Friederike Gieseke

Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM

BACKGROUND Multipotent mesenchymal stromal cells (MSC) have become important tools in regenerative and transplantation medicine. Rapidly increasing numbers of patients are receiving in vitro-expanded MSC. Culture conditions typically include FSC because human serum does not fully support growth of human MSC in vitro (MSC(FCS)). Concerns regarding BSE, other infectious complications and host immune reactions have fueled investigation of alternative culture supplements. METHODS As PDGF has long been identified as a growth factor for MSC, we tested media supplementation with platelet lysate for support of MSC proliferation. RESULTS We found that primary cultures of BM-derived MSC can be established with animal serum-free media containing fresh frozen plasma and platelets (MSC(FFPP)). Moreover, MSC(FFPP) showed vigorous proliferation that was superior to classical culture conditions containing FCS. MSC(FFPP) morphology was equivalent to MSC(FCS), and MSC(FFPP) expressed CD73, CD90, CD105, CD106, CD146 and HLA-ABC while being negative for CD34, CD45 and surface HLA-DR, as expected. In addition to being phenotypically identical, MSC(FFPP) could efficiently differentiate into adipocytes and osteoblasts. In terms of immune regulatory properties, MSC(FFPP) were indistinguishable from MSC(FCS). Proliferation of PBMC induced by IL-2 in combination with OKT-3 or by PHA was inhibited in the presence of MSC(FFPP). DISCUSSION Taken together, FCS can be replaced safely by FFPP in cultures of MSC for clinical purposes.

Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

BackgroundHuman multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.ResultsAfter transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.ConclusionPhysiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

HUMAN MULTIPOTENT MESENCHYMAL STROMAL CELLS USE GALECTIN-1 TO INHIBIT IMMUNE EFFECTOR CELLS

Human multipotent mesenchymal stromal cells (MSCs) suppress proliferation and alloreactivity of T cells. Several signaling molecules and enzymes contribute to this effect. We focused on carbohydrate-protein interactions and investigated whether lectins are involved in immune modulation by MSC. Gene expression profiling of MSCs revealed that one of the most important lectins in this setting, galectin-1, was highly expressed. Galectin-1 protein was detected intracellularly and on the cell surface of MSCs. In addition, galectin-1 was released into the cell culture supernatant by MSCs. To analyze the functional role of galectin-1, a stable knockdown of galectin-1 in MSCs with use of a retroviral transfection system was established. Galectin-1 knockdown in MSCs resulted in a significant loss of their immunomodulatory properties, compared with MSCs infected with nontargeting control sequences. The galectin-1 knockdown partially restored the proliferation of CD4(+) and CD8(+) T cells. By contrast, the effect of MSCs on nonalloreactive natural killer (NK) cells was unaffected by down-regulation of galectin-1 expression. Furthermore, MSC-derived galectin-1 significantly modulated the release of cytokines involved in graft-versus-host disease (GVHD) and autoimmunity (eg, tumor necrosis factor-α [TNFα], IFNγ, interleukin-2 [IL-2], and IL-10. These results identify galectin-1 as the first lectin mediating the immunomodulatory effect of MSCs on allogeneic T cells.

Activated human hepatic stellate cells induce myeloid derived suppressor cells from peripheral blood monocytes in a CD44-dependent fashion

BACKGROUND & AIMS Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of cells associated with the suppression of immunity. However, little is known about how or where MDSCs are induced and from which cells they originate. The liver is known for its immune regulatory functions. Here, we investigated the capacity of human hepatic stellate cells (HSCs) to transform peripheral blood monocytes into MDSCs. METHODS We cultured freshly isolated human monocytes from healthy donors on primary human HSCs or an HSC cell-line and characterized the phenotype and function of resulting CD14(+)HLA-DR(-/low) monocytes by flow cytometry, quantitative PCR, and functional assays. We analyzed the molecular mechanisms underlying the induction and function of the CD14(+)HLA-DR(-/low) cells by using blocking antibodies or knock-down technology. RESULTS Mature peripheral blood monocytes co-cultured with HSCs downregulated HLA-DR and developed a phenotypic and functional profile similar to MDSCs. Only activated but not freshly isolated HSCs were capable of inducing CD14(+)HLA-DR(-/low) cells. Such CD14(+)HLA-DR(-/low) monocyte-derived MDSCs suppressed T-cell proliferation in an arginase-1 dependent fashion. HSC-induced development of CD14(+)HLA-DR(-/low) monocyte-derived MDSCs was not mediated by soluble factors, but required physical interaction and was abrogated by blocking CD44. CONCLUSIONS Our study shows that activated human HSCs convert mature peripheral blood monocytes into MDSCs. As HSCs are activated during chronic inflammation, the subsequent local induction of MDSCs may prevent ensuing excessive liver injury. HSC-induced MDSCs functionally and phenotypically resemble those isolated from liver cancer patients. Thus, our data suggest that local generation of MDSCs by liver-resident HSCs may contribute to immune suppression during inflammation and cancer in the liver.

Proinflammatory stimuli induce galectin-9 in human mesenchymal stromal cells to suppress T-cell proliferation.

Human multipotent mesenchymal stromal cells (MSCs) are clinically applied to treat autoimmune diseases and graft‐versus‐host disease due to their immunomodulatory properties. Several molecules have been identified to mediate these effects, including constitutively expressed galectin‐1. However, there are indications in the literature that MSCs exert enhanced immunosuppressive functions after interaction with an inflammatory environment. Therefore, we analyzed how inflammatory stimuli influence the expression of the galectin network in MSCs and functionally tested the relevance for the immunomodulatory effects of MSCs. We found that galectin‐9 was strongly induced in MSCs upon interaction with activated PBMCs. Proinflammatory cytokines, such as interferon‐gamma (IFN‐γ) and tumor necrosis factor‐alpha (TNF‐α), and also ligands of the Toll‐like receptors (TLRs) TLR2, TLR3, and TLR4 elicited similar induction of galectin‐9 in activated PBMCs. Galectin‐9 was not only upregulated intracellularly, but also released by MSCs in significant amounts into the supernatant after exposure to proinflammatory stimuli. In proliferation assays, MSCs with a galectin‐9 knockdown lost a significant portion of their antiproliferative effects on T cells. In conclusion, we found that unlike constitutively expressed galectin‐1, galectin‐9 is induced by several proinflammatory stimuli and released by MSCs. Thus, galectin‐9 contributes to the inducible immunomodulatory functions of MSCs.

Tumour stromal cells derived from paediatric malignancies display MSC-like properties and impair NK cell cytotoxicity

BackgroundTumour growth and metastatic infiltration are favoured by several components of the tumour microenvironment. Bone marrow-derived multipotent mesenchymal stromal cells (MSC) are known to contribute to the tumour stroma. When isolated from healthy bone marrow, MSC exert potent antiproliferative effects on immune effector cells. Due to phenotypic and morphological similarities of MSC and tumour stromal cells (TStrC), we speculated that immunotherapeutic approaches may be hampered if TStrC may still exhibit immunomodulatory properties of MSC.MethodsIn order to compare immunomodulatory properties of MSC and tumour stromal cells (TStrC), we established and analyzed TStrC cultures from eleven paediatric tumours and MSC preparations from bone marrow aspirates. Immunophenotyping, proliferation assays and NK cell cytotoxicity assays were employed to address the issue.ResultsWhile TStrC differed from MSC in terms of plasticity, they shared surface expression of CD105, CD73 and other markers used for MSC characterization. Furthermore, TStrC displayed a strong antiproliferative effect on peripheral blood mononuclear cells (PBMC) in coculture experiments similar to MSC. NK cell cytotoxicity was significantly impaired after co-culture with TStrC and expression of the activating NK cell receptors NKp44 and NKp46 was reduced.ConclusionsOur data show that TStrC and MSC share important phenotypic and functional characteristics. The inhibitory effect of TStrC on PBMC and especially on NK cells may facilitate the immune evasion of paediatric tumours.

Dendritic cells: functional aspects of glycosylation and lectins.

Dendritic cells (DC) direct immune responses either toward tolerance to a presented antigen or toward inflammatory reactions of effector cells. Many crucial cytokines and cell surface proteins have been identified in this process using gene expression profiling. However, it is becoming evident that important steps involve carbohydrate-protein interactions, which cannot be anticipated by gene expression profiling in most cases. These contacts are crucial for the uptake of certain antigens, migration, and homing, but also for infection by viruses. On one hand, DC use numerous C-type lectins, such as DC-SIGN, dectin-1, langerin, and DEC-205, for antigen uptake. Other lectins, such as CD83, siglecs, and galectins, may be involved in regulation of the immune response to a given antigen. On the other hand, cell surface glycosylation of DC themselves changes significantly depending on the environment and the functional state, generating different signals by altered glycans. Because DC occur at the interface between innate and acquired immunity, it may not be surprising that glycans and lectins play an important role in many biological functions of DC. In this review, we focus on glycobiological aspects of antigen uptake and processing, immune modulation, and viral infections in the context of DC biology.

Siglec‐7 tetramers characterize B‐cell subpopulations and leukemic blasts

Cell surface glycosylation has important regulatory functions in the maturation, act‐ivation, and homeostasis of lymphocytes. The family of human sialic acid‐binding immunoglobulin‐like lectins (siglecs) comprises inhibitory as well as activating receptors intimately involved in the regulation of immune responses. Analyses of the interaction between siglecs and glycans are hampered by the low affinity of this interaction. Therefore, we expressed siglec‐7 in eukaryotic cells, allowing for glycosylation, and oligomerized the protein in analogy to MHC tetramers. Using this tool, flow cytometric analysis of lymphocytes became possible. Sialic acid‐dependent binding of siglec‐7 tetramers was confirmed by glycan array analysis and loss of siglec tetramer binding after neuraminidase treatment of lymphocytes. In contrast to most lymphocyte subpopulations, which showed high siglec‐7 ligand expression, B‐cell subpopulations could be further subdivided according to different siglec‐7 ligand expression levels. We also analyzed blasts from acute lymphoblastic leukemias of the B‐cell lineage as well as the T‐cell lineage, since malignant transformation is often associated with aberrant cell surface glycosylation. While pediatric T‐ALL blasts highly expressed siglec‐7 ligands, siglec‐7 ligands were barely detectable on cALL blasts.

Enzymatic characterization of novel arylsulfatase A variants using human arylsulfatase A-deficient immortalized mesenchymal stromal cells.

Metachromatic leukodystrophy (MLD) is an autosomal‐recessive lysosomal storage disease caused by mutations in the ARSA gene leading to arylsulfatase A (ARSA) deficiency and causing sulfatide accumulation. Main symptoms of the disease are progressive demyelination, neurological dysfunction, and reduced life expectancy. To date, more than 200 different ARSA variants have been reported in MLD patients. Here, we report the biochemical characterization of seven novel pathogenic variants (c.98T > C, c.195delC, c.229G > C, c.545C > G, c.674A > G, c.852T > A, and c.1274A > G), which were found when sequencing a cohort of 31 German MLD families. For that purpose, the ARSA cDNAs carrying the respective mutations inserted by site‐directed mutagenesis were cloned into a MigR1 (MSCV, IRES, GFP, retrovirus‐1) vector. The constructs were overexpressed using retroviral gene transfer in immortalized, human multipotent mesenchymal stromal cells prepared from a patient deficient in ARSA activity (late infantile MLD). In this novel ARSA−/− cell system, the seven ARSA mutants showed ARSA activity of less than 10% when compared with wild type, which is evidence for the pathogenicity of all seven variants. In conclusion, the system of ARSA−/−‐immortalized MSC turned out to be a helpful novel tool for the biochemical characterization of ARSA variants.