Recently, the antagonizing effect on the differentiation of mesenchymal stem cells (MSCs) by toll‐like receptor (TLR) ligands, was described. Our study shows that on more primitive cord blood derived ...MSCs, the expression of TLRs and ligand‐induced triggering differs from that of bone marrow derived MSCs. At the RNA level, cord blood MSCs (unrestricted somatic stem cells; USSCs) express low levels of TLR1,3,5,9 and high levels of TLR4 and TLR6. At the protein level expression of TLR5 and very low expression of TLR4 was observed. NF‐κB translocation studies revealed that both TLR4 and TLR5 are functional, although signalling kinetics induced by the individual ligands differed. Stimulation of USSCs with either lipopolysaccharide (LPS) or flagellin resulted in a marked increase of interleukin (IL)‐6 and/or IL‐8 production although levels differed significantly between both stimuli. Interestingly, tumour necrosis factor (TNF)‐α was undetectable after TLR stimulation, which appeared to be due to an inactivated TNF‐α promoter in USSCs. Moreover, osteoblastic differentiation was enhanced after triggering USSCs with LPS and flagellin. In summary, TLR4 and 5 signalling in USSCs is slow and results in the up‐regulation of a restricted number of pro‐inflammatory cytokines and enhanced osteoblastic differentiation. Apparently, the outcome of TLR signalling depends on the cell type that expresses them.
The TNF promoter is silenced in mesenchymal stem cells able to respond to LPS by NFκB translocation and cytokine production yet without TNF.
Previously, we demonstrated that several TLRs are ...expressed on cord blood‐derived USSC. Stimulation of USSC with TLR agonists resulted in a marked increase of IL‐6 and IL‐8 production. Interestingly, TNF was undetectable after TLR stimulation, which appeared to be a result of an inactivated TNF promoter in USSC. Here, we elaborate this study by demonstrating that although USSC do not produce TNF, they are susceptible to TNF stimulation, resulting in NF‐κB translocation and cytokine production. Additionally, we compared different stem cell sources for their ability to produce TNF. Interestingly, we found that the TNF promoter in BM‐MSC is inactivated as well. Like USSC, they are able to respond to TNF stimulation, but they are not able to produce TNF, even not after LPS stimulation. This limited cytokine response in combination with the well‐studied immunosuppressive properties of MSC makes these cells ideal for immune‐suppressive treatment modalities such as graft‐versus‐host disease.
Implantable three-dimensional (3D) constructs to engineer tissue have great therapeutic potential in regenerative medicine and immunotherapy. However, autonomous recruitment of cells into the ...engineered scaffold in vivo is hampered by lack of attracting scaffolds. As a first step to engineering immune tissue, 3D collagen scaffolds were investigated for their ability to enhance in vivo recruitment and growth of various hematopoietic cells. Scaffolds containing immobilized heparin to trap the stem cell chemo-attractant stromal cell-derived factor 1 alpha (SDF1alpha) were implanted subcutaneously into C57Bl6 mice, and influx of cells was monitored using immunohistochemistry. Five weeks post-implantation, heparinized scaffolds were always populated by cells, but incorporating SDF1alpha considerably stimulated recruitment of cells. SDF1alpha could not exert this effect when the formation of a SDF1alpha gradient was abrogated. Scaffolds were mainly populated by CD11b+ and CD11c+ myeloid cells and fibroblasts. One week after implantation, scaffolds harbored only low numbers of cells. Apparently, not all CXCR4-expressing cells, like large numbers of granulocytes, migrate into the scaffold, but retransplantation of a 1-week-old scaffold from a CD45.2(+) into a CD45.1(+) mouse yielded a scaffold harboring mainly CD45.2(+) cells after 5 weeks. These data confirm that only a few progenitor cells are recruited early after implantation. These cells then proliferate and differentiate along different lineages and determine the outcome after 5 weeks.
Immune suppression is a very stable property of multipotent stromal cells also known as mesenchymal stem cells (MSCs). All cell lines tested showed robust immune suppression not affected by a long ...culture history. Several mechanisms were described to account for this capability. Since several of the described mechanisms were not causing the immune suppression, the expression pattern of cord-blood-derived MSCs by microarray experiments was determined. Dendritic cells cocultured with cord blood MSCs were compared with cord blood MSCs. Putative immune suppressive candidates were tested to explain this inhibition. We find that cord blood MSCs themselves are hardly immunogenic as tested with allogeneic T-cells. Dendritic cells cocultured with second-party T-cells evoked abundant proliferation that was inhibited by third-party cord blood MSCs. Optimal inhibition was seen with one cord blood MSC for every dendritic cell. Blocking human leukocyte antigen G only saw partial recovery of proliferation. Several cytokines, gangliosides, enzymes like arginase, NO synthase, and indole amine 2,3-dioxygenase as well as the induction of Treg were not involved in the inhibition. The inhibiting moiety was identified as prostaglandin B2 by lipid metabolite analysis of the culture supernatant and confirmed with purified prostaglandin B2.
Pathogen-derived entities force the tissue-resident dendritic cells (DCs) towards a mature state, followed by migration to the draining lymph node to present antigens to T cells. Bone marrow ...mesenchymal stem cells (MSCs) modulate the differentiation, maturation and function of DCs. In umbilical cord blood an immature MSC population was identified. Remarkably, these immature stem cells modulated DCs in a different way. Marker expression was unchanged during the differentiation of monocytes towards immature DCs (iDCs) when cocultured with cord blood MSC unrestricted somatic stem cells (USSCs). The maturation to mature DCs (mDCs) was enhanced when DCs were co-cultured with USSC, as evidenced by the up-regulation of costimulatory molecules. Endocytosis of dextran by iDCs was hampered in the presence of USSCs, which is indicative for the maturation of iDCs. Despite this maturation, the migration of iDCs cocultured with USSCs appeared to be identical to iDCs cultured alone. However, USSCs increased the migration of mDCs towards CCL21 and boosted interleukin-12 production. So, USSCs mature iDCs, thereby redirecting the antigen-uptake phenotype towards a mature phenotype. Furthermore, DC maturation by lipopolysaccharide (LPS) or USSCs reflects two distinct pathways because migration was unaffected when iDCs were matured by coculture with USSCs, while it was strongly enhanced in the presence of LPS. DCs are able to discriminate the different MSC subtypes, resulting in diverse differentiation programmes.
Recently, the antagonizing effect on the differentiation of mesenchymal stem cells (MSCs) by toll-like receptor (TLR) ligands, was described. Our study shows that on more primitive cord blood derived ...MSCs, the expression of TLRs and ligand-induced triggering differs from that of bone marrow derived MSCs. At the RNA level, cord blood MSCs (unrestricted somatic stem cells; USSCs) express low levels of TLR1,3,5,9 and high levels of TLR4 and TLR6. At the protein level expression of TLR5 and very low expression of TLR4 was observed. NF-kappaB translocation studies revealed that both TLR4 and TLR5 are functional, although signalling kinetics induced by the individual ligands differed. Stimulation of USSCs with either lipopolysaccharide (LPS) or flagellin resulted in a marked increase of interleukin (IL)-6 and/or IL-8 production although levels differed significantly between both stimuli. Interestingly, tumour necrosis factor (TNF)-alpha was undetectable after TLR stimulation, which appeared to be due to an inactivated TNF-alpha promoter in USSCs. Moreover, osteoblastic differentiation was enhanced after triggering USSCs with LPS and flagellin. In summary, TLR4 and 5 signalling in USSCs is slow and results in the up-regulation of a restricted number of pro-inflammatory cytokines and enhanced osteoblastic differentiation. Apparently, the outcome of TLR signalling depends on the cell type that expresses them.
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