Hatim Hemeda

Interferon-γ and Tumor Necrosis Factor-α Differentially Affect Cytokine Expression and Migration Properties of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with the capacity to differentiate into different tissue cell types such as chondrocytes, osteocytes, and adipocytes. In addition, they can home to damaged, in-flamed, and malignant tissues and display immunomodulatory properties. Since tissue-derived factors might modulate these properties, we decided to explore the impact of prototypic tissue-derived inflammatory cytokines such as TNF-alpha and IFN-gamma on immunomodulatory MSCs functions. To this end, we used primary bone marrow and cord blood-derived MSCs as well as an immortalized MSC line (V54/2) as model systems. We demonstrate that under unstimulated conditions, V54/2 cells constitutively express low levels of indoleamine 2,3-dioxygenase (IDO), exert an immunosuppressive effect on activated T-lymphocyte proliferation, secrete a distinct set of cytokines, and express a wide range of chemokine receptors. Upon stimulation, the proinflammatory cytokines IFN-gamma and TNF-alpha did not inhibit suppression of T-cell proliferation, although IDO expression was up-regulated by IFN-gamma. In contrast, TNF-alpha but not IFN-gamma amplified the cytokine production of V54/2 and primary MSCs. Interestingly, IFN-gamma was superior to TNF-alpha in up-regulating expression of chemokine receptors and migration of the V54/2 cell line, while TNF-alpha was the predominant regulator of migration in primary MSCs. Altogether, our data show that properties of MSCs depend on local environmental factors. In particular, we have shown that IFN-gamma and TNF-alpha differentially regulate cytokine expression and migration of MSCs.

Donor Age of Human Platelet Lysate Affects Proliferation and Differentiation of Mesenchymal Stem Cells

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

Tissue-resident mesenchymal stem cells attract peripheral blood neutrophils and enhance their inflammatory activity in response to microbial challenge.

Human MSCs may respond to TLR ligation, and recent research has suggested that many tissues contain tissue‐specific MSCs, possibly located in periendothelial and perivascular regions. At present, the functional consequences of these findings are unclear. We hypothesized that tissue‐specific MSCs could play an instructional role during early phases of bacterial challenge. To investigate this hypothesis further, we set up a coculture system of glandular MSCs and peripheral blood neutrophils so that we could analyze the cellular interactions of these cells in response to LPS challenge. We found that stimulation with bacterial endotoxin induced chemokine receptor expression and mobility of MSCs. Activated MSCs secreted large amounts of inflammatory cytokines and recruited neutrophils in an IL‐8‐ and MIF‐dependent manner. Recruited and activated neutrophils showed a prolonged lifespan, an increased expression of inflammatory chemokines, and an enhanced responsiveness toward subsequent challenge with LPS. Our findings demonstrate a complex, functional interaction between tissue‐resident MSCs and peripheral blood neutrophils upon bacterial challenge and suggest a role for MSCs in the early phases of pathogen challenge, when classical immune cells have not been recruited yet.

Epigenetic Rejuvenation of Mesenchymal Stromal Cells Derived from Induced Pluripotent Stem Cells

Summary Standardization of mesenchymal stromal cells (MSCs) remains a major obstacle in regenerative medicine. Starting material and culture expansion affect cell preparations and render comparison between studies difficult. In contrast, induced pluripotent stem cells (iPSCs) assimilate toward a ground state and may therefore give rise to more standardized cell preparations. We reprogrammed MSCs into iPSCs, which were subsequently redifferentiated toward MSCs. These iPS-MSCs revealed similar morphology, immunophenotype, in vitro differentiation potential, and gene expression profiles as primary MSCs. However, iPS-MSCs were impaired in suppressing T cell proliferation. DNA methylation (DNAm) profiles of iPSCs maintained donor-specific characteristics, whereas tissue-specific, senescence-associated, and age-related DNAm patterns were erased during reprogramming. iPS-MSCs reacquired senescence-associated DNAm during culture expansion, but they remained rejuvenated with regard to age-related DNAm. Overall, iPS-MSCs are similar to MSCs, but they reveal incomplete reacquisition of immunomodulatory function and MSC-specific DNAm patterns—particularly of DNAm patterns associated with tissue type and aging.

Human Nasal Mucosa Contains Tissue-Resident Immunologically Responsive Mesenchymal Stromal Cells

Multipotent mesenchymal stromal cells (MSC) are present in bone marrow and other tissues such as adipose tissue, muscle, pancreas, liver, and so on. Recent evidence suggests that MSC migrate to sites of infection, inflammation, and cancer, and interact with different immune cell subsets. Here, we report for the first time on the isolation and characterization of multipotent nasal mucosa-derived mesenchymal stromal cells (nm-MSC). nm-MSC showed a plastic adherent and fibroblast-like morphology and were able to form colonies. They expressed the typical bone marrow MSC marker antigens CD29, CD44, CD73, CD90, and CD105 and were able to differentiate along the adipogenic, chondrogenic, and osteogenic pathways. nm-MSC produced a set of inflammatory cytokines, expressed chemokine receptors, and were responsive to stimulation with cytokines, chemokines, and TLR4 ligand LPS. Thus, these cells may serve as an alternative adult stromal cell resource for regenerative tissue repair and may represent important regulators of local mucosal immunity.

Heparin concentration is critical for cell culture with human platelet lysate

BACKGROUND AIMS Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. METHODS Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. RESULTS At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. CONCLUSIONS Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized.

Matrix elasticity, replicative senescence and DNA methylation patterns of mesenchymal stem cells

Matrix elasticity guides 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 tissue culture plastic (TCP) or on polydimethylsiloxane (PDMS) gels of different elasticity 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 behavior while the cells reside on the substrate, but it does not have major impact on cell-intrinsic lineage determination, replicative senescence or DNAm patterns.

The bidirectional tumor - mesenchymal stromal cell interaction promotes the progression of head and neck cancer

IntroductionMesenchymal stromal cells (MSC) are an integral cellular component of the tumor microenvironment. Nevertheless, very little is known about MSC originating from human malignant tissue and modulation of these cells by tumor-derived factors. The aim of this study was to isolate and characterize MSC from head and neck squamous cell carcinoma (HNSCC) and to investigate their interaction with tumor cells.MethodsMSC were isolated from tumor tissues of HNSCC patients during routine oncological surgery. Immunophenotyping, immunofluorescence and in vitro differentiation were performed to determine whether the isolated cells met the consensus criteria for MSC. The cytokine profile of tumor-derived MSC was determined by enzyme-linked immunosorbent assay (ELISA). Activation of MSC by tumor-conditioned media was assessed by measuring cytokine release and expression of CD54. The impact of MSC on tumor growth in vivo was analyzed in a HNSCC xenograft model.ResultsCells isolated from HNSCC tissue met the consensus criteria for MSC. Tumor-derived MSC constitutively produced high amounts of interleukin (IL)-6, IL-8 and stromal cell-derived factor (SDF)-1α. HNSCC-derived factors activated MSC and enhanced secretion of IL-8 and expression of CD54. Furthermore, MSC provided stromal support for human HNSCC cell lines in vivo and enhanced their growth in a murine xenograft model.ConclusionsThis is the first study to isolate and characterize MSC from malignant tissues of patients with HNSCC. We observed cross-talk of stromal cells and tumor cells resulting in enhanced growth of HNSCC in vivo.

To clone or not to clone? Induced pluripotent stem cells can be generated in bulk culture

Induced pluripotent stem cells (iPSCs) are usually clonally derived. The selection of fully reprogrammed cells generally involves picking of individual colonies with morphology similar to embryonic stem cells (ESCs). Given that fully reprogrammed cells are highly proliferative and escape from cellular senescence, it is conceivable that they outgrow non-pluripotent and partially reprogrammed cells during culture expansion without the need of clonal selection. In this study, we have reprogrammed human dermal fibroblasts (HDFs) with episomal plasmid vectors. Colony frequency was higher and size was larger when using murine embryonic fibroblasts (MEFs) as stromal support instead of HDFs or human mesenchymal stromal cells (MSCs). We have then compared iPSCs which were either clonally derived by manual selection of a single colony, or derived from bulk-cultures of all initial colonies. After few passages their morphology, expression of pluripotency markers, and gene expression profiles did not reveal any significant differences. Furthermore, clonally-derived and bulk-cultured iPSCs revealed similar in vitro differentiation potential towards the three germ layers. Therefore, manual selection of individual colonies does not appear to be necessary for the generation of iPSCs – this is of relevance for standardization and automation of cell culture procedures.

Tracking of Replicative Senescence in Mesenchymal Stem Cells by Colony-Forming Unit Frequency

Long-term culture of mesenchymal stem cells (MSC) has major impact on cellular characteristics and differentiation potential. Numerous clinical trials raise high hopes in regenerative medicine and this necessitates reliable quality control of the cellular products-also with regard to replicative senescence. The maximum number of population doublings before entering the senescent state depends on the cell type, tissue of origin, culture medium as well as cell culture methods. Therefore, it would be valuable to predict the remaining proliferative potential in the course of culture expansion. Here, we describe a refined fibroblastic colony forming unit (CFU-f) assay which can be performed at any passage during culture expansion with simple cell culture techniques. This method is based on limiting dilutions in the 96-well format to determine the proportion of highly proliferative and clonogenic cells. The number of CFU-f declines rapidly during culture expansion. Especially at higher passages the CFU-f frequency correlates very well with the remaining cumulative population doublings. This approach can be used as quality measure to estimate the remaining proliferative potential of MSC in culture.