Final Report Summary - HYALSTEMAGE (Effect of controlled hyaluronan synthesis on the stemness of aged mesenchymal stem cells)
Stem cells are essential for regeneration and repair of worn-out or injured tissue. A particular stem cell type in the adult human-being is the ‘Mesenchymal stem cell’ (MSC). This special type of stem cell is present in many tissues and restores bone, connective tissues and fat. MSC reside in a quiescent state at a special site referred to as the stem cell niche. In the bone marrow MSC share the niche with haematopoietic stem cells (HSC).
In the case of overwhelming and long-lasting stress or damage MSC get activated, e.g. by inflammatory signals, and in turn start to divide and to migrate to the sites of injury. There, MSC differentiate and regenerate tissue integrity. A major component outside of the cells, which notably is distinctly involved in controlling and guiding many cellular processes, is the polysaccharide hyaluronan (HA). HA is an unbranched, anionic biopolymer of high molecular mass. Due to its physicochemical properties, HA can associate with large volumes of water, thereby supporting proper tissue functions. Sites of loosely packed extracellular matrix (ECM) also offer stem and progenitor cell optimal space to proliferate and differentiate.
In this project we investigated the role of HA on MSC stemness during aging. Previous results indicated that MSC express HA-synthesizing enzymes, so called HA synthases (HAS). In MSC of elderly donors some HAS isozymes are upregulated compared to MSC from young persons, leading to the assumption that elevated amounts of high molecular mass HA are secreted to the pericellular space. We hypothesized that such a tightly packed HA-rich ECM functions as an enclosure and thus interferes with proper cellular activity and presumably restrains MSC from exerting their regenerative potential. On the other hand, increased HAS transcript levels of MSC from elderly could also be interpreted as an attempt of MSC to compensate for age-related reduction of stemness that has emerged over time. For a better understanding of this phenomenon we manipulated the extracellular HA content by extrinsic and intrinsic means.
Supplementation of HA to the MSC culture supernatant resulted in an increased proliferation rate. Colony-forming unit assays yielded denser cell colonies in the HA treated samples compared to the controls, yet no difference in the number of colonies was detected, indicating that the number of stem cells remained unchanged. In contrast, addition of the known HAS inhibitor 4-methylumbelliferone (4-MU) to MSC led to a reversible proliferation arrest. In the literature this growth arrest was so far described being caused by the depletion of one of the two uridine diphosphate (UDP) precursor sugars needed for HA synthesis. In order to eliminate 4-MU from the cytosol, the xenobiotic-detoxification machinery of MSC covalently attaches glucuronic acid (GlcUA) to 4-MU, thereby depriving HAS from UDP-GlcUA thus ceasing HA production. In turn, cellular levels of the second HA building block, i.e. UDP-N-acetylglucosamine (GlcNAc) are rising. We now could demonstrate that this augmented availability of UDP-GlcNAc is altering the O-glycosylation levels of cellular proteins. This posttranslational modification on serine or threonine residues is catalysed by the enzyme O-linked N-acetylglucosamine transferase (OGT) and shows increased occurrence in 4-MU treated MSC, presumably due to an overly high intracellular UDP-GlcNAc concentration.
O-linked glycosylation is known to be a key player in controlling protein translation via regulating the formation of mRNA containing compartments termed stress granules (SG). In addition to mRNAs, SG contain ribosomal proteins. This allows the cells to quickly adapt the translational activity in response to environmental changes. Our results now suggest that 4-MU treatment of MSC not only affects the formation of SG via increased levels of O-linked glycosylation of cellular proteins but also that MSC cultivated under osteogenic stimulation develop SG, which in turn support MSC differentiation into osteogenic precursor cells. Moreover, we could demonstrate that supplementation of 4-MU to the osteogenic differentiation medium brings forth not just osteoblasts but notably proceeds further to osteocytes. To our knowledge this is the first time osteocytogenesis was observed in vitro.
In conclusion, we were able to show that components of the stem cell niche are pertinently involved in controlling stem cell behaviour. Based on our experimental data and interpretation thereof, we were able putting forward a novel concept for extracellular crowding: intracellular signalling processes are distinctly regulated by O-glycosylation of specific protein moieties which are often found phosphorylated by receptor kinases of major signalling pathways. This strongly argues for a novel sensing mechanisms, in which cells can readily decide at the absence of specific growth factors or hormones whether to proliferate, migrate or differentiate due to changing spatial information.
In the case of overwhelming and long-lasting stress or damage MSC get activated, e.g. by inflammatory signals, and in turn start to divide and to migrate to the sites of injury. There, MSC differentiate and regenerate tissue integrity. A major component outside of the cells, which notably is distinctly involved in controlling and guiding many cellular processes, is the polysaccharide hyaluronan (HA). HA is an unbranched, anionic biopolymer of high molecular mass. Due to its physicochemical properties, HA can associate with large volumes of water, thereby supporting proper tissue functions. Sites of loosely packed extracellular matrix (ECM) also offer stem and progenitor cell optimal space to proliferate and differentiate.
In this project we investigated the role of HA on MSC stemness during aging. Previous results indicated that MSC express HA-synthesizing enzymes, so called HA synthases (HAS). In MSC of elderly donors some HAS isozymes are upregulated compared to MSC from young persons, leading to the assumption that elevated amounts of high molecular mass HA are secreted to the pericellular space. We hypothesized that such a tightly packed HA-rich ECM functions as an enclosure and thus interferes with proper cellular activity and presumably restrains MSC from exerting their regenerative potential. On the other hand, increased HAS transcript levels of MSC from elderly could also be interpreted as an attempt of MSC to compensate for age-related reduction of stemness that has emerged over time. For a better understanding of this phenomenon we manipulated the extracellular HA content by extrinsic and intrinsic means.
Supplementation of HA to the MSC culture supernatant resulted in an increased proliferation rate. Colony-forming unit assays yielded denser cell colonies in the HA treated samples compared to the controls, yet no difference in the number of colonies was detected, indicating that the number of stem cells remained unchanged. In contrast, addition of the known HAS inhibitor 4-methylumbelliferone (4-MU) to MSC led to a reversible proliferation arrest. In the literature this growth arrest was so far described being caused by the depletion of one of the two uridine diphosphate (UDP) precursor sugars needed for HA synthesis. In order to eliminate 4-MU from the cytosol, the xenobiotic-detoxification machinery of MSC covalently attaches glucuronic acid (GlcUA) to 4-MU, thereby depriving HAS from UDP-GlcUA thus ceasing HA production. In turn, cellular levels of the second HA building block, i.e. UDP-N-acetylglucosamine (GlcNAc) are rising. We now could demonstrate that this augmented availability of UDP-GlcNAc is altering the O-glycosylation levels of cellular proteins. This posttranslational modification on serine or threonine residues is catalysed by the enzyme O-linked N-acetylglucosamine transferase (OGT) and shows increased occurrence in 4-MU treated MSC, presumably due to an overly high intracellular UDP-GlcNAc concentration.
O-linked glycosylation is known to be a key player in controlling protein translation via regulating the formation of mRNA containing compartments termed stress granules (SG). In addition to mRNAs, SG contain ribosomal proteins. This allows the cells to quickly adapt the translational activity in response to environmental changes. Our results now suggest that 4-MU treatment of MSC not only affects the formation of SG via increased levels of O-linked glycosylation of cellular proteins but also that MSC cultivated under osteogenic stimulation develop SG, which in turn support MSC differentiation into osteogenic precursor cells. Moreover, we could demonstrate that supplementation of 4-MU to the osteogenic differentiation medium brings forth not just osteoblasts but notably proceeds further to osteocytes. To our knowledge this is the first time osteocytogenesis was observed in vitro.
In conclusion, we were able to show that components of the stem cell niche are pertinently involved in controlling stem cell behaviour. Based on our experimental data and interpretation thereof, we were able putting forward a novel concept for extracellular crowding: intracellular signalling processes are distinctly regulated by O-glycosylation of specific protein moieties which are often found phosphorylated by receptor kinases of major signalling pathways. This strongly argues for a novel sensing mechanisms, in which cells can readily decide at the absence of specific growth factors or hormones whether to proliferate, migrate or differentiate due to changing spatial information.