Effect of Short-Term Stimulation with Interleukin-1? and Differentiation Medium on Human Mesenchymal Stromal Cell Paracrine Activity in Coculture with Osteoblasts.
ABSTRACT: Human mesenchymal stromal cells (hMSCs) exhibit the potential to accelerate bone healing by enhanced osteogenic differentiation. Interleukin-1? is highly expressed during fracture healing and has been demonstrated to exert a significant impact on the differentiation behaviour of hMSCs. Here, we investigate the effect of 2-hour IL-1? stimulation on the differentiation and paracrine activity of hMSCs in coculture with osteosarcoma cells in vitro.hMSCs from 3 donors were incubated for 2 hours with 10 ng/mL IL-1? and subsequently cocultured with MG63-GFP cells either in control or in differentiation medium in a transwell system for 28 days. Genetic and functional effects were investigated.hMSCs cultured in control medium exhibited a regulatory effect on cocultured MG63-GFP cells, resulting in upregulation of osteogenic gene expression in combination with increased ALP activity. However, while stimulated hMSCs cultured under differentiation conditions exhibit signs of osteogenic differentiation, osteogenic differentiation also caused an impaired regulatory effect on the cocultured MG63-GFP cells.Short stimulation of hMSCs has the potential to modify their long-term behaviour. In addition, undifferentiated hMSCs are able to regulate osteoblast differentiation; however, this regulatory function is lost upon osteogenic differentiation in vitro. This offers a novel approach for clinical cell therapy protocols.
Project description:The effects of immune cells, in particular macrophages, on the behaviour of mesenchymal stromal cells (MSCs) have recently gained much attention for MSCs-based tissue-engineered constructs. This study aimed to evaluate the effect of monocytes/macrophages on the osteogenic differentiation of adipose-derived mesenchymal stromal cells (ADMSCs) in three-dimensional (3D) cocultures. For this, we cocultured THP-1 monocytes, M1 macrophages, or M2 macrophages with ADMSCs on 3D poly(lactic-co-glycolic) acid (PLGA)/polycaprolactone (PCL) scaffolds using osteogenic medium for up to 42 days. We found that osteogenic differentiation of ADMSCs was inhibited by monocytes and both macrophage subtypes in 3D scaffolds. Furthermore, coculture of monocytes/macrophages with ADMSCs resulted in downregulated secretion of oncostatin M (OSM) and bone morphogenetic protein 2 (BMP-2) and inhibited expression of osteogenic markers alkaline phosphatase (ALP), bone sialoprotein (BSP), and runt-related transcription factor 2 (RUNX2). Compared with both macrophage subtypes, monocytes inhibited osteogenic differentiation of ADMSCs more significantly. These data suggest that the mutual interactions between monocytes/macrophages and ADMSCs negatively affect MSC osteogenic differentiation and thus possibly bone healing capacity, which highlights the importance of the micro-environment in influencing cell-based constructs to treat bone defects and the potential to improve their performance by resolving the inflammation ahead of treatment.
Project description:Shockwave treatment promotes bone healing of nonunion fractures. In this study, we investigated whether this effect could be due to adenosine 5'-triphosphate (ATP) release-induced differentiation of human mesenchymal stem cells (hMSCs) into osteoprogenitor cells. Cultured bone marrow-derived hMSCs were subjected to shockwave treatment and ATP release was assessed. Osteogenic differentiation and mineralization of hMSCs were evaluated by examining alkaline phosphatase activity, osteocalcin production, and calcium nodule formation. Expression of P2X7 receptors and c-fos and c-jun mRNA was determined with real-time reverse transcription polymerase chain reaction and Western blotting. P2X7-siRNA, apyrase, P2 receptor antagonists, and p38 MAPK inhibitors were used to evaluate the roles of ATP release, P2X7 receptors, and p38 MAPK signaling in shockwave-induced osteogenic hMSCs differentiation. Shockwave treatment released significant amounts (? 7 ?M) of ATP from hMSCs. Shockwaves and exogenous ATP induced c-fos and c-jun mRNA transcription, p38 MAPK activation, and hMSC differentiation. Removal of ATP with apyrase, targeting of P2X7 receptors with P2X7-siRNA or selective antagonists, or blockade of p38 MAPK with SB203580 prevented osteogenic differentiation of hMSCs. Our findings indicate that shockwaves release cellular ATP that activates P2X7 receptors and downstream signaling events that caused osteogenic differentiation of hMSCs. We conclude that shockwave therapy promotes bone healing through P2X7 receptor signaling, which contributes to hMSC differentiation.
Project description:To date, bone tissue regeneration strategies lack an approach that effectively provides an osteogenic and angiogenic environment conducive to bone growth. In the current study, we evaluated the osteogenic and angiogenic response of human mesenchymal stem cells (hMSCs) and green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) cocultured on a self-assembled, peptide amphiphile nanomatrix functionalized with the cell adhesive ligand RGDS (PA-RGDS). Analysis of alkaline phosphatase activity, von Kossa staining, Alizarin Red quantification, and osteogenic gene expression, indicates a significant synergistic effect between the PA-RGDS nanomatrix and coculture that promoted hMSC osteogenesis. In addition, coculturing on PA-RGDS resulted in enhanced HUVEC network formation and upregulated vascular endothelial growth factor gene and protein expression. Though PA-RGDS and coculturing hMSCs with HUVECs were each previously reported to individually enhance hMSC osteogenesis, this study is the first to demonstrate a synergistic promotion of HUVEC angiogenesis and hMSC osteogenesis by integrating coculturing with the PA-RGDS nanomatrix. We believe that using the combination of hMSC/HUVEC coculture and PA-RGDS substrate is an efficient method for promoting osteogenesis and angiogenesis, which has immense potential as an efficacious, engineered platform for bone tissue regeneration.
Project description:The instructive capabilities of extracellular matrix-inspired materials for osteoprogenitor differentiation have sparked interest in understanding modulation of other cell types within the bone regenerative microenvironment. We previously demonstrated that nanoparticulate mineralized collagen glycosaminoglycan (MC-GAG) scaffolds efficiently induced osteoprogenitor differentiation and bone healing. In this work, we combined adenovirus-mediated delivery of osteoprotegerin (AdOPG), an endogenous anti-osteoclastogenic decoy receptor, in primary human mesenchymal stem cells (hMSCs) with MC-GAG to understand the role of osteoclast inactivation in augmentation of bone regeneration. Simultaneous differentiation of osteoprogenitors on MC-GAG and osteoclast progenitors resulted in bidirectional positive regulation. AdOPG expression did not affect osteogenic differentiation alone. In the presence of both cell types, AdOPG-transduced hMSCs on MC-GAG diminished osteoclast-mediated resorption in direct contact; however, osteoclast-mediated augmentation of osteogenic differentiation was unaffected. Thus, the combination of OPG with MC-GAG may represent a method for uncoupling osteogenic and osteoclastogenic differentiation to augment bone regeneration.
Project description:Human mesenchymal stem cell (hMSC)-derived exosomes have shown regenerative effects, but their role in osteogenesis and the underlying mechanism are yet to be determined. In this study, we examined the time-course secretion of exosomes by hMSCs during the entire process of osteogenic differentiation. Exosomes derived from hMSCs in various stages of osteogenic differentiation committed homotypic cells to differentiate towards osteogenic lineage, but only exosomes from late stages of osteogenic differentiation induced extracellular matrix mineralisation. Exosomes from expansion and early and late stages of osteogenic differentiation were internalised by a subpopulation of hMSCs. MicroRNA profiling revealed a set of differentially expressed exosomal microRNAs from the late stage of osteogenic differentiation, which were osteogenesis related. Target prediction demonstrated that these microRNAs enriched pathways involved in regulation of osteogenic differentiation and general mechanisms how exosomes exert their functions, such as "Wnt signalling pathway" and "endocytosis". Taken together, the results show that MSCs secrete exosomes with different biological properties depending on differentiation stage of their parent cells. The exosomal cargo transferred from MSCs in the late stage of differentiation induces osteogenic differentiation and mineralisation. Moreover, it is suggested that the regulatory effect on osteogenesis by exosomes is at least partly exerted by exosomal microRNA.
Project description:Bone marrow-derived human mesenchymal stem cells (hMSCs) possess multipotent differentiation capabilities and are a potent source of paracrine factors. We show how the epidermal keratinocyte can direct hMSC differentiation selectively. Keratinocytes and hMSCs were either cocultured in physical contact (contact cocultures), or separated without physical contact using a transwell insert (noncontact cocultures). We also delivered hMSCs into an ex vivo human excisional wound where subpopulations of the hMSCs were either in contact or were physically separated from the epidermal keratinocytes. In comparison to control hMSCs that were not cocultured, contact cocultured hMSCs adopted an epithelial morphology and expressed keratinocyte markers while noncontact cocultured hMSCs, surprisingly, adopted phenotypes that resembled myofibroblast and early neural lineage, both of which are of dermal origin. Cell fusion was not a requirement in in vitro contact cocultures, as determined by fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization analysis (FISH). To the best of our knowledge, this work provides the first example of hMSC differentiation into different lineages depending on their proximity to a single cell type.
Project description:Small fractures in bone tissue can heal by themselves, but in case of larger defects current therapies are not completely successful due to several drawbacks. A possible strategy relies on the combination of additive manufactured polymeric scaffolds and human mesenchymal stromal cells (hMSCs). The architecture of bone tissue is characterized by a structural gradient. Long bones display a structural gradient in the radial direction, while flat bones in the axial direction. Such gradient presents a variation in bone density from the cancellous bone to the cortical bone. Therefore, scaffolds presenting a gradient in porosity could be ideal candidates to improve bone tissue regeneration. In this study, we present a construct with a discrete gradient in pore size and characterize its ability to further support the osteogenic differentiation of hMSCs. Furthermore, we studied the behaviour of hMSCs within the different compartments of the gradient scaffolds, showing a correlation between osteogenic differentiation and ECM mineralization, and pore dimensions. Alkaline phosphatase activity and calcium content increased with increasing pore dimensions. Our results indicate that designing structural porosity gradients may be an appealing strategy to support gradual osteogenic differentiation of adult stem cells.
Project description:BACKGROUND:The differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts (OBs) is a prerequisite for bone formation. However, little is known about the definitive surface markers for OBs during osteogenesis. METHODS:To study the surface markers on OBs, we generated and used monoclonal antibodies (MAbs) against surface molecules on transforming growth factor-?1 (TGF-?1)-treated cancer cells. The generated MAbs were further selected toward expression changes on hMSCs cultured with TGF-?1/bone morphogenetic protein-2 (BMP-2) or osteogenic differentiation medium (ODM) by flow cytometry. Immunoprecipitation and mass spectrometry were performed to identify target antigens of selected MAbs. Expression changes of the target antigens were evaluated in hMSCs, human periodontal ligament cells (hPDLCs), and human dental pulp cells (hDPCs) during osteogenic and adipogenic differentiation by quantitative polymerase chain reaction (qPCR) and flow cytometry. hMSCs were also sorted by the MAbs using magnetic-activated cell sorting system, and osteogenic potential of sorted cells was evaluated via Alizarin Red S (ARS) staining and qPCR. RESULTS:The binding reactivity of MR14-E5, one of the MAbs, was downregulated in hMSCs with ODM while the binding reactivity of ER7-A7, ER7-A8, and MR1-B1 MAbs was upregulated. Mass spectrometry and overexpression identified that MR14-E5, ER7-A7/ER7-A8, and MR1-B1 recognized integrin ?2, ?3, and ?V, respectively. Upon osteogenic differentiation of hMSCs, the expression of integrin ?2 was drastically downregulated, but the expression of integrin ?3 and ?V was upregulated in accordance with upregulation of osteogenic markers. Expression of integrin ?3 and ?V was also upregulated in hPDLCs and hDPCs during osteogenic differentiation. Cell sorting showed that integrin ?V-high hMSCs have a greater osteogenic potential than integrin ?V-low hMSCs upon the osteogenic differentiation of hMSCs. Cell sorting further revealed that the surface expression of integrin ?V is more dramatically induced even in integrin ?V-low hMSCs. CONCLUSION:These findings suggest that integrin ?3 and ?V induction is a good indicator of OB differentiation. These findings also shed insight into the expression dynamics of integrins upon osteogenic differentiation of hMSCs and provide the reason why different integrin ligands are required for OB differentiation of hMSCs.
Project description:RNA interference (RNAi) may be an effective and valuable tool for promoting the growth of functional tissue, as short interfering RNA (siRNA) and microRNA (miRNA) can block the expression of genes that have negative effects on tissue regeneration. Our group has recently reported that the localized and sustained presentation of siRNA against noggin (siNoggin) and miRNA-20a from in situ forming poly(ethylene glycol) (PEG) hydrogels enhanced osteogenic differentiation of encapsulated human bone marrow-derived mesenchymal stem cells (hMSCs). Here, the capacity of the hydrogel system to accelerate bone formation in a rat calvarial bone defect model is presented. After 12?weeks post-implantation, the hydrogels containing encapsulated hMSCs and miRNA-20a resulted in more bone formation in the defects than the hydrogels containing hMSCs without siRNA or with negative control siRNA. This localized and sustained RNA interfering molecule delivery system may provide an excellent platform for healing bony defects and other tissues. STATEMENT OF SIGNIFICANCE:Delivery of RNAi molecules may be a valuable strategy to guide cell behavior for tissue engineering applications, but to date there have been no reports of a biomaterial system capable of both encapsulation of cells and controlled delivery of incorporated RNA. Here, we present PEG hydrogels that form in situ via Michael type reaction, and that permit encapsulation of hMSCs and the concomitant controlled delivery of siNoggin and/or miRNA-20a. These RNAs were chosen to suppress noggin, a BMP-2 antagonist, and/or PPAR-?, a negative regulator of BMP-2-mediated osteogenesis, and therefore promote osteogenic differentiation of hMSCs and subsequent bone repair in critical-sized rat calvarial defects. Simultaneous delivery of hMSCs and miRNA-20a enhanced repair of these defects compared to hydrogels containing hMSCs without siRNA or with negative control siRNA. This in situ forming PEG hydrogel system offers an exciting platform for healing critical-sized bone defects by localized, controlled delivery of RNAi molecules to encapsulated hMSCs and surrounding cells.
Project description:The response of human bone marrow derived human mesenchymal stem cells (hMSCs) encapsulated in silk ionomer hydrogels was studied. Silk aqueous solutions with silk-poly-L-lysine or silk-poly-L-glutamate were formed into hydrogels via ultrasonication in situ with different net charges. hMSCs were encapsulated within the hydrogels and the impact of matrix charge was assessed over weeks in osteogenic, adipogenic and maintenance growth media. These modified silk charged polymers supported cell viability and proliferative potential, and the hMSCs were able to differentiate toward osteogenic or adipogenic lineages in the corresponding differentiation media. The silk/silk-poly-L-lysine hydrogels exhibited a positive effect on selective osteogenesis of hMSCs, inducing differentiation toward an osteogenic lineage even in the absence of osteogenic supplements, while also inhibiting adipogenesis. In contrast, silk/silk fibroin-poly-L-glutamate hydrogels supported both osteogenic and adipogenic differentiation of hMSCs when cultured under induction conditions. The results demonstrate the potential utility of silk-based ionomers in gel formats for hMSCs encapsulation and for directing hMSCs long term functional differentiation toward specific lineages.