Calcium chloride declotted human platelet lysate promotes the expansion of mesenchymal stromal cells and allows manufacturing of immunomodulatory active extracellular vesicle products.

BACKGROUND: Mesenchymal stromal cells (MSCs) exert immunomodulatory effects, primarily through released extracellular vesicles (EVs). For the clinical-grade manufacturing of MSC-EV products culture conditions need to support MSC expansion and allow the manufacturing of potent MSC-EV products. Traditionally, MSCs are expanded in fetal bovine serum-supplemented media. However, according to good manufacturing practice (GMP) guidelines the use of animal sera should be avoided. To this end, human platelet lysate (hPL) has been qualified as an animal serum replacement. Although hPL outcompetes animal sera in promoting MSC expansion, hPL typically contains components of the coagulation system that need to be inhibited or removed to avoid coagulation reactions in the cell culture. Commonly, heparin is utilized as an anticoagulant; however, higher concentrations of heparin can negatively impact MSC viability, and conventional concentrations alone do not sufficiently prevent clot formation in prepared media. METHODS: To circumvent unwanted coagulation processes, this study compared various clotting prevention strategies, including different anticoagulants and calcium chloride (CaCl2)-mediated declotting methods, which in combination with heparin addition was found effective. We evaluated the influence of the differently treated hPLs on the proliferation and phenotype of primary bone marrow-derived MSCs and identified the CaCl2-mediated declotting method as the most effective option. To determine whether CaCl2 declotted hPL allows the manufacturing of immunomodulatory MSC-EV products, EVs were prepared from conditioned media of MSCs expanded with either conventional or CaCl2 declotted hPL. In addition to metric analyses, the immunomodulatory potential of resulting MSC-EV products was assessed in a recently established multi-donor mixed lymphocyte reaction assay. RESULTS AND CONCLUSIONS: Our findings conclusively show that CaCl2-declotted hPLs support the production of immunomodulatory-active MSC-EV products.

From 3D to 3D: isolation of mesenchymal stem/stromal cells into a three-dimensional human platelet lysate matrix.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) are considered an important candidate in cell therapy and tissue engineering approaches. The culture of stem cells in a 3D environment is known to better resemble the in vivo situation and to promote therapeutically relevant effects in isolated cells. Therefore, the aim of this study was to develop an approach for the direct isolation of MSCs from adipose tissue into a 3D environment, avoiding contact to a 2D plastic surface. Furthermore, the use of a cryoprotective medium for the cryopreservation of whole adipose tissue was evaluated. MATERIALS AND METHODS: Cryopreservation of fresh adipose tissue with and without a cryoprotective medium was compared with regard to the viability and metabolic activity of cells. After thawing, the tissue was embedded in a novel human platelet lysate-based hydrogel for the isolation of MSCs. The migration, yield, viability, and metabolic activity of cells from the 3D matrix were compared to cells from 2D explant culture. Also, the surface marker profile and differentiation capacity of MSCs from the 3D matrix were evaluated and compared to MSCs from isolation by enzymatic treatment or 2D explant culture. RESULTS: The cryopreservation of whole adipose tissue was found to be feasible, and therefore, adipose tissue can be stored and is available for MSC isolation on demand. Also, we demonstrate the isolation of MSCs from adipose tissue into the 3D matrix. The cells derived from this isolation procedure display a similar phenotype and differentiation capacity like MSCs derived by traditional procedures. CONCLUSIONS: The presented approach allows to cryopreserve adipose tissue. Furthermore, for the first time, MSCs were directly isolated from the tissue into a soft 3D hydrogel environment, avoiding any contact to a 2D plastic culture surface.

Epigenetic biomarker to support classification into pluripotent and non-pluripotent cells.

Quality control of human induced pluripotent stem cells (iPSCs) can be performed by several methods. These methods are usually relatively labor-intensive, difficult to standardize, or they do not facilitate reliable quantification. Here, we describe a biomarker to distinguish between pluripotent and non-pluripotent cells based on DNA methylation (DNAm) levels at only three specific CpG sites. Two of these CpG sites were selected by their discriminatory power in 258 DNAm profiles - they were either methylated in pluripotent or non-pluripotent cells. The difference between these two ß-values provides an Epi-Pluri-Score that was validated on independent DNAm-datasets (264 pluripotent and 1,951 non-pluripotent samples) with 99.9% specificity and 98.9% sensitivity. This score was complemented by a third CpG within the gene POU5F1 (OCT4), which better demarcates early differentiation events. We established pyrosequencing assays for the three relevant CpG sites and thereby correctly classified DNA of 12 pluripotent cell lines and 31 non-pluripotent cell lines. Furthermore, DNAm changes at these three CpGs were tracked in the course of differentiation of iPSCs towards mesenchymal stromal cells. The Epi-Pluri-Score does not give information on lineage-specific differentiation potential, but it provides a simple, reliable, and robust biomarker to support high-throughput classification into either pluripotent or non-pluripotent cells.

Epigenetic rejuvenation of mesenchymal stromal cells derived from induced pluripotent stem cells.

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.

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

INTRODUCTION: Mesenchymal 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. METHODS: MSC 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. RESULTS: Cells 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. CONCLUSIONS: This 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.

Stimulation of mesenchymal stromal cells (MSCs) via TLR3 reveals a novel mechanism of autocrine priming.

Mesenchymal stem/stromal cells (MSCs) are emerging as important regulators of innate and adaptive immunity. In this context, both proinflammatory and anti-inflammatory effects have been described for MSCs. The mechanisms mediating this functional plasticity are poorly characterized at present. Here, we investigated the inflammatory responses of MSCs isolated from human nasal mucosa (nmMSCs) upon challenge with different Toll-like receptor (TLR) ligands. We found that TLR3 ligands induced the strongest release of both proinflammatory cytokines [interleukin (IL)-6 and IL-8] and type I interferon by nmMSCs compared with other TLR ligands. Notably, TLR3 ligands triggered a biphasic cytokine response, with an early peak of type I interferon at 4 h poststimulation and a late release of proinflammatory cytokines at 24 h poststimulation. While the early interferon response was subject to direct stimulation, the proinflammatory response was regulated by factors released during the early cytokine response, which subsequently enhanced sensitivity to TLR3 ligation and amplified the production of IL-6 and IL-8 but not that of interferon. Taken together, our findings indicate that TLR3 ligands polarize the inflammatory phenotype of MSCs in a time-dependent manner. Thus, our study proposes a novel model that helps to explain the strikingly dichotomous functionality of MSCs in inflammation and immunoregulation.

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.

Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations.

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.

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.

Comparative functional cell biological analysis of mesenchymal stem cells of the head and neck region: potential impact on wound healing, trauma, and infection.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotent mesenchymal progenitor cells, originally identified in bone-marrow. Little is known about MSCs of the head and neck region. We investigated cell biological properties with a potential impact on wound healing of 2 different tissue-resident MSC populations. METHODS: MSCs were isolated from human nasal mucosa (nmMSCs) and parotid gland (pgMSCs). Clonogenic potential, cell surface markers, cytokine secretion, chemokine receptor expression, mobility, and adhesion to extracellular matrix were examined in unstimulated and stimulated MSCs. RESULTS: NmMSCs had the higher clonogenic potential. PgMSCs showed a broader panel of chemokine receptor expression and displayed higher mobility, especially after challenge with bacterial lipopolysaccharide (LPS). NmMSCs were less mobile and showed increased LPS-induced secretion of the inflammatory cytokine interleukin-8 (IL-8) compared with pgMSCs. CONCLUSION: These data highlight functional differences between tissue-resident MSCs of the head and neck region, which may impact functional properties of these cells in response to trauma or infection.

Human platelet lysate gel provides a novel three dimensional-matrix for enhanced culture expansion of mesenchymal stromal cells.

Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that-apart from heparin-consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix-this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion 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-ß1, 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-ßgal). 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-ß1, 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.

An additional glucose dehydrogenase from Sulfolobus solfataricus: fine-tuning of sugar degradation?

Within the SulfoSYS (Sulfolobus Systems Biology) project, the effect of temperature on a metabolic network is investigated at the systems level. Sulfolobus solfataricus utilizes an unusual branched ED (Entner-Doudoroff) pathway for sugar degradation that is promiscuous for glucose and galactose. In the course of metabolic pathway reconstruction, a glucose dehydrogenase isoenzyme (GDH-2, SSO3204) was identified. GDH-2 exhibits high similarity to the previously characterized GDH-1 (SSO3003, 61% amino acid identity), but possesses different enzymatic properties, particularly regarding substrate specificity and catalytic efficiency. In contrast with GDH-1, which exhibits broad substrate specificity for C5 and C6 sugars, GDH-2 is absolutely specific for glucose. The comparison of kinetic parameters suggests that GDH-2 might represent the major player in glucose catabolism via the branched ED pathway, whereas GDH-1 might have a dominant role in galactose degradation via the same pathway as well as in different sugar-degradation pathways.

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.

Interferon-gamma and tumor necrosis factor-alpha 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.

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.