ScRNAsequencing of in vitro expanded limbal stem cells of aniridia donors
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ABSTRACT:
PROVIDER: EGAS00001007397 | EGA |
REPOSITORIES: EGA
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Project description:Adipose-derived stem cells (ASCs) and their small extracellular vesicles (sEVs) hold significant potential for regenerative medicine due to their capabilities for tissue repair. However, the effects of ASC heterogeneity on sEV characteristics and microRNA (miRNA) profiles remain unclear. We investigated ASCs heterogeneity and its changes with aging and cell expansion using bulk RNA-seq from young and old donors across various passages. Non-negative matrix factorization (NMF) was used to identify distinct ASC clusters. Small RNA sequencing was performed on sEVs collected from ASC culture supernatants to analyze miRNA profiles. NMF analysis revealed ASC heterogeneity, identifying clusters characterized by ISLR and genes associated with collagen and smooth muscle cells. The miRNA profiles of sEVs matched these ASC clusters, showing variations based on donor age and cell expansion. Functional enrichment analysis suggested that these miRNAs may have distinct functional roles. Our findings show that ASC heterogeneity and sEV miRNA content are significantly influenced by donor age and cell expansion. These variations underscore the necessity of selecting specific ASC-derived sEVs.
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Project description:Adipose-derived stem cells (ASCs) and their small extracellular vesicles (sEVs) hold significant potential for regenerative medicine due to their capabilities for tissue repair. However, the effects of ASC heterogeneity on sEV characteristics and microRNA (miRNA) profiles remain unclear. We investigated ASCs heterogeneity and its changes with aging and cell expansion using bulk RNA-seq from young and old donors across various passages. Non-negative matrix factorization (NMF) was used to identify distinct ASC clusters. Small RNA sequencing was performed on sEVs collected from ASC culture supernatants to analyze miRNA profiles. NMF analysis revealed ASC heterogeneity, identifying clusters characterized by ISLR and genes associated with collagen and smooth muscle cells. The miRNA profiles of sEVs matched these ASC clusters, showing variations based on donor age and cell expansion. Functional enrichment analysis suggested that these miRNAs may have distinct functional roles. Our findings show that ASC heterogeneity and sEV miRNA content are significantly influenced by donor age and cell expansion. These variations underscore the necessity of selecting specific ASC-derived sEVs.
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Project description:We would like to check differences among stem cells derived from various donors. The purpose of this project is to investigate donor dependency for stem cell implantation.
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Project description:One of the promising approaches to slow down or treat neurodegenerative diseases or spinal cord injuries represent cell therapies. The grafted cells could either integrate into the damaged tissue to replace dead or damaged cells or by secretion of different factors modulate inflammatory reaction, reduce tissue damage and support neuronal survival. However, due to the heterogeneity of in vitro cultured cells, comprehensive characterization of such cells is absolutely crucial to prevent safety risks.
Here, we performed SWATH-MS analysis to characterize changes in proteome of human neural stem cells (NSCs) during their differentiation either spontaneously by withdrawal of EGF and FGF2 in cell culture media or by trophic support of BDNF/GDNF growth factors. We quantified about 2800 proteins over the 28 days of differentiation and showed that changes in cellular proteomes are caused mostly by differentiation time course, rather than type of differentiation itself and that the major changes in protein expression occurred between day 0 and day 7 of both differentiations.
2021-01-07 | PXD021860 | panorama
Project description:Lung Cancer (LC), colorectal cancer (CRC) and breast cancer (BC) are the major killers in oncology, accounting for about 40% of cancer deaths. Although progresses have been made in the last few years, unfortunately no patient with metastatic disease can obtain a definitive cure.
A recent hypothesis is that cancer is driven by a small subpopulation of cells called "cancer stem cells" (CSCs) or "tumor initiating cells" with an unlimited proliferative potential and the ability to reproduce the original human tumor in experimental animal models. These cells are thought to be responsible for the development of the tumor and represent the only cell population able to sustain tumor growth and progression.
Therefore, CSCs represent the elective target for new targeted therapies, endowed with high and selective toxicity towards the tumor but harmless towards normal cells.
Current technologies allow us to isolate and expand in vitro the CSCs from tumor specimens, testing their sensitivity to different anticancer drugs in a short period of time.
Therefore, there is the potential opportunity to identify LC, CRC and BC CSCs.This is a prospective study assessing feasibility of CSCS isolation in LC, CRC and BC.
Patients with a previously performed diagnosis of LC, colon cancer or breast cancer with no further standard therapy options, with a Karnofsky performance status of 100% and with tumor tissue available will be considered eligible for the study. Tumor tissue will be collected before study entry, i.e tissue obtained during a diagnostic or therapeutical procedure, like surgery or biopsies with other purposes than the protocol. In vitro tumor sensitivity to chemotherapy drugs will be tested on tumor cell cultures per each patient.
Drugs and their combination will be considered effective and if they kill ≥ 60% of tumor stem cells in vitro test. By using cancer spheres the investigators will also generate orthotopic xenograft models that recapitulate the parental tumor behaviour, including the aggressive features and the invasiveness potential. Orthotopic injection technique will be assessed in 5 weeks-old NOD/SCID mice
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Project description:Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. See the Data Processing section of the published paper concerning the bioinformatics pipeline used. PMCID: PMC3261715 PMID: 22108792 Mol Syst Biol. 2011 Nov 22;7:550. doi: 10.1038/msb.2011.84. The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells. Munoz J, Low TY, Kok YJ, Chin A, Frese CK, Ding V, Choo A, Heck AJ. Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
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Project description:Human embryonic stem cells (hESC) display substantial heterogeneity in gene expression, implying the existence of discrete substates within the stem cell compartment. To determine whether these substates impact fate decisions of hESC we used a GFP reporter line to investigate the properties of fractions of putative undifferentiated cells defined by their differential expression of the endoderm transcription factor, GATA6, together with the hESC surface marker, SSEA3. By single cell cloning, we confirmed that substates characterized by expression of GATA6 and SSEA3 include pluripotent stem cells capable of long term self-renewal. When clonal stem cell colonies were formed from GATA6-positive and GATA6-negative cells, more of those derived from GATA6-positive cells contained spontaneously differentiated endoderm cells than similar colonies derived from the GATA6-negative cells. We characterized these discrete cellular states using single cell transcriptomic analysis, identifying a potential role for SOX17 in the establishment of the endoderm biased stem cell state.
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Project description:The heterogeneous nature of glioblastoma stem-like cells (GSCs) creates hurdles in developing effective therapies against glioblastoma, making it extremely difficult to eradicate. In this part of the project, we investigated the potential role of GSCs-derived small extracellular vesicles (sEVs) in glioblastoma heterogeneity, plasticity, and aggressiveness with the particular focus on their complexity in term of proteins. Proteome profiling of sEVs and their respective cell lines showed that GSCs-derived sEVs retain their subtype characteristics and are enriched in proteins playing a role in amino acids, carboxylic acids, organic acids transmembrane transport, and growth factor binding. In summary, we revealed the protein content of GSCs-derived sEVs and unveiled their potential contribution to tumor heterogeneity and critical cellular processes commonly deregulated in glioblastoma.
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Project description:Patients with acute myeloid leukemia (AML) suffer dismal prognosis and the most adverse subpopulation within each tumor determines patient’s prognosis. To better understand challenging features in AML, we studied individual stem cells from a single AML sample, complementing genomic with in vivo functional studies. Primary tumor cells from an AML patient’s first and second relapse were transplanted into NSG mice to establish serially transplantable patient derived xenografts (PDX). In an innovative approach, twelve derivative PDX clones were generated thereof, each derived from a single AML stem cell as proven by molecular barcoding, and were color-marked to facilitate multiplex competitive in vivo assays. PDX clones consisted of four different genomic clusters; one cluster displayed resistance against Cytarabine treatment, while two other clusters harbored increased stem cell potential, indicating that stemness and treatment resistance had evolved independently in the sample. In vivo functional data correlated closely with the phylogenetic tree calculated from exome data. The Cytarabine-resistant cluster was characterized by a distinct gene expression profile, and a score thereof predicted outcome in large clinical patient data cohorts. Taken together, we provide proof of concept that intra-sample heterogeneity mimics inter-sample heterogeneity in AML. Stem cell disparities within a single sample allow insights into adverse characteristics of general importance for AML.
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