Fibroblasts direct differentiation of human breast epithelial progenitors
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ABSTRACT:
PROVIDER: PRJNA643540 | ENA |
REPOSITORIES: ENA
Dataset's files
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| SRR12128883.fastq.gz | Fastqsanger.gz | |||
| SRR12128884.fastq.gz | Fastqsanger.gz | |||
| SRR12128885.fastq.gz | Fastqsanger.gz | |||
| SRR12128886.fastq.gz | Fastqsanger.gz | |||
| SRR12128887.fastq.gz | Fastqsanger.gz |
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Project description:Background: Breast cancer arises within specific regions in the human breast referred to as the terminal duct lobular units (TDLUs). These are relatively dynamic structures characterized by sex hormone driven cyclic epithelial turnover. TDLUs consist of unique parenchymal entities embedded within a fibroblast-rich lobular stroma. Here, we established and characterized a new human breast lobular fibroblast cell line against its interlobular counterpart with a view to assessing the role of region-specific stromal cues in the control of TDLU dynamics. Methods: Primary lobular and interlobular fibroblasts were transduced to express human telomerase reverse transcriptase (hTERT). Differentiation of the established cell lines along lobular and interlobular pathways was determined by immunocytochemical staining and genome-wide RNA sequencing. Their functional properties were further characterized by analysis of mesenchymal stem cell (MSC) differentiation repertoire in culture and in vivo. The cells’ physiological relevance for parenchymal differentiation was examined in heterotypic co-culture with fluorescence-activated cell sorting (FACS)-purified normal breast primary luminal or myoepithelial progenitors. The co-cultures were immunostained for quantitative assessment of epithelial branching morphogenesis, polarization, growth, and luminal epithelial maturation. In extension, myoepithelial progenitors were tested for luminal differentiation capacity in culture and in mouse xenografts. To unravel the significance of transforming growth factor-beta (TGF-β)-mediated crosstalk in TDLU-like morphogenesis and differentiation, fibroblasts were incubated with the TGF-β signaling inhibitor, SB431542, prior to heterotypic co-culture with luminal cells. Results: hTERT immortalized fibroblast cell lines retained critical phenotypic traits in culture and linked to primary fibroblasts. Cell culture assays and transplantation to mice showed that the origin of fibroblasts determines TDLU-like and ductal-like differentiation of epithelial progenitors. Whereas lobular fibroblasts supported a high level of branching morphogenesis by luminal cells, interlobular fibroblasts supported ductal-like myoepithelial characteristics. TDLU-like morphogenesis, at least in part, relied on intact TGF-β signaling. Conclusions: The significance of the most prominent cell type in normal breast stroma, the fibroblast, in directing epithelial differentiation is largely unknown. Through establishment of lobular and interlobular fibroblast cell lines, we here demonstrate that epithelial progenitors are submitted to stromal cues for site-specific differentiation. Our findings lend credence to considering stromal subtleties of crucial importance in the development of normal breast and, in turn, breast cancer.
2020-09-30 | GSE153646 | GEO
Project description:BACKGROUND:Breast cancer arises within specific regions in the human breast referred to as the terminal duct lobular units (TDLUs). These are relatively dynamic structures characterized by sex hormone driven cyclic epithelial turnover. TDLUs consist of unique parenchymal entities embedded within a fibroblast-rich lobular stroma. Here, we established and characterized a new human breast lobular fibroblast cell line against its interlobular counterpart with a view to assessing the role of region-specific stromal cues in the control of TDLU dynamics. METHODS:Primary lobular and interlobular fibroblasts were transduced to express human telomerase reverse transcriptase (hTERT). Differentiation of the established cell lines along lobular and interlobular pathways was determined by immunocytochemical staining and genome-wide RNA sequencing. Their functional properties were further characterized by analysis of mesenchymal stem cell (MSC) differentiation repertoire in culture and in vivo. The cells' physiological relevance for parenchymal differentiation was examined in heterotypic co-culture with fluorescence-activated cell sorting (FACS)-purified normal breast primary luminal or myoepithelial progenitors. The co-cultures were immunostained for quantitative assessment of epithelial branching morphogenesis, polarization, growth, and luminal epithelial maturation. In extension, myoepithelial progenitors were tested for luminal differentiation capacity in culture and in mouse xenografts. To unravel the significance of transforming growth factor-beta (TGF-?)-mediated crosstalk in TDLU-like morphogenesis and differentiation, fibroblasts were incubated with the TGF-? signaling inhibitor, SB431542, prior to heterotypic co-culture with luminal cells. RESULTS:hTERT immortalized fibroblast cell lines retained critical phenotypic traits in culture and linked to primary fibroblasts. Cell culture assays and transplantation to mice showed that the origin of fibroblasts determines TDLU-like and ductal-like differentiation of epithelial progenitors. Whereas lobular fibroblasts supported a high level of branching morphogenesis by luminal cells, interlobular fibroblasts supported ductal-like myoepithelial characteristics. TDLU-like morphogenesis, at least in part, relied on intact TGF-? signaling. CONCLUSIONS:The significance of the most prominent cell type in normal breast stroma, the fibroblast, in directing epithelial differentiation is largely unknown. Through establishment of lobular and interlobular fibroblast cell lines, we here demonstrate that epithelial progenitors are submitted to stromal cues for site-specific differentiation. Our findings lend credence to considering stromal subtleties of crucial importance in the development of normal breast and, in turn, breast cancer.
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Project description:Neuronal restricted progenitors (NRPs) represent a type of transitional intermediate cells that lie between multipotent neural progenitors (NPs) and terminal differentiated neurons during neurogenesis. These NRPs have the ability to self-renew and differentiate into neurons, but not into glial cells, which is considered as an advantage for cellular therapy of human neurodegenerative diseases. However, difficulty in the extraction of highly purified NPRs from normal nervous tissue prevents further studies and applications. In this study, we reported conversion of human fetal dermal fibroblasts into human induced neuronal restricted progenitors (hiNRPs) in seven days by using just three defined factors: Sox2, c-Myc, and either Brn2 or Brn4. The hiNRPs exhibited distinct neuronal characteristics, including cell morphology, multiple neuronal markers expression, self-renewal capacity, and genome-wide transcriptional profile. Moreover, hiNRPs were able to differentiate into various terminal neurons with functional membrane properties, but not glial cells. Direct generation of hiNRPs from somatic cells will provide a new source of cells for cellular replacement therapy of human neurodegenerative diseases. This is a general expression microarray design (NimbleGen platform). It includes 5 samples.
2013-10-24 | E-GEOD-51580 | biostudies-arrayexpress
Project description:Neuronal restricted progenitors (NRPs) represent a type of transitional intermediate cells that lie between multipotent neural progenitors (NPs) and terminal differentiated neurons during neurogenesis. These NRPs have the ability to self-renew and differentiate into neurons, but not into glial cells, which is considered as an advantage for cellular therapy of human neurodegenerative diseases. However, difficulty in the extraction of highly purified NPRs from normal nervous tissue prevents further studies and applications. In this study, we reported conversion of human fetal dermal fibroblasts into human induced neuronal restricted progenitors (hiNRPs) in seven days by using just three defined factors: Sox2, c-Myc, and either Brn2 or Brn4. The hiNRPs exhibited distinct neuronal characteristics, including cell morphology, multiple neuronal markers expression, self-renewal capacity, and genome-wide transcriptional profile. Moreover, hiNRPs were able to differentiate into various terminal neurons with functional membrane properties, but not glial cells. Direct generation of hiNRPs from somatic cells will provide a new source of cells for cellular replacement therapy of human neurodegenerative diseases.
2013-10-24 | GSE51580 | GEO
Project description:Enhancement of direct reprogramming from fibroblasts to epithelial lineages by OVOL2-induced mesenchymal-to-epithelial transition
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Project description:Enhancement of direct reprogramming from fibroblasts to epithelial lineages by OVOL2-induced mesenchymal-to-epithelial transition [CAGE]
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Project description:Fibrotic interstitial lung disease (ILD) are lung disorders characterized by the accumulation of extracellular matrix, ultimately resulting in the destruction of the pulmonary scaffold. Continuous pro-fibrotic signaling perpetuates the remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Studies that address this detrimental crosstalk between lung epithelial cells and fibroblasts are key to understanding ILD. With the aim of identifying functionally relevant targets that drive mesenchymal-epithelial crosstalk and their potential as new avenues to therapeutic strategies, we developed an organoid co-culture system based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells and lung fibroblasts from ILD patients as well as IMR-90 controls. While organoid formation capacity and organoid size was comparable in the presence of ILD or control lung fibroblasts, metabolic activity was significantly increased in ILD co-cultures. Alveolar organoids cultured with ILD fibroblasts further demonstrated reduced stem cell function supported by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. In order to identify key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used secretome mass spectrometry to identify key signals secreted by end stage ILD lung fibroblasts. Over 2000 proteins were detected in a single-shot experiment with 47 differentially upregulated proteins when comparing ILD and non-chronic lung disease control fibroblasts. The secretome profile was dominated by chemokines of the C-X-C motif family, including CXCL1, -3, and -8, all interfering with (epithelial) growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating 3D monocultures of iAT2s with IL11 we recapitulated the co-culture results obtained with primary ILD fibroblasts including changes in metabolic activity as well as organoid formation capacity and size. In summary, our analysis identified mesenchyme-derived mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD by using sophisticated alveolar organoid co-cultures indicating the importance of cytokine-driven aberrant epithelial differentiation and confirmed IL11 as a key player in ILD using an unbiased approach.
2023-04-19 | PXD039554 | Pride
Project description:Abstract The interplay between histone modifications and promoter hypermethylation provides a causative explanation for epigenetic gene silencing in cancer. Less is known about the upstream initiators that direct this process. Here, we report that the Cystatin M (CST6) tumor suppressor gene is concurrently down-regulated with other loci in breast epithelial cells co-cultured with cancer-associated fibroblasts (CAFs). Promoter hypermethylation of CST6 is associated with aberrant AKT1 activation in epithelial cells, as well as the disabled INNP4B regulator resulted from the suppression by CAFs. Repressive chromatin, marked by trimethyl-H3K27 and dimethyl-H3K9, and de novo DNA methylation is established at the promoter. The findings suggest that microenvironmental stimuli are triggers in this epigenetic cascade, leading to the long-term silencing of CST6 in breast tumors. Our present findings implicate a causal mechanism defining how tumor stromal fibroblasts support neoplastic progression by manipulating the epigenome of mammary epithelial cells. The result also highlights the importance of direct cell-cell contract between epithelial cells and the surrounding fibroblasts that confer this epigenetic perturbation. Since this two-way interaction is anticipated, the described co-culture system can be used to determine the effect of epithelial factors on fibroblasts in future studies.
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2023-11-15 | GSE167422 | GEO