Signal Factors Secreted by 2D and Spheroid Mesenchymal Stem Cells and by Cocultures of Mesenchymal Stem Cells Derived Microvesicles and Retinal Photoreceptor Neurons

We aim to identify levels of signal factors secreted by MSCs cultured in 2D monolayers (2D-MSCs), spheroids (spheroids MSCs), and cocultures of microvesicles (MVs) derived from 2D-MSCs or spheroid MSCs and retinal photoreceptor neurons. We seeded 2D-MSCs, spheroid MSCs, and cells derived from spheroids MSCs at equal numbers. MVs isolated from all 3 culture conditions were incubated with 661W cells. Levels of 51 signal factors in conditioned medium from those cultured conditions were quantified with bead-based assay. We found that IL-8, IL-6, and GROαwere the top three most abundant signal factors. Moreover, compared to 2D-MSCs, levels of 11 cytokines and IL-2Rαwere significantly increased in conditioned medium from spheroid MSCs. Finally, to test if enhanced expression of these factors reflects altered immunomodulating activities, we assessed the effect of 2D-MSC-MVs and 3D-MSC-MVs on CD14+ cell chemoattraction. Compared to 2D-MSC-MVs, 3D-MSC-MVs significantly decreased the chemotactic index of CD14+ cells. Our results suggest that spheroid culture conditions improve the ability of MSCs to selectively secrete signal factors. Moreover, 3D-MSC-MVs also possessed an enhanced capability to promote signal factors secretion compared to 2D-MSC-MVs and may possess enhanced immunomodulating activities and might be a better regenerative therapy for retinal degenerative diseases.

Evidence of an Evolutionarily Conserved LMBR1 Domain-Containing Protein That Associates with Endocytic Cups and Plays a Role in Cell Migration in Dictyostelium discoideum

ABSTRACT The ampA gene plays a role in Dictyostelium discoideum cell migration. Loss of ampA function results in reduced ability of growing cells to migrate to folic acid and causes small plaques on bacterial lawns, while overexpression of AmpA results in a rapid-migration phenotype and correspondingly larger plaques than seen with wild-type cells. To help understand how the ampA gene functions, second-site suppressors were created by restriction enzyme-mediated integration (REMI) mutagenesis. These mutants were selected for their ability to reduce the large plaque size of the AmpA overexpresser strain. The lmbd2B gene was identified as a suppressor of an AmpA-overexpressing strain. The lmbd2B gene product belongs to the evolutionarily conserved LMBR1 protein family, some of whose known members are endocytic receptors associated with human diseases, such as anemia. In order to understand lmbd2B function, mRFP fusion proteins were created and lmbd2B knockout cell lines were established. Our findings indicate that the LMBD2B protein is found associated with endocytic cups. It colocalizes with proteins that play key roles in endocytic events and is localized to ruffles on the dorsal surfaces of growing cells. Vegetative lmbd2B -null cells display defects in cell migration. These cells have difficulty sensing the chemoattractant folic acid, as indicated by a decrease in their chemotactic index. lmbd2B -null cells also appear to have difficulty establishing a front/back orientation to facilitate migration. A role for lmbd2B in development is also suggested. Our research gives insight into the function of a previously uncharacterized branch of the LMBR1 family of proteins. We provide evidence of an LMBR1 family plasma membrane protein that associates with endocytic cups and plays a role in chemotaxis.

Different Roles of Membrane Potentials in Electrotaxis and Chemotaxis of Dictyostelium Cells

ABSTRACT Many types of cells migrate directionally in direct current (DC) electric fields (EFs), a phenomenon termed galvanotaxis or electrotaxis. The directional sensing mechanisms responsible for this response to EFs, however, remain unknown. Exposing cells to an EF causes changes in plasma membrane potentials ( V m ). Exploiting the ability of Dictyostelium cells to tolerate drastic V m changes, we investigated the role of V m in electrotaxis and, in parallel, in chemotaxis. We used three independent factors to control V m : extracellular pH, extracellular [K + ], and electroporation. Changes in V m were monitored with microelectrode recording techniques. Depolarized V m was observed under acidic (pH 5.0) and alkaline (pH 9.0) conditions as well as under higher extracellular [K + ] conditions. Electroporation permeabilized the cell membrane and significantly reduced the V m , which gradually recovered over 40 min. We then recorded the electrotactic behaviors of Dictyostelium cells with a defined V m using these three techniques. The directionality (directedness of electrotaxis) was quantified and compared to that of chemotaxis (chemotactic index). We found that a reduced V m significantly impaired electrotaxis without significantly affecting random motility or chemotaxis. We conclude that extracellular pH, [K + ], and electroporation all significantly affected electrotaxis, which appeared to be mediated by the changes in V m . The initial directional sensing mechanisms for electrotaxis therefore differ from those of chemotaxis and may be mediated by changes in resting V m .

Effects of Garlic Oil on the Migration of Neutrophil-Like Cell Studied by Using a Chemotactic Gradient Labchip

We have designed and fabricated a novel chemotactic gradient Labchip for studying cell migration quantitatively. Owing to the great potential of garlic and its preparations in developing antiinflammatory drugs, the aim of the present study is to investigate the effect of garlic oil on the locomotion of a neutrophil-like cell by measuring the dynamic features of cell migration including migration direction, average migration speed, chemotactic index (CI), and motility index (MI) with the newly designed Labchip. We found that garlic oil treatment lowered the values of CI and MI and reduced the average speed of cell migration from 13 to 8 μm/min. The results indicate that garlic oil is a potential inhibitor for neutrophil-like cell migration and chemotactic responsiveness. By comparing with the effects of nocodazole and cytochalasin B, we also suggest that the antiinflammatory activity exhibited by garlic oil was mainly through inhibiting the assembly-disassembly processes of the cytoskeleton.

In Vitro Chemotactic Index and Response to CXCR4 Antagonist AMD3100 Correlate with CD34+ Cell Yield from Leukapheresis Products: Implications for Poor Mobilizers.

While autologous hematopoietic stem/progenitor cell (HSPC) transplantation has been used successfully for many years, new approaches are being explored to boost the number of HSPC (CD34+ cells) collected by leukapheresis to permit more rapid hematopoietic recovery. Still, a considerable number of patients may be excluded from this procedure because a sufficient quantity of HSPC cannot be obtained by standard mobilization agents. Among factors regulating mobilization is stromal cell-derived factor (SDF)-1 constitutively produced by bone marrow (BM) stromal cells and which is known to retain HSPC in the BM. Responsiveness to an SDF-1 gradient can be measured in vitro using the chemotactic index, defined as the ratio of the number of cells that migrate towards SDF-1 and the number of cells that migrate towards media alone. We hypothesized that (i) a high chemotactic index would indicate a greater ability of the HSPC to be retained in their BM niches and therefore more difficulty in coaxing them out into the circulation, as could be the case in poor mobilizers; and that (ii) AMD3100, an antagonist of the SDF-1 receptor CXCR4, will have a greater disruptive effect on SDF-1-dependent signalling in poor mobilizers. Leukapheresis products were obtained from patients diagnosed with Hodgkin’s or non-Hodgkin’s lymphoma and CD34+ cells isolated by positive selection were loaded onto Boyden chambers and allowed to migrate across bare filters towards a gradient of SDF-1 (200 ng/mL). In some experiments cells were incubated with AMD3100 (10 mg/mL) for the duration of the assay. We observed broad inter-patient differences in in vitro migratory ability of CD34+ cells, ranging from 3.1 ± 0.6 to 21.7 ± 2.1 % for spontaneous or passive percentage migration; and from 11.1 ± 0.7 to 58.6 ± 10.7 % for SDF-1-directed chemotaxis, neither of which bore any correlation with the number of CD34+ cells/kg obtained from the leukapheresis product. However, a negative correlation (r=−0.7) was found between the chemotactic index and the percentage of CD34+ cells obtained from the leukapheresis product, i.e., the good mobilizers responded poorly to an SDF-1 gradient whereas the poor mobilizers responded better. Also a higher expression of CXCR4 was observed (by flow cytometry) in samples from poor mobilizers. Chemotaxis towards SDF-1 was reduced by 10 to 80% by AMD3100 and there was a positive correlation (r=0.6) between chemotactic index and percentage inhibition, i.e., chemotaxis of poor mobilizers was significantly more inhibited by this CXCR4 antagonist. Thus our results suggest that the chemotactic index could be employed as a predictor of good vs. poor mobilization; optimal mobilization especially in poor mobilizers may be achieved by a protocol with both G-CSF and AMD3100; for good mobilizers, including AMD3100 for mobilization could lead to a reduced requirement for volume of leukapheresis product and number of collections; and finally in an allogeneic transplant setting the chemotactic index could be used to predict whether a normal donor of HSPC will be a good or poor mobilizer.

The release of eosinophil chemotactic activity and eosinophil chemokinesis inhibitory activity by mononuclear cells from atopic asthmatic and non-atopic subjects

The goal of our study was to assess the chemotactic activity for eosinophils (ECA) and neutrophils (NCA) and histamine releasing activity (HRA) in crude supernatants of mononuclear cells in monosensitized atopic asthmatics and healthy controls. Chemotactic activity for ECA and neutrophils was measured in supernatants of cultured mononuclear cells with modified Boyden’s chamber and HRA was assessed on healthy donor basophils. With respect to ECA generation two distinct subgroups of subjects were distinguished: releasers [ECA (+)] and non-releasers [ECA (–)]. In atopic and non-atopic ECA (+) the mean ECA index was 3.78 ± 0.49 and 2.47 ± 0.27 respectively (P> 0.05). Supernatants from the remaining subjects (seven of 22 atopic and five of 11 non-atopic) did not express ECA, but revealed significant inhibitory activity for chemokinesis of eosinophils (mean chemotactic index 0.25 ± 0.16 and 0.48 ± 0.22 for atopic and non-atopic non-releasers respectively). Stimulation with antigen of MNC from atopic and with PHA from non-atopic ECA (–) restored cells ability to release ECA. Sephadex gel chromatography revealed that supernatants of MNC contained chemotactic and chemokinesis inhibitory activity in different fractions. The spontaneous productions of NCA and HRA by mononuclear cells was sim ilar in ECA releasers and non-releasers, although the HRA was higher following stimulation with PHA in the non-atopic ECA (+) subgroup. Our study demonstrated, for the first time, that MNC are capable of generating not only chemotactic activity but also chemokinesis inhibitory activity for eosinophils.

Exposure To Dextran During Isolation Enhances The Chem0- Tactic Response Of Human Peripheral Polymorphonuclear Leukocytes To Synthetic N-F-Met-Leu-Phe But Not To Zymosan Activated Serum (ZAS)

The effect of the method of isolation of human peripheral polymorphonuclear leukocytes (PMN) on their chemotactic response to ZAS and N-f-met-leu-phe was studied. Chemotoxis was measured in Boyden chambers by counting the cells at consecutive 10 μM depths of the filters and calculating the chemotactic index (CI). PMN isolated by two-step ficoll-hypaque gradients and resuspended in medium 199 with 0.035% human albuman had a control CI of 9.75. With ZAS (1:2) as the chemoattractant in the lower chamber (1 hr at 37°C, CO2 incubator) the CI was 31.44 (222% increase). Exposure of gradient isoalted PMS to a 1-1000 dilution of DMSO increased the baseline CI to 24.72 when the medium remained the same. N-f-met-leu-Phe as the chemoattractant in the lower chamber illicited a very modest (19% increase) over a range of 10-10 to 10-5 M. In contrast, PMN isolated by a combination if ficoll- hypaque cushion and dextran sedimentation responded to N-f-met-leu-phe with an increase from baseline CI of 18.75 to stimulated CI of 33.18 at 10-8 M (77% increase). After exposure to dextran PMN isolated by the two-step gradient also responded to N-f-met-leu-phe. Thus, a low concentration of dextran or other glucose polymer may be required for the response of PMS to synthetic chemotactic peptides. This is consistent with the wide spread use of PMN isolated by dextran sedimentation or PMN from pero-tineal exudate elicited by the injection of glycogen solution. Glucose polymers might replace bacterial surface carbohydrates and function to absorb the peptides thereby making an active polyvalent ligand.