Gene expression analysis of basal cells expanded with UK-5099 or dual SMAD inhibitors
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PROVIDER: PRJNA1003919 | ENA |
REPOSITORIES: ENA
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Project description:Dual SMAD inhibitors (TGF-beta inhibitor A-8301 and BMP inhibitor DMH-1) were previously reported to promote long-term expansion of basal cells. Since UK-5099 showed a comparable effect to dual SMAD inhibitors in promoting basal cells expansion, we performed RNA-seq to determine if cells expanded by these two methods exhibit a similar transcriptional profile. The Pierson correlation test indicated that cells expanded by UK5099 displayed a transcriptional profile similar to those expanded with dual-SMAD inhibitors. The expression levels of canonical basal cell marker genes, such as Krt5 and p63, were comparable in cells expanded using both methods, as well as genes related to the TCA cycle and glycolysis. Interestingly, despite the similarity in the expression of canonical basal cell markers, cells expanded via UK-5099 exhibited higher activity in differentiation-related signaling pathways, such as the Hippo and TGF-β signaling pathways.This observation suggests that mitochondrial pyruvate transport is a critical metabolic checkpoint for basal cell differentiation.
2025-01-08 | GSE240499 | GEO
Project description:SUMMARY of the associated publication: Many adult epithelial stem cells cannot undergo prolonged expansion. This may be due to a cell autonomous limitation or a lack of necessary culture conditions. Thus far, the long-term culture of adult stem cells has required the use of Matrigel organoid culture or feeder co-culture systems. We demonstrate that the TGFbetaBMP/SMAD signaling pathway is highly active in the luminal cells of diverse p63+ basal cell-containing epithelia. Indeed, the activation of SMAD signaling in airway stem cells causes their differentiation, while conversely, inhibition of SMAD signaling leads to stem cell hyperplasia and compromised differentiation. Consistently, TGFbetaBMP/SMAD signaling activation prevents mature cell dedifferentiation. Using pharmacologic dual SMAD inhibition, we developed a feeder-free culture system that allows the cloning of single airway basal stem cells and the expansion of airway stem cells from clinical non-invasively obtained samples with high efficiency. Furthermore, the expansion protocols are applicable not only to human cells but also to those of mouse, ferret, and pig. When differentiated epithelia are produced from expanded cells, they exhibit proper epithelial physiology, and respond to clinically relevant pharmacologic agents. Most remarkably, a very wide variety of functional adult epithelial basal stem cells can be expanded despite their differing origins. The goal of this microarray analysis: It has been shown that inhibition of TGFbeta signaling leads to tumorigenesis (e.g. Guasch et al., 2007, and others). It will be important to show whether the dual inhibited population assumes a tumorigenic status in the same or different manner compared to directly isolated progenitor cells. To address this concern, we have done comprehensive transcriptome analysis comparing gene expression signature of airway stem cells at their early and late passages. We do not find tumor-associated gene signatures during stem cell expansion.
2016-04-19 | GSE80408 | GEO
Project description:Discovery of therapies that are able to correct defective CFTR requires patient derived in vitro pre-clinical cell models to be developed. Two main approaches exist to expand bronchial and nasal cultures, such as conditionally reprogrammed cells (CRC) and feeder free dual SMAD inhibition (SMADi) to overcome senescence, low cell count, and increase passages. To mimic airway epithelium the expanded cells are differentiated at air liquid Interface (ALI). This project focused to compare the global proteome of ALI differentiated CRC and SMADi expanded HNECs both treated and untreated with CFTR corrector VX-809.
2021-12-05 | PXD018386 | Pride
Project description:Underdeveloped lungs are a primary cause of morbidity and mortality in premature infants, but our ability to help these patients by speeding up lung development are hindered by a lack of understanding of human lung developmental biology. Here, we performed single cell RNA sequencing of the human fetal lung from samples spanning from 11.5 weeks gestation to 21 weeks gestation from the distal lung, middle airways, and the tracheal epithelium. The primary goal of this experiment was to define fetal cell states to serve as a gold standard for pluripotent stem cell-derived lung cells and tissues, and to identify potential signaling pathways that drive differentiation of lung progenitor cells to mature cell types. Additionally, we generated bud tip progenitor organoids from 12 week human fetal lung bud tip progenitors. We show that treatment of bud tip progenitor organoids with a short pulse of dual SMAD activation (BMP4+TGFb1) led to the upregulation of lung basal cell markers, a cell type that serves as a critical stem cell for the adult airway, and that further treatment with dual SMAD inhibition leads to the generation of airway-like organoids containing differentiated cell types of the adult airway, including basal stem cells.
2020-04-17 | E-MTAB-8221 | biostudies-arrayexpress
Project description:COVID-19 Genomics UK (COG-UK) consortium
| PRJEB37886 | ENA
Project description:Gene-specific transcription factors (GSTFs) control of gene transcription by DNA binding and specific protein complex recruitment, which regulates promoter accessibility for transcription initiation by RNA polymerase II. GSTFs that are frequently mutated in colon and rectal carcinomas are Suppressor of Mothers Against Decapentaplegic 2 (SMAD2) and SMAD4, which play an important role in the TGF-β signaling pathways controlling cell fate and proliferation (ref.). The SMAD protein family is a diverse and it can be divided into three subclasses: receptor activated SMADs, inhibitory SMADs and the common SMAD4 co-activator. To study protein interactors of the SMAD protein family we generated a quantitative proteomics pipeline that allows for inducible expression of GFP-tagged SMAD proteins followed by affinity purification and MS analysis. The nuclear importin IPO5 was identified as a novel interacting protein of SMAD1. Overexpression of IPO5 shows forced BMP R-SMAD nuclear localization confirming a functional relationship between BMP but not TGF-β R-SMADs and IPO5. Finally we provide evidence that the length of the lysine stretch in the NLS is involved in importin selection.
2016-10-10 | PXD004529 | Pride
Project description:Functional modeling of many adult epithelia is limited by the difficulty in maintaining relevant stem cell populations in culture. Here, we show that dual inhibition of SMAD signaling pathways enables robust expansion of primary epithelial basal cell populations. We find that TGFβ/BMP/SMAD pathway signaling is strongly activated in luminal and suprabasal cells of several epithelia, but suppressed in p63+ basal cells. In airway epithelium, SMAD signaling promotes differentiation, and its inhibition leads to stem cell hyperplasia. Using dual SMAD signaling inhibition in a feeder-free culture system, we have been able to expand airway basal stem cells from multiple species. Expanded cells can produce functional airway epithelium physiologically responsive to clinically relevant drugs, such as CFTR modulators. This approach is effective for the clonal expansion of single human cells and for basal cell populations from epithelial tissues from all three germ layers and therefore may be broadly applicable for modeling of epithelia.
| S-EPMC4975684 | biostudies-literature