Expression of HLA-DR by mesenchymal stromal cells in the platelet lysate era: an obsolete release criterion for MSCs?

BACKGROUND: According to the definition of the International Society for Cell and Gene Therapy (ISCT), mesenchymal stromal cells (MSCs) do not express HLA-DR. This phenotypic marker as a release criterion for clinical use was established at a time when MSCs were expanded in fetal bovine serum (FBS)-containing media. Replacement of FBS with platelet lysate (PLs) as a medium supplement induced a significantly higher fraction of MSCs to express MHC class II antigens. METHODS: As this raised concerns that such MSCs may play the role of antigen-presenting cells for T cells, in the current study, we studied major factors that may induce HLA-DR on MSCs by means of flow cytometry and real-time polymerase chain reaction. The immunomodulatory potential of MSCs was assessed by a mixed lymphocyte reaction. RESULTS: Our results demonstrated that a very low percentage of generated and expanded MSCs in FBS express HLA-DR (median: 1.1%, range: 0.3-22%) compared to MSCs generated and expanded in PLs (median: 28.4%, range: 3.3-73.7%). Analysis of the cytokine composition of ten PLs showed a significant positive correlation between the levels of IL-1ß, IL-4, IL-10, IL-17, bFGF and expression of HLA-DR, in contrast to no correlation with the age of MSC donors and HLA-DR (r = 0.21). Both MSCs expressing low and high levels of HLA-DR expressed class II transactivator (CIITA), a master gene coding for these molecules. Our results demonstrate for the first time that MSCs with constitutively high levels of HLA-DR also express moderate levels of indoleamine 2,3-dioxygenase (IDO). Treatment of MSCs with multiple doses of TGF-ß1 at passage 0 (P0) and passage 1 (P1) completely abrogated HLA-DR and IDO expression. In contrast, treatment of MSCs with a single dose of TGF-ß1 after P0 only partially reduced the expression of HLA-DR and CIITA. Remarkably, increased expression of HLA-DR on MSCs that constitutively express high levels of this antigen after overnight incubation with IFN-γ was rather unaffected by incubation with TGF-ß1. However, treatment of MSCs with TGF-ß1 for 24 h completely abrogated constitutive expression of IDO. CONCLUSIONS: Irrespective of HLA-DR expression at the population level, all MSC preparations significantly inhibited the proliferation of stimulated peripheral blood mononuclear cells, indicating that HLA-DR represents an obsolete release marker for the clinical use of MSCs.

Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation.

Mesenchymal stromal cells (MSCs) have the potential to suppress pathological activation of immune cells and have therefore been considered for the treatment of Graft-versus-Host-Disease. The clinical application of MSCs requires a process validation to ensure consistent quality. A flow cytometry-based mixed lymphocyte reaction (MLR) was developed to analyse the inhibitory effect of MSCs on T cell proliferation. Monoclonal antibodies were used to stimulate T cell expansion and determine the effect of MSCs after four days of co-culture based on proliferation tracking with the violet proliferation dye VPD450. Following the guidelines of the International Council for Harmonisation (ICH) Q2 (R1), the performance of n = 30 peripheral blood mononuclear cell (PBMC) donor pairs was assessed. The specific inhibition of T cells by viable MSCs was determined and precision values of <10% variation for repeatability and <15% for intermediate precision were found. Compared to a non-compendial reference method, a linear correlation of r = 0.9021 was shown. Serial dilution experiments demonstrated a linear range for PBMC:MSC ratios from 1:1 to 1:0.01. The assay was unaffected by PBMC inter-donor variability. In conclusion, the presented MLR can be used as part of quality control tests for the validation of MSCs as a clinical product.

Cell penetrating peptides are versatile tools for enhancing multimodal uptake into cells from pest insects.

Cell penetrating peptides (CPPs) are small peptides defined by their ability to deliver molecular cargo into cells. While the subject of frequent investigation for pharmaceutical drug delivery, little consideration has been given to the possibility of CPPs for use as insecticides or insecticide enhancers. Here, we characterize the entry of four fluorescently tagged CPPs into two insect cell lines and dissected midgut tissues in terms of both total quantity and mode of penetration. Fluorescent microscopy showed that substantial amounts of CPPs penetrate the plasma membrane via endosomal uptake in ovarian (Sf9) and midgut derived (AW1) lepidopteran cells and that this process was sensitive to selected endocytosis inhibitors. Differences in the quantity of uptake was observed between CPPs, and further differences were found in the ability CPP-1838 to efficiently penetrate membranes through passive diffusion. These findings were extended to primary midgut derived cells and dissected tissues suggesting that CPPs show a preference for goblet cells and that CPP-1838 shows far higher rates of penetration. CPP-1838 thus shows extraordinary abilities to penetrate cells efficiency in both a diffusional and endocytotic manner. From these results more sophisticated delivery methods based on the utilization of CPPs can be developed.

Characterization of a novel pesticide transporter and P-glycoprotein orthologues in Drosophila melanogaster.

Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge about mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect Drosophila melanogaster. An RNAi screen was performed, which knocked down individual ABCs in specific epithelial tissues and examined the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This implicated a novel ABC drug transporter, CG4562, in spinosad transport, but also highlighted the P-glycoprotein orthologue Mdr65 as the most impactful ABC in terms of chemoprotection. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, transgenic Drosophila lines expressing P-glycoprotein from the major malaria vector Anopheles gambiae were used to establish a system for in vivo characterization of this transporter in non-model insects. This study provides the basis for establishing Drosophila as a model for toxicology research on drug transporters.

Can the mammalian organoid technology be applied to the insect gut?

Mammalian intestinal organoids are multicellular structures that closely resemble the structure of the intestinal epithelium and can be generated in vitro from intestinal stem cells under appropriate culture conditions. This technology has transformed pharmaceutical research and drug development in human medicine. For the insect gut, no biotechnological platform equivalent to organoid cultures has been described yet. Comparison of the regulation of intestinal homeostasis and growth between insects and mammals has revealed significant similarities but also important differences. In contrast to mammals, the differentiation potential of available insect cell lines is limited and can not be exploited for in vitro permeability assays to measure the uptake of insecticides. The successful development of in vitro models could be a result of the emergence of molecular mechanisms of self-organization and signaling in the intestine that are unique to mammals. It is nevertheless considered that the technology gap is a consequence of vast differences in knowledge, particularly with respect to culture conditions that maintain the differentation potential of insect midgut cells. From the viewpoint of pest control, advanced in vitro models of the insect midgut would be very desirable because of its key barrier function for orally ingested insecticides with hemolymphatic target and its role in insecticide resistance. © 2020 Society of Chemical Industry.

Functional characterization and transcriptomic profiling of a spheroid-forming midgut cell line from Helicoverpa zea (Lepidoptera: Noctuidae).

Insect cell lines have been frequently used in insect science research in recent years. Establishment of cell lines from specialized tissues like the lepidopteran midgut is expected to facilitate research efforts towards the understanding of uptake and metabolic properties, as well as the design of assays for use in pesticide discovery. However, the number of available lines from specialized tissues of insects and the level of understanding of the biological processes taking place in insect cells is far behind mammalian systems. In this study we examine two established cell lines of insect midgut origin, investigate their growth parameters and amenability to transfection and genetic manipulation, and test their potential to form spheroid-like 3D structures. Our results indicate that a midgut-derived cell line from Helicoverpa zea, RP-HzGUT-AW1, is amenable to genetic manipulation by transfection with a standard insect expression vector and has excellent ability to form spheroids. To further investigate the differentiation status of this line, we examined for expression of several candidate marker genes from different midgut cell types, enterocytes (ECs), Goblet cells (GCs), enteroendocrine cells (EEs) and intestinal stem cells (ISCs), indicating that both certain ISC and certain differentiated cell markers were present. To acquire a more detailed perspective of the differentiation landscape of the specific cells, we performed an RNAseq analysis of RP-HzGUT-AW1 grown either in 2D or 3D cultures. We hypothesize that RP-HzGUT-AW1 are in an "arrested" developmental stage between ISC and terminal differentiation. Furthermore, an enrichment of stress response and oxidoreductase genes was observed in the spheroid samples while no significant difference was evident in differentiation markers between cells grown in 2D and 3D. These results render RP-HzGUT-AW1 as the most well-characterized insect gut derived cell line so far, and lay the groundwork for future work investigating midgut cell lines application potential.

Receptor assay guided structure-activity studies of helicokinin insect neuropeptides and peptidomimetic analogues.

Neuropeptides control numerous physiological processes in insects. The regulation of water balance is a crucial aspect of homeostasis in terrestrial insects and has been shown to be under endocrine control, primarily by corticotrophin releasing factor (CRF)-related peptides and kinins. For helicokinin I, a diuretic neuropeptide from the economically important insect pest Heliothis virescens, detailed structure-activity relationships have been established based on truncated structures, diverse amino acid scans and peptidomimetic analogues. The activities of selected compounds on functional expressed helicokinin receptors are compared with the results of a Malphigian tubule assay. Implications for further peptidomimetic variations are provided.

Insecticidal heterolignans--tubuline polymerization inhibitors with activity against chewing pests.

Starting from natural product podophyllotoxin 1 substituted heterolignans were identified with promising insecticidal in vivo activity. The impact of substitution in each segment of the core structure was investigated in a detailed SAR study, and variation of substituents in both aromatic moieties afforded derivatives 5 and 43 with broad insecticidal activity against lepidopteran and coleopteran species. In vitro measurements supported by modeling studies indicate that heterolignans 3-134 act as tubuline polymerization inhibitors interacting with the colchicine-binding site. Insect specific structure-activity effects were observed showing that the insecticidal SAR described herein differs from reported cytotoxicity studies.