Abstract:Mesenchymal stem cell (MSC) differentiation is regulated intrinsically by transcription factors and extrinsically by the extracellular matrix. We tested whether matrix metalloproteinase-2 (MMP-2) and its inhibitor TIMP-2, MEF2a and TAZ transcription factors are involved in porcine MSC differentiation towards adipocytes and osteocytes. Flow cytometry and immunofluorescence were used to investigate the expression levels of multipotent cell surface markers CD73 and CD105. Real-time PCR was performed to detect the… Show more
“…For example, NFIL3 regulated by an adipocytes-selective SE has been reported as a key pro-adipogenic TF to promote adipogenesis and related to glucocorticoid-regulated adipogenesis [ 40 , 41 ]. MEF2A targeted by an osteoblasts-selective SE, which was in consistent with previous study [ 39 ], was accompanied with expression of osteogenic markers during osteogenesis [ 28 ]. Fig.…”
Section: Resultssupporting
confidence: 90%
“…3D ). All these TFs have been reported to be involved in imbalance of hMSCs lineages commitment or the resulting phenotypes/diseases [ 28 , 38 , 39 ]. For example, NFIL3 regulated by an adipocytes-selective SE has been reported as a key pro-adipogenic TF to promote adipogenesis and related to glucocorticoid-regulated adipogenesis [ 40 , 41 ].…”
Human mesenchymal stem cells (hMSCs) can be differentiated into osteoblasts and adipocytes. During these processes, super enhancers (SEs) play important roles. Here, we performed comprehensive characterization of the SEs changes associated with adipogenic and osteogenic differentiation of hMSCs, and revealed that SEs changed more dramatically compared with typical enhancers. We identified a set of lineage-selective SEs, whose target genes were enriched with cell type-specific functions. Functional experiments in lineage-selective SEs demonstrated their specific roles in directed differentiation of hMSCs. We also found that some key transcription factors regulated by lineage-selective SEs could form core regulatory circuitry (CRC) to regulate each other’s expression and control the hMSCs fate determination. In addition, we found that GWAS SNPs of osteoporosis and obesity were significantly enriched in osteoblasts-selective SEs or adipocytes-selective SEs, respectively. Taken together, our studies unveiled important roles of lineage-selective SEs in hMSCs differentiation into osteoblasts and adipocytes.
“…For example, NFIL3 regulated by an adipocytes-selective SE has been reported as a key pro-adipogenic TF to promote adipogenesis and related to glucocorticoid-regulated adipogenesis [ 40 , 41 ]. MEF2A targeted by an osteoblasts-selective SE, which was in consistent with previous study [ 39 ], was accompanied with expression of osteogenic markers during osteogenesis [ 28 ]. Fig.…”
Section: Resultssupporting
confidence: 90%
“…3D ). All these TFs have been reported to be involved in imbalance of hMSCs lineages commitment or the resulting phenotypes/diseases [ 28 , 38 , 39 ]. For example, NFIL3 regulated by an adipocytes-selective SE has been reported as a key pro-adipogenic TF to promote adipogenesis and related to glucocorticoid-regulated adipogenesis [ 40 , 41 ].…”
Human mesenchymal stem cells (hMSCs) can be differentiated into osteoblasts and adipocytes. During these processes, super enhancers (SEs) play important roles. Here, we performed comprehensive characterization of the SEs changes associated with adipogenic and osteogenic differentiation of hMSCs, and revealed that SEs changed more dramatically compared with typical enhancers. We identified a set of lineage-selective SEs, whose target genes were enriched with cell type-specific functions. Functional experiments in lineage-selective SEs demonstrated their specific roles in directed differentiation of hMSCs. We also found that some key transcription factors regulated by lineage-selective SEs could form core regulatory circuitry (CRC) to regulate each other’s expression and control the hMSCs fate determination. In addition, we found that GWAS SNPs of osteoporosis and obesity were significantly enriched in osteoblasts-selective SEs or adipocytes-selective SEs, respectively. Taken together, our studies unveiled important roles of lineage-selective SEs in hMSCs differentiation into osteoblasts and adipocytes.
“…The analysis was performed on isolated MSCs and TSA-modulated and unmodulated MSCs not subjected to freezing/thawing procedure. Moreover, we proved the mesenchymal origin of the porcine bone-marrow derived cells by performing their differentiation into osteoblasts and adipocytes [ 31 ].…”
Cryopreservation is an important procedure in maintenance and clinical applications of mesenchymal stem/stromal cells (MSCs). Although the methods of cell freezing using various cryoprotectants are well developed and allow preserving structurally intact living cells, the freezing process can be considered as a severe cellular stress associated with ice formation, osmotic damage, cryoprotectants migration/cytotoxicity or rapid cell shrinkage. The cellular response to freezing stress is aimed at the restoring of homeostasis and repair of cell damage and is crucial for cell viability. In this study we evaluated the changes arising in the pig mesenchymal stromal cell transcriptome following cryopreservation and showed the vast alterations in cell transcriptional activity (5,575 genes with altered expression) suggesting the engagement in post-thawing cell recovery of processes connected with cell membrane tension regulation, membrane damage repair, cell shape maintenance, mitochondria-connected energy homeostasis and apoptosis mediation. We also evaluated the effect of known gene expression stimulator—Trichostain A (TSA) on the frozen/thawed cells transcriptome and showed that TSA is able to counteract to a certain extent transcriptome alterations, however, its specificity and advantages for cell recovery after cryopreservation require further studies.
“…To confirm the mesenchymal stemness origin of isolated cells, Western blotting-based and flow cytometry-based approaches to the detection (immunophenotyping) of positive and/or negative expression profiles of specific surface cluster of differentiation (CD) antigens were accomplished as described by Gurgul et al (2017) and Gurgul, Romanek, Pawlina-Tyszko, Szmatoła, and Opiela (2018), respectively. Moreover, for that reason, the procedures indispensable for extracorporeal differentiation of established BM-MSC lines into adipocytes and osteoblasts/osteocytes were performed as previously described by Opiela et al (2016).…”
Section: Isolation and In Vitro Culture Of Porcine Bm-mscsmentioning
The present study sought to examine whether trichostatin A (TSA)‐assisted epigenetic transformation of porcine bone marrow (BM)‐derived mesenchymal stem cells (BM‐MSCs) affects the transcriptional activities of pluripotency‐related genes (Oct4, Nanog, c‐Myc, Sox2 and Rex1), multipotent stemness‐related gene (Nestin) and anti‐apoptotic/anti‐senescence‐related gene (Survivin). Epigenetically transformed or non‐transformed BM‐MSCs that had been transcriptionally profiled by qRT‐PCR and had been analysed for different stages of apoptosis progression provided a source of nuclear donor cells for the in vitro production of cloned pig embryos. TSA‐mediated epigenomic modulation has been found to enhance the multipotency extent, stemness and intracellular anti‐ageing properties of porcine BM‐MSCs. This has been confirmed by the relative abundances for Nanog, c‐Myc Rex1, Sox2 and Survivin mRNAs in TSA‐exposed BM‐MSCs that turned out to be significantly higher than those of TSA‐unexposed BM‐MSCs. Additionally, TSA‐assisted epigenomic modulation of BM‐MSCs did not impact the caspase‐8 activity, Bax protein expression and the incidence of TUNEL‐positive cells. In conclusion, the considerably elevated quantitative profiles of Sox2, Rex1, c‐Myc, Nanog and Survivin mRNA transcripts seem to trigger improved reprogrammability of TSA‐treated BM‐MSC nuclei in cloned pig embryos that thereby displayed remarkably increased blastocyst formation rates as compared to those noticed for embryos derived from TSA‐untreated BM‐MSCs.
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