The inherent immunomodulatory capacity of mesenchymal stem/progenitor cells (MSPCs) encouraged initiation of multiple clinical trials. Release criteria for therapeutic MSPCs cover identity, purity and safety but appropriate potency assessment is often missing. Reports on functional heterogeneity of MSPCs created additional uncertainty regarding donor and organ/source selection. We established a robust immunomodulation potency assay based on pooling responder leukocytes to minimize individual immune response variability. Comparing various MSPCs revealed significant potency inconsistency and generally diminished allo-immunosuppression compared to dose-dependent inhibition of mitogenesis. Gamma-irradiation to block unintended MSPC proliferation did not prohibit chondrogenesis and osteogenesis in vivo, indicating the need for alternative safety strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0233-8) contains supplementary material, which is available to authorized users.

Abstract:The regenerative and immunomodulatory activity of mesenchymal stromal cells (MSCs) is partially mediated by secreted vesicular factors. Extracellular vesicles (EVs) exocytosed by MSCs are gaining increased attention as prospective non-cellular therapeutics for a variety of diseases. However, the lack of suitable in vitro assays to monitor the therapeutic potential of EVs currently restricts their application in clinical studies. We have evaluated a dual in vitro immunomodulation potency assay that reproducibly reports the inhibitory effect of MSCs on induced T-cell proliferation and the alloantigen-driven mixed leukocyte reaction of pooled peripheral blood mononuclear cells in a dose-dependent manner. Phytohemagglutinin-stimulated T-cell proliferation was inhibited by MSC-derived EVs in a dose-dependent manner comparable to MSCs. In contrast, inhibition of alloantigen-driven mixed leukocyte reaction was only observed for MSCs, but not for EVs. Our results support the application of a cell-based in vitro potency assay for reproducibly determining the immunomodulatory potential of EVs. Validation of this assay can help establish reliable release criteria for EVs for future clinical studies.

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation.Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC.Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells.Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.

Background/Aims: The function of β-cells is regulated by nutrient uptake and metabolism. The cells′ metabolic state can be expressed as concentration ratios of AMP, ADP and ATP. Relative changes in these ratios regulate insulin release. An increase in the intracellular ATP concentration causes closure of K_ATP channels and cell membrane depolarization, which triggers stimulus-secretion coupling (SSC). In addition to K_ATP channels, the AMP-dependent protein kinase (AMPK), a major cellular fuel sensor in a variety of cells and tissues, also affects insulin secretion and β-cell survival. In a previous study we found that the widely used AMPK inhibitor compound C retards proliferation and induces apoptosis in the rat β-cell line INS-1E. We therefore tested the effects of AMPK activators (AICAR and metformin), and compound C on AMPK phosphorylation, insulin secretion, K_ATP channel currents, cell membrane potential, intracellular calcium concentration, apoptosis and cell cycle distribution of INS-1E cells under standard cell culture conditions (11 mM glucose). Methods: Western blotting, ELISA, patch-clamp, calcium imaging and flow cytometry. Results: We found that basal AMPK phosphorylation is enhanced by AICAR (1 mM) and metformin (1 mM) but remained unaffected by compound C (10 µM). Both AICAR and compound C stimulated basal insulin secretion whereas metformin had no effect. Pre-incubation with AICAR (1 mM) caused an inhibition of K_ATP currents but did not significantly alter the average cell membrane potential (Vm) or the threshold potential of electrical activity. Acute administration of AICAR (300 µM) led to a depolarization of Vm, which was not due to an inhibition of the basal- or glucose-induced chloride conductance, and was not accompanied by elevations of intracellular calcium (Ca_i). AICAR had no additive blocking effect on K_ATP currents when applied together with tolbutamide. Compound C applied over 24 hours induced an increase in the percentage of cells positive for caspase activity, whereas AICAR (1 mM) applied for 48 hours was without effect. Medium glucose concentration <3 mM caused cell cycle arrest, caspase activation and an increase of cell granularity. Conclusion: We conclude that under standard cell culture conditions the AMPK modulators AICAR and compound C, but not metformin, stimulate insulin secretion by AMPK-independent mechanisms.

Background/Aims: Glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells involves glucose uptake and metabolism, closure of K_ATP channels and depolarization of the cell membrane potential (V_mem), activation of voltage-activated Ca²⁺ currents (ICa_v) and influx of Ca²⁺, which eventually triggers hormone exocytosis. Beside this classical pathway, K_ATP-independent mechanisms such as changes in intracellular pH (pH_i) or cell volume, which also affect β-cell viability, can elicit or modify insulin release. In β-cells the regulation of pH_i is mainly accomplished by Na⁺/H⁺ exchangers (NHEs). To investigate if other proton extrusion mechanisms than NHEs are involved in pH regulation, we tested for the presence of the non-gastric H⁺/K⁺ ATPase in rat insulinoma cells and assessed effects of the H⁺/K⁺ ATPase inhibitor SCH-28080 on insulin secretion, cell viability and apoptosis. Methods: In INS-1E cell cultures, H⁺/K⁺ ATPase gene and protein expression was analyzed by reverse transcription PCR and Western blotting. Intracellular pH (pH_i) recovery after acute acidic load was measured by NH₄Cl prepulsing using BCECF. Insulin secretion was determined by ELISA from the cell culture supernatant. V_mem, K⁺ and Ca²⁺ currents were recorded using patch clamp. Overall cell responses were determined using resazurin (viability) and cytotoxicity assays. The mean cell volume (MCV), cell granularity (side-scatter; SSC), phosphatidylserine (PS) exposure, cell membrane integrity, caspase activity and the mitochondrial membrane potential (ΔΨ_m) were measured by flow cytometry. Results: We found that the α-subunit of the non-gastric H⁺/K⁺ ATPase (HKα2) is expressed on mRNA and protein level. However, compared to rat colon tissue, in INS-1E cells mRNA abundance was very low. In NH₄Cl prepulsing experiments no K⁺-dependent pH_i recovery was observed under Na⁺-free extracellular conditions. Nonetheless within 1 h, 20 µM SCH-28080 inhibited GSIS by ∼50%, while basal release was unaffected. The L-type ICa_v blocker nifedipine caused a full inhibition of GSIS at 10 and 20 µM. At 20 µM, SCH-28080 inhibited ICa_v comparable to 20 µM nifedipine and in addition augmented IK_ATP recorded at -60 mV and hyperpolarized V_mem by ∼15 mV. Cell viability 2 and 24 h post treatment with SCH-28080 was dose-dependently inhibited with IC₅₀ values of 22.9 µM and 15.3 µM, respectively. At 20 µM the percentages of Annexin-V+, caspase+ and propidium iodide+ cells were significantly increased after 24 and 48 h. Concurrently, the MCV was significantly decreased (apoptotic volume decrease, AVD) and the SSC signal was increased. At concentrations >40–50 µM, SCH-28080 became progressively cytotoxic causing a steep i...