self-renewal of glioblastoma stem cells by inhibiting ZDHHC15-mediated GP130 palmitoylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xiaoqing Fan ◽  
Haoran Yang ◽  
Chenggang Zhao ◽  
Lizhu Hu ◽  
Delong Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Western Blot ◽  
Local Anesthetics ◽  
Molecular Mechanisms ◽  
Biological Activities ◽  
Xenograft Model ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Self Renewal

Abstract Background A large number of preclinical studies have shown that local anesthetics have a direct inhibitory effect on tumor biological activities, including cell survival, proliferation, migration, and invasion. There are few studies on the role of local anesthetics in cancer stem cells. This study aimed to determine the possible role of local anesthetics in glioblastoma stem cell (GSC) self-renewal and the underlying molecular mechanisms. Methods The effects of local anesthetics in GSCs were investigated through in vitro and in vivo assays (i.e., Cell Counting Kit 8, spheroidal formation assay, double immunofluorescence, western blot, and xenograft model). The acyl-biotin exchange method (ABE) assay was identified proteins that are S-acylated by zinc finger Asp-His-His-Cys-type palmitoyltransferase 15 (ZDHHC15). Western blot, co-immunoprecipitation, and liquid chromatograph mass spectrometer-mass spectrometry assays were used to explore the mechanisms of ZDHHC15 in effects of local anesthetics in GSCs. Results In this study, we identified a novel mechanism through which local anesthetics can damage the malignant phenotype of glioma. We found that local anesthetics prilocaine, lidocaine, procaine, and ropivacaine can impair the survival and self-renewal of GSCs, especially the classic glioblastoma subtype. These findings suggest that local anesthetics may weaken ZDHHC15 transcripts and decrease GP130 palmitoylation levels and membrane localization, thus inhibiting the activation of IL-6/STAT3 signaling. Conclusions In conclusion, our work emphasizes that ZDHHC15 is a candidate therapeutic target, and local anesthetics are potential therapeutic options for glioblastoma.

scholarly journals Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors

2021 ◽  
Vol 22 (4) ◽  
pp. 2047
Author(s):  
Nina Schmid ◽  
Kim-Gwendolyn Dietrich ◽  
Ignasi Forne ◽  
Alexander Burges ◽  
Magdalena Szymanska ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cell ◽  
Granulosa Cells ◽  
Drug Targets ◽  
Growth Regulation ◽  
Sirtuin 1 ◽  
Ki 67 ◽  
Granulosa Cell Tumors ◽  
Novel Drug

Sirtuins (SIRTs) are NAD+-dependent deacetylases that regulate proliferation and cell death. In the human ovary, granulosa cells express sirtuin 1 (SIRT1), which has also been detected in human tumors derived from granulosa cells, i.e., granulosa cell tumors (GCTs), and in KGN cells. KGN cells are an established cellular model for the majority of GCTs and were used to explore the role of SIRT1. The SIRT1 activator SRT2104 increased cell proliferation. By contrast, the inhibitor EX527 reduced cell numbers, without inducing apoptosis. These results were supported by the outcome of siRNA-mediated silencing studies. A tissue microarray containing 92 GCTs revealed nuclear and/or cytoplasmic SIRT1 staining in the majority of the samples, and also, SIRT2-7 were detected in most samples. The expression of SIRT1–7 was not correlated with the survival of the patients; however, SIRT3 and SIRT7 expression was significantly correlated with the proliferation marker Ki-67, implying roles in tumor cell proliferation. SIRT3 was identified by a proteomic analysis as the most abundant SIRT in KGN. The results of the siRNA-silencing experiments indicate involvement of SIRT3 in proliferation. Thus, several SIRTs are expressed by GCTs, and SIRT1 and SIRT3 are involved in the growth regulation of KGN. If transferable to GCTs, these SIRTs may represent novel drug targets.

scholarly journals The Act of Controlling Adult Stem Cell Dynamics: Insights from Animal Models

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 667
Author(s):  
Meera Krishnan ◽  
Sahil Kumar ◽  
Luis Johnson Kangale ◽  
Eric Ghigo ◽  
Prasad Abnave
Keyword(s):  
Stem Cells ◽  
Animal Models ◽  
Adult Stem Cells ◽  
The Body ◽  
In Vivo Studies ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Organ Systems

Adult stem cells (ASCs) are the undifferentiated cells that possess self-renewal and differentiation abilities. They are present in all major organ systems of the body and are uniquely reserved there during development for tissue maintenance during homeostasis, injury, and infection. They do so by promptly modulating the dynamics of proliferation, differentiation, survival, and migration. Any imbalance in these processes may result in regeneration failure or developing cancer. Hence, the dynamics of these various behaviors of ASCs need to always be precisely controlled. Several genetic and epigenetic factors have been demonstrated to be involved in tightly regulating the proliferation, differentiation, and self-renewal of ASCs. Understanding these mechanisms is of great importance, given the role of stem cells in regenerative medicine. Investigations on various animal models have played a significant part in enriching our knowledge and giving In Vivo in-sight into such ASCs regulatory mechanisms. In this review, we have discussed the recent In Vivo studies demonstrating the role of various genetic factors in regulating dynamics of different ASCs viz. intestinal stem cells (ISCs), neural stem cells (NSCs), hematopoietic stem cells (HSCs), and epidermal stem cells (Ep-SCs).

scholarly journals HAUSP Stabilizes SOX2 through Deubiquitination to Maintain Self-renewal and Tumorigenic Potential of Glioma Stem Cells

2021 ◽  
Author(s):  
Zhi Huang ◽  
Kui Zhai ◽  
Qiulian Wu ◽  
Xiaoguang Fang ◽  
Qian Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Glioma Stem Cells ◽  
Posttranslational Regulation ◽  
Malignant Growth ◽  
Self Renewal ◽  
Animal Survival ◽  
Promising Target ◽  
Tumorigenic Potential ◽  
Therapeutic Opportunity

Glioblastoma (GBM) is the most lethal brain tumor containing glioma stem cells (GSCs) that promote malignant growth and therapeutic resistance. The self-renewal and tumorigenic potential of GSCs are maintained by core stem cell transcription factors including SOX2. Defining the posttranslational regulation of SOX2 may offer new insights into GSC biology and potential therapeutic opportunity. Here, we discover that HAUSP stabilizes SOX2 through deubiquitination to maintain GSC self-renewal and tumorigenic potential. HAUSP is preferentially expressed in GSCs in perivascular niches in GBMs. Disrupting HAUSP by shRNA or its inhibitor P22077 promoted SOX2 degradation, induced GSC differentiation, impaired GSC tumorigenic potential, and suppressed GBM tumor growth. Importantly, pharmacological inhibition of HAUSP synergized with radiation to inhibit GBM growth and extended animal survival, indicating that targeting HAUSP may overcome GSC-mediated radioresistance. Our findings reveal an unappreciated crucial role of HAUSP in the GSC maintenance and provide a promising target for developing effective anti-GSC therapeutics to improve GBM treatment.

scholarly journals Long noncoding RNA LINC00261 upregulates ITIH5 to impair tumorigenic ability of pancreatic cancer stem cells

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Lijuan Zou ◽  
Hengpeng He ◽  
Zhiguo Li ◽  
Ou Chen ◽  
Xiukun Jia ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Stem Cells ◽  
Cell Proliferation ◽  
Drug Resistance ◽  
Cancer Stem Cells ◽  
Noncoding Rna ◽  
Cell Subset ◽  
Luciferase Reporter ◽  
Stem Cell Markers ◽  
Self Renewal

AbstractLong noncoding RNAs (lncRNAs) are implicated tumor development in a range of different cancers, including pancreatic cancer (PC). Cancer stem cells (CSCs), a drug-resistant cancer cell subset, drive tumor progression in PC. In this work, we aimed to investigate the mechanism by which lncRNA LINC00261 affects the biological functions of CSCs during the progression of PC. Microarray analysis of differentially expressed genes and lncRNAs suggested that LINC00261 is downregulated in PC. Both LINC00261 and ITIH5 were confirmed to be downregulated in PC cells and PC stem cells. Gain-of-function and loss-of-function investigations were performed to analyze their effects on cell proliferation, drug resistance, cell cycle distribution, self-renewal, invasion, and ultimately overall tumorigenicity. These experiments revealed that the expression of stem cell markers was reduced, and cell proliferation, self-renewal ability, cell invasion, drug resistance, and tumorigenicity were all suppressed by upregulation of LINC00261 or ITIH5. The results of dual-luciferase reporter gene, ChIP, and RIP assays indicated that LINC00261 binds directly to GATA6, increasing its activity at the ITIH5 promoter. The presence of LINC00261 and GATA6 inhibited the self-renewal and tumorigenesis of PC stem cells, while silence of ITIH5 rescued those functions. Collectively, this study identifies the tumor suppressive activity of LINC00261 in PC, showing that this lncRNA limits the functions of PC stem through an ITIH5/GATA6 regulatory pathway.

scholarly journals YAP regulates porcine skin-derived stem cells self-renewal partly by repressing Wnt/β-catenin signaling pathway

2021 ◽  
Author(s):  
Hong-Chen Yan ◽  
Yu Sun ◽  
Ming-Yu Zhang ◽  
Shu-Er Zhang ◽  
Jia-Dong Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Signaling Pathway ◽  
Adult Stem Cells ◽  
Self Renewal ◽  
Human Ascs ◽  
Regulation Mechanisms ◽  
Interfering Rna ◽  
Pluripotency Genes

Abstract Background Skin-derived stem cells (SDSCs) are a class of adult stem cells (ASCs) that have the ability to self-renew and differentiate. The regulation mechanisms involved in the differentiation of ASCs is a hot topic. Porcine models have close similarities to humans and porcine SDSCs (pSDSCs) offer an ideal in vitro model to investigate human ASCs. To date, studies concerning the role of yes-associated protein (YAP) in ASCs are limited, and the mechanism of its influence on self-renewal and differentiation of ASCs remain unclear. In this paper, we explore the link between the transcriptional regulator YAP and the fate of pSDSCs. Results We found that YAP promotes the pluripotent state of pSDSCs by maintaining the high expression of the pluripotency genes Sox2, Oct4. The overexpression of YAP prevented the differentiation of pSDSCs and the depletion of YAP by small interfering RNA (siRNAs) suppressed the self-renewal of pSDSCs. In addition, we found that YAP regulates the fate of pSDSCs through a mechanism related to the Wnt/β-catenin signaling pathway. When an activator of the Wnt/β-catenin signaling pathway, CHIR99021, was added to pSDSCs overexpressing YAP the ability of pSDSCs to differentiate was partially restored. Conversely, when XAV939 an inhibitor of Wnt/β-catenin signaling pathway, was added to YAP knockdown pSDSCs a higher self-renewal ability resulted. Conclusions our results suggested that, YAP and the Wnt/β-catenin signaling pathway interact to regulate the fate of pSDSCs.

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