Project description:Induced pluripotent stem cells (iPSCs) can be differentiated toward mesenchymal stromal cells (MSCs), but at least on epigenetic level this transition remains incomplete with the current culture conditions. Hydrogels provide a more physiologic three-dimensional environment for in vitro cell culture than conventional tissue culture plastic (TCP). In this study, we followed the hypothesis that growth and differentiation of primary MSCs and of iPSC-derived MSCs (iMSCs) can be enhanced on hydrogels. To this end, we used a hydrogel made of human platelet lysate (hPL). MSCs were effectively cultured on and inside hPL-gel and demonstrated more structured deposition of extracellular matrix (ECM) components than TCP. Furthermore, hPL-gel supported differentiation of iPSCs toward MSCs. Unexpectedly, the differentiation process seemed to be hardly affected by the substrate: iMSCs generated either on TCP or hPL-gel did not reveal differences in morphology, immunophenotype, or differentiation potential. Moreover, global gene expression and DNA-methylation profiles were almost identical in iMSCs generated on TCP or hPL-gel. Our results indicate that matrix elasticity is less crucial for directed lineage-specific differentiation toward MSCs than expected.
Project description:Induced pluripotent stem cells (iPSCs) can be differentiated toward mesenchymal stromal cells (MSCs), but at least on epigenetic level this transition remains incomplete with the current culture conditions. Hydrogels provide a more physiologic three-dimensional environment for in vitro cell culture than conventional tissue culture plastic (TCP). In this study, we followed the hypothesis that growth and differentiation of primary MSCs and of iPSC-derived MSCs (iMSCs) can be enhanced on hydrogels. To this end, we used a hydrogel made of human platelet lysate (hPL). MSCs were effectively cultured on and inside hPL-gel and demonstrated more structured deposition of extracellular matrix (ECM) components than TCP. Furthermore, hPL-gel supported differentiation of iPSCs toward MSCs. Unexpectedly, the differentiation process seemed to be hardly affected by the substrate: iMSCs generated either on TCP or hPL-gel did not reveal differences in morphology, immunophenotype, or differentiation potential. Moreover, global gene expression and DNA-methylation profiles were almost identical in iMSCs generated on TCP or hPL-gel. Our results indicate that matrix elasticity is less crucial for directed lineage-specific differentiation toward MSCs than expected.
Project description:Matrix elasticity influences differentiation of mesenchymal stem cells (MSCs) but it is unclear if these effects are only transient - while the cells reside on the substrate - or if they reflect persistent lineage commitment. In this study, MSCs were continuously culture-expanded in parallel either on polydimethylsiloxane (PDMS) gels of different elasticity or on tissue culture plastic (TCP) to compare impact on replicative senescence, in vitro differentiation, gene expression, and DNA methylation (DNAm) profiles. The maximal number of cumulative population doublings was not affected by matrix elasticity. Differentiation towards adipogenic and osteogenic lineage was increased on soft and rigid biomaterials, respectively - but this propensity was no more evident if cells were transferred to TCP. Global gene expression profiles and DNAm profiles revealed relatively few differences in MSCs cultured on soft or rigid matrices. Furthermore, only moderate DNAm changes were observed upon culture on very soft hydrogels of human platelet lysate (hPL-gel). Our results support the notion that matrix elasticity influences cellular differentiation while the cells are organized on the substrate, but it does not have major impact on cell-intrinsic lineage determination, replicative senescence or DNAm patterns. 20 samples were hybridized to the Illumina Infinium 450k Human Methylation Beadchip
Project description:Differentiation of Induced Pluripotent Stem Cells towards Mesenchymal Stromal Cells is Hampered by Culture in 3D Hydrogels [RNA-Seq]
Project description:Matrix elasticity influences differentiation of mesenchymal stem cells (MSCs) but it is unclear if these effects are only transient - while the cells reside on the substrate - or if they reflect persistent lineage commitment. In this study, MSCs were continuously culture-expanded in parallel either on polydimethylsiloxane (PDMS) gels of different elasticity or on tissue culture plastic (TCP) to compare impact on replicative senescence, in vitro differentiation, gene expression, and DNA methylation (DNAm) profiles. The maximal number of cumulative population doublings was not affected by matrix elasticity. Differentiation towards adipogenic and osteogenic lineage was increased on soft and rigid biomaterials, respectively - but this propensity was no more evident if cells were transferred to TCP. Global gene expression profiles and DNAm profiles revealed relatively few differences in MSCs cultured on soft or rigid matrices. Furthermore, only moderate DNAm changes were observed upon culture on very soft hydrogels of human platelet lysate. Our results support the notion that matrix elasticity influences cellular differentiation while the cells are organized on the substrate, but it does not have major impact on cell-intrinsic lineage determination, replicative senescence or DNAm patterns. MSCs cultivated either on polydimethylsiloxane (PDMS) gels of different elasticity or on tissue culture plastic (TCP) to compare impact on gene expression profiles.
Project description:Differentiation of Induced Pluripotent Stem Cells towards Mesenchymal Stromal Cells is Hampered by Culture in 3D Hydrogels [DNA methylation array]
Project description:Mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of multipotent progenitors that contribute to tissue regeneration and homeostasis. MSCs assess extracellular elasticity by probing resistance to applied forces via adhesion, cytoskeletal, and nuclear mechanotransducers, that direct differentiation toward soft or stiff tissue lineages. Even under controlled conditions, MSC differentiation exhibits substantial cell-to-cell variation that remains poorly characterized. By single-cell transcriptional profiling of naïve, matrix-conditioned, and early differentiation state cells, we identified distinct MSC subpopulations with distinct mechanosensitivities, differentiation capacities, and cell cycling. We showed that soft matrices support adipogenesis of multipotent cells and endochondral ossification of non-adipogenic cells, whereas intramembranous ossification and pre-osteoblast proliferation are enhanced by stiff matrices. Using diffusion pseudotime mapping, we delineated hierarchical matrix-directed differentiation and identified mechanoresponsive genes. We found that tropomyosin-1 (TPM1) is highly sensitive to stiffness cues both at RNA and protein levels and that changes in expression of TPM1 determine adipogenic or osteogenic fates. Thus, cell-to-cell variation in tropomyosin-mediated matrix-sensing contributes to impaired differentiation with implications to the biomedical potential of MSCs.
