Chromatin accessibility identifies diversity in mesenchymal stem cells from different tissue origins.
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ABSTRACT:
SUBMITTER: Ho YT
PROVIDER: S-EPMC6288149 | biostudies-literature | 2018 Dec
REPOSITORIES: biostudies-literature
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Project description:IntroductionStem cell therapy with mesenchymal stem cells (MSCs) has been widely used in many clinical trials, and therapy with MSC sheets shows promise for patients. However, there are few reports characterizing MSC sheets. In the present study, the properties of MSC sheets derived from bone marrow, adipose tissue, and umbilical cord were evaluated.MethodsCell sheets were fabricated with MSCs from different tissue origins in temperature-responsive cell culture dishes with and without pre-coating of fetal bovine serum (FBS). MSC adhesion behavior in the culture dish was observed. Secretion of cytokines related to cell proliferation and immune regulation from MSC sheets was investigated by ELISA. The adhesion properties of the MSC sheets were investigated by time-lapse microscopy.ResultsDifferent cell adhesion and proliferation rates in temperature-responsive cell culture dishes were observed among the three types of MSCs. FBS pre-coating of the dishes enhanced cell attachment and proliferation in all cell types. Harvested cell sheets showed high attachment capacity to tissue culture polystyrene dish surfaces.ConclusionsMSC sheets can be fabricated from MSCs from different tissue origins using temperature-responsive cell culture dishes. The fabricated MSC sheets could be useful in cell transplantation therapies by choosing appropriate types of MSCs that secrete therapeutic cytokines for the targeted diseases.
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Project description:Murine regulatory T (Treg) cells in tissues promote tissue homeostasis and regeneration. We sought to identify features that characterize human Treg cells with these functions in healthy tissues. Single-cell chromatin accessibility profiles of murine and human tissue Treg cells defined a conserved, microbiota-independent tissue-repair Treg signature with a prevailing footprint of the transcription factor BATF. This signature, combined with gene expression profiling and TCR fate mapping, identified a population of tissue-like Treg cells in human peripheral blood that expressed BATF, chemokine receptor CCR8 and HLA-DR. Human BATF+CCR8+ Treg cells from normal skin and adipose tissue shared features with nonlymphoid T follicular helper-like (Tfh-like) cells, and induction of a Tfh-like differentiation program in naive human Treg cells partially recapitulated tissue Treg regenerative characteristics, including wound healing potential. Human BATF+CCR8+ Treg cells from healthy tissue share features with tumor-resident Treg cells, highlighting the importance of understanding the context-specific functions of these cells.
| S-EPMC8050210 | biostudies-literature
Project description:Mesenchymal stem cells (MSCs) have been widely used in regenerative medicine and cellular therapy due to their multi-lineage differentiation potential and immunomodulatory function. The applicability of MSCs also depends on their cellular sources and in vivo functions. Here in this study, we systematically compared the morphologic characteristics, immunophenotypes and the adipogenic differentiation of MSCs derived from umbilical cord (UC), adipose tissue (Ad) and bone marrow (BM). We found that the three tissues-derived MSCs displayed decreased adipogenic capacity in the order: Ad-MSC > BM-MSC > UC-MSC, and no morphologic and immunophenotypic differences were observed. Mechanistic investigation revealed a miR-301b~miR-130b-PPARγ axis, whose expression pattern in UC-MSC, Ad-MSC and BM-MSC significantly correlates with their adipogenic capacity. Our results come up with a potential mechanism to elucidate the differential adipogenesis of Ad-MSC, BM-MSC and UC-MSC, which would provide instructional advice for which source of MSCs to choose according to a certain clinical purpose. Furthermore, the miR-301b~miR-130b-PPARγ axis may also be used as a potential therapeutic target for the disorders associated with MSCs-mediated abnormal adipogenesis.
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Project description:Although differential expression of genes is apparent during the adipogenic/osteogenic differentiation of marrow mesenchymal stem cells (MSCs), it is not known whether this is associated with changes in chromosomal structure. In this study, we used ATAC-sequencing technology to observe variations in chromatin assembly during the early stages of MSC differentiation. This showed significant changes in the number and distribution of chromosome accessibility at different time points of adipogenic/osteogenic differentiation. Sequencing of differential peaks indicated alterations in transcription factor motifs involved in MSC differentiation. Gene Ontology (GO) and pathway analysis indicated that changes in biological function resulted from the alterations in chromatin accessibility. We then integrated ATAC-seq and RNA-seq and found that only a small proportion of the overlapping genes were screened out from ATAC-seq and RNA-seq overlapping. Through GO and pathway analysis of these overlapped genes, we not only observed some known biological functions related to adipogenic/osteogenic differentiation but also noticed some unusual biological clustering during MSC differentiation. In summary, our work not only presents the landscape of chromatin accessibility of MSC during differentiation but also helps to further our understanding of the underlying mechanisms of gene expression in these processes.
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Project description:It has been previously demonstrated that uniaxial cyclic stretching (UCS) induces differentiation of mesenchymal stem cells (MSCs) into osteoblasts in vitro. It is also known that interactions between cells and external forces occur at various aspects including cell-matrix, cytoskeleton, nucleus membrane, and chromatin. However, changes in chromatin landscape during this process are still not clear. The present study was aimed to determine changes of chromatin accessibility under cyclic stretch. The influence of cyclic stretching on the morphology, proliferation, and differentiation of hMSCs was characterized. Changes of open chromatin sites were determined by assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq). Our results showed that UCS induced cell reorientation and actin stress fibers realignment, and in turn caused nuclear reorientation and deformation. Compared with unstrained group, the expression of osteogenic and chondrogenic marker genes were the highest in group of 1 Hz + 8% strain; this condition also led to lower cell proliferation rate. Furthermore, there were 2022 gene loci with upregulated chromatin accessibility in 1 Hz + 8% groups based on the analysis of chromatin accessibility. These genes are associated with regulation of cell morphogenesis, cell-substrate adhesion, and ossification. Signaling pathways involved in osteogenic differentiation were found in up-regulated GO biological processes. These findings demonstrated that UCS increased the openness of gene loci associated with regulation of cell morphogenesis and osteogenesis as well as the corresponding transcription activities. Moreover, the findings also connect the changes in chromatin accessibility with cell reorientation, nuclear reorientation, and deformation. Our study may provide reference for directed differentiation of stem cells induced by mechanical microenvironments.
| S-EPMC8294092 | biostudies-literature
Project description:BackgroundEver since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferrable regarding the differentiation of osteoclasts.MethodsIn this study we compare the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. Results were validated using qRT-PCR throughout the differentiation stages.ResultsEmbryoid-body based differentiation yielded CD45+, CD14+, CD11b+ subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34+ and did not subsequently differentiate into osteoclasts.ConclusionsThe outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines.
| S-EPMC10350192 | biostudies-literature
Project description:BackgroundEver since their discovery, induced pluripotent stem cells (iPSCs) have been extensively differentiated into a large variety of cell types. However, a limited amount of work has been dedicated to differentiating iPSCs into osteoclasts. While several differentiation protocols have been published, it remains unclear which protocols or differentiation methods are preferable regarding the differentiation of osteoclasts.MethodsIn this study, we compared the osteoclastogenesis capacity of a peripheral blood mononuclear cell (PBMC)-derived iPSC line to a fibroblast-derived iPSC line in conjunction with either embryoid body-based or monolayer-based differentiation strategies. Both cell lines and differentiation protocols were investigated regarding their ability to generate osteoclasts and their inherent robustness and ease of use. The ability of both cell lines to remain undifferentiated while propagating using a feeder-free system was assessed using alkaline phosphatase staining. This was followed by evaluating mesodermal differentiation and the characterization of hematopoietic progenitor cells using flow cytometry. Finally, osteoclast yield and functionality based on resorptive activity, Cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression were assessed. The results were validated using qRT-PCR throughout the differentiation stages.ResultsEmbryoid body-based differentiation yielded CD45+, CD14+, CD11b+ subpopulations which in turn differentiated into osteoclasts which demonstrated TRAP positivity, Cathepsin K expression and mineral resorptive capabilities. This was regardless of which iPSC line was used. Monolayer-based differentiation yielded lower quantities of hematopoietic cells that were mostly CD34+ and did not subsequently differentiate into osteoclasts.ConclusionsThe outcome of this study demonstrates the successful differentiation of osteoclasts from iPSCs in conjunction with the embryoid-based differentiation method, while the monolayer-based method did not yield osteoclasts. No differences were observed regarding osteoclast differentiation between the PBMC and fibroblast-derived iPSC lines.
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Project description:Satellite cells represent the main myogenic population accounting for skeletal muscle homeostasis and regeneration. While our knowledge of the signaling pathways controlling satellite cell regenerative capability is increasing, the underlying epigenetic mechanisms are still not clear, especially in the case of human satellite cells. Here, by performing chromatin accessibility profiling (ATAC-seq) in samples isolated from human and murine muscles, we investigated the changes in the epigenetic landscape occurring during the transition from activated satellite cells to myoblasts. Our analysis identifies a compendium of putative regulatory elements defining human activated satellite cells and myoblasts, respectively. A subset of these differentially accessible loci is shared by both murine and human satellite cells, includes elements associated with known self-renewal regulators, and is enriched for motifs bound by transcription factors participating in satellite cell regulation. Integration of transcriptional and epigenetic data reveals that known regulators of metabolic gene expression, such as PPARGC1A, represent potential PAX7 targets. Through characterization of genomic networks and the underlying effectors, our data represent an important starting point for decoding and manipulating the molecular mechanisms underlying human satellite cell muscle regenerative potential.
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Project description:Epithelial-to-mesenchymal transition (EMT) is physiological in embryogenesis and wound healing but also associated with the formation of cancer stem cells (CSCs). Many EMT signaling pathways are implicated in CSC formation, but the precise underlying mechanisms of CSC formation remain elusive. We have previously demonstrated that PKC is critical for EMT induction and CSC formation in inducible breast EMT/CSC models. Here, we used formaldehyde-assisted isolation of regulatory elements-sequencing (FAIRE-seq) to investigate DNA accessibility changes after PKC activation and determine how they influence EMT and CSC formation. During EMT, DNA accessibility principally increased in regions distant from transcription start sites, low in CpG content, and enriched with chromatin enhancer marks. ChIP-sequencing revealed that a subset of these regions changed from poised to active enhancers upon stimulation, with some even more acteylated in CSCs. While regions with increased accessibility were enriched for FOX, AP-1, TEAD, and TFAP2 motifs, those containing FOX and AP-1 motif were associated with increased expression of CSC-associated genes, while those with TFAP2 were associated with genes with increased expression in non-CSCs. Silencing of 2 members of the FOX family, FOXN2 and FOXQ1, repressed CSCs and the mesenchymal phenotype and inhibited the CSC gene signature. These novel, PKC-induced DNA accessibility regions help explain how the epigenomic plasticity of cells undergoing EMT leads to CSC gene activation.
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Project description:Hematopoietic stem cells (HSCs) exhibit considerable cell-intrinsic changes with age. Here, we present an integrated analysis of transcriptome and chromatin accessibility of aged HSCs and downstream progenitors. Alterations in chromatin accessibility preferentially take place in HSCs with aging, which gradually resolve with differentiation. Differentially open accessible regions (open DARs) in aged HSCs are enriched for enhancers and show enrichment of binding motifs of the STAT, ATF, and CNC family transcription factors that are activated in response to external stresses. Genes linked to open DARs show significantly higher levels of basal expression and their expression reaches significantly higher peaks after cytokine stimulation in aged HSCs than in young HSCs, suggesting that open DARs contribute to augmented transcriptional responses under stress conditions. However, a short-term stress challenge that mimics infection is not sufficient to induce persistent chromatin accessibility changes in young HSCs. These results indicate that the ongoing and/or history of exposure to external stresses may be epigenetically inscribed in HSCs to augment their responses to external stimuli.
| S-EPMC9110722 | biostudies-literature