Long-term in vitro expansion of epithelial stem cells enabled by pharmacological inhibition of PAK1-ROCK-Myosin II and TGF-β signaling
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE103759 | GEO | 2018/10/01
REPOSITORIES: GEO
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Project description:Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here, we describe the EpiX method, which utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary, and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole-genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.
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Project description:Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here we describe the EpiX™ method that utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.
2018-10-01 | GSE103758 | GEO
Project description:Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here we describe the EpiX™ method that utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.
2018-10-01 | GSE103757 | GEO
Project description:Despite substantial self-renewal capability in vivo, epithelial stem and progenitor cells located in various tissues expand for a few passages in vitro in feeder-free condition before they succumb to growth arrest. Here we describe the EpiX™ method that utilizes small molecules that inhibit PAK1-ROCK-Myosin II and TGF-β signaling to achieve over one trillion-fold expansion of human epithelial stem and progenitor cells from skin, airway, mammary and prostate glands in the absence of feeder cells. Transcriptomic and epigenomic studies show that this condition helps epithelial cells to overcome stresses for continuous proliferation. EpiX-expanded basal epithelial cells differentiate into mature epithelial cells consistent with their tissue origins. Whole genome sequencing reveals that the cells retain remarkable genome integrity after extensive in vitro expansion without acquiring tumorigenicity. EpiX technology provides a solution to exploit the potential of tissue-resident epithelial stem and progenitor cells for regenerative medicine.
2018-10-01 | GSE103756 | GEO
Project description:Long-term in vitro expansion of epithelial stem cells enabled by pharmacological inhibition of PAK1-ROCK-Myosin II and TGF-β signaling
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Project description:Neural crest is a population of multipotent progenitor cells that form at the border of neural and non-neural ectoderm in vertebrate embryos, and undergo epithelial-mesenchymal transition and migration. According to the traditional view, the neural crest is specified in early embryos by signaling molecules including BMP, FGF, and Wnt proteins. Here, we identify a novel signaling pathway leading to neural crest specification, which involves Rho-associated kinase (ROCK) and its downstream target nonmuscle Myosin II. We show that ROCK inhibitors promote differentiation of human embryonic stem cells (hESCs) into neural crest-like progenitors (NCPs) that are characterized by specific molecular markers and ability to differentiate into multiple cell types, including neurons, chondrocytes, osteocytes, and smooth muscle cells. Moreover, inhibition of Myosin II was sufficient for generating NCPs at high efficiency. Whereas Myosin II has been previously implicated in the self-renewal and survival of hESCs, we demonstrate its role in neural crest development during ESC differentiation. Inhibition of this pathway in Xenopus embryos expanded neural crest in vivo, further indicating that neural crest specification is controlled by ROCK-dependent Myosin II activity. We propose that changes in cell morphology in response to ROCK and Myosin II inhibition initiate mechanical signaling leading to neural crest fates.
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Project description:Tissues are maintained by homeostatic feedback mechanisms in which cells can respond to, but also modify, the chemical and mechanical properties of the surrounding extracellular matrix (ECM). Mechano-sensitive mesenchymal stem cells (MSCs) resident in the marrow niche experience a diverse mechanical environment, but ageing can affect the composition and quality of bone and marrow tissues. Here we quantified the compounded effects of substrate stiffness and replication-induced senescence on MSC morphology and their ability to modify their environments through secretion of ECM proteins. The ECM proteome was found to be sensitive to substrate stiffness, but pharmacological inhibition of cellular contractility perturbed this response, decreasing levels of tenascin-C, fibulins and fibronectin. A corresponding change in the ECM of senescent cells, concomitant with a loss of mechano-responsive morphological features, suggested a decoupling of mechanotransduction pathways. Intracellular proteomic and transcriptomic analyses confirmed a decrease in all components of the cytoskeletal protein homeostasis machinery in senescent MSCs. These results demonstrate a senescence-mediated perturbation to cytoskeletal homeostasis, pathways of mechanotransduction and the secretion of ECM proteins considered necessary for tissue maintenance.
2022-10-12 | PXD013239 | Pride
Project description:Due to their stem-like characteristics and their resistance to existing chemo- and radiation therapies, there is a growing appreciation that cancer stem cells (CSCs) are the root cause behind cancer metastasis and recurrence. However, these cells represent a small subpopulation of cancer cells and are difficult to propagate in vitro. Glioblastoma is an extremely deadly form of brain cancer that is hypothesized to have a subpopulation of CSCs called glioblastoma stem cells (GSCs; also called brain tumor initiating cells, BTICs). We propose the use of selective Rho-kinase (ROCK) inhibitors, Y-27632 and fasudil, to promote GSC/BTIC-like cell survival and propagation in vitro. ROCK inhibitors have been implicated in suppressing apoptosis, and it was hypothesized that they would increase the number of GSC/BTIC-like cells grown in vitro and improve cloning efficiencies. Indeed, our data demonstrate that transient and continuous supplementation of non-toxic concentrations of Y-27632 and fasudil inhibited apoptosis, enhanced the cells' ability to form spheres, and increased stem cell marker expressing GSC/BTIC-like cell subpopulation. Our data indicated that pharmacological and genetic (siRNA) inhibitions of the ROCK pathway facilitates in vitro expansion of GSC/BTIC-like cells. Thus, ROCK pathway inhibition shows promise for future optimization of CSC culture media.
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Project description:Current approaches to cancer treatment focus on targeting signal transduction pathways. Here, we develop an alternative system for targeting cell mechanics for the discovery of novel therapeutics. We designed a live-cell, high-throughput chemical screen to identify mechanical modulators. We characterized 4-hydroxyacetophenone (4-HAP), which enhances the cortical localization of the mechanoenzyme myosin II, independent of myosin heavy-chain phosphorylation, thus increasing cellular cortical tension. To shift cell mechanics, 4-HAP requires myosin II, including its full power stroke, specifically activating human myosin IIB (MYH10) and human myosin IIC (MYH14), but not human myosin IIA (MYH9). We further demonstrated that invasive pancreatic cancer cells are more deformable than normal pancreatic ductal epithelial cells, a mechanical profile that was partially corrected with 4-HAP, which also decreased the invasion and migration of these cancer cells. Overall, 4-HAP modifies nonmuscle myosin II-based cell mechanics across phylogeny and disease states and provides proof of concept that cell mechanics offer a rich drug target space, allowing for possible corrective modulation of tumor cell behavior.
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Project description:In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.
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