Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

Shantou University

Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B-1, that express CD5. Here, we describe our studies with a representative CD5+ B-cell-derived natural autoantibody, the VH11Vkappa9 B-cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5-10% of the CD5+ B-cell subset, with a large portion accounted for by two novel BCRs, VH11Vkappa9 and VH12Vkappa4. We have found that the development of B-lineage cells with a VH11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i). one of the most common VH11 rearrangements generates a heavy-chain protein that only inefficiently assembles a pre-BCR, key for recombinase-activating gene downregulation/allelic exclusion and pre-B-clonal expansion; (ii). cells containing VH11- micro chains lacking N-addition are favored for progression to the B-cell stage, eliminating most bone marrow VH11 rearrangements; and (iii). only a subset of Vkappa-light chains combine with VH11 heavy chain to foster progression to the mature B-cell stage. Together, these constrain VH11 generation to fetal development and may favor production of B cells with the prototype VH11Vkappa9 BCR.

Selection during development of VH11+ B cells: a model for natural autoantibody-producing CD5+ B cells.

Islet transplantation represents an important alternative for the treatment of diabetes. However, the selection of suitable materials is critical for the success of such an implantation application. In this study, cellular migration, aggregation, and insulin production of a murine islet beta-cell line, NIT-1 cells on microbially produced polyesters poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) or polylactic acid (PLA) films were investigated. Spherical islet-like structures were only detected on PHBHHx films after 48 h cultivation. To understand the mechanism underlying the formation of cell aggregates, NIT-1-GFP, a stable transfectant of the green fluorescent protein was used in a time-lapse imaging study. Cell aggregation began on PHBHHx at 2 h, and became obvious at 4 h. Furthermore, cells on PHBHHx displayed higher metabolic activities measured by MTT assay than that on tissue culture plate. More importantly, insulin gene expression as well as extracellular secretion was upregulated after growth on PHBHHx for 72 h. Thus, PHBHHx can be a strong candidate for islet transplantation.

Enhanced insulin production from murine islet beta cells incubated on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate).

Understanding the mechanisms of beta-cell dynamics in postnatal animals is central to cure diabetes. A major obstacle in evaluating the status of pancreatic cells is the lack of surface markers. Here we performed quantitative measurements of two internal markers to follow the developmental history of islets. One marker, cell-cycle activity, was established by measuring expression of Ki67 and the incorporation of 5-bromodeoxyuridine. The other marker, the aging process, was delineated by the determination of telomere length. Moreover, islet neogenesis, possibly from ductal precursors, was monitored by pancreatic duct labeling with an enhanced green fluorescence protein (EGFP) transgene. We found that islets from younger animals, on average, expressed higher Ki67 transcripts, displayed elevated 5-bromodeoxyuridine incorporation, and had longer telomeres. However, significant heterogeneity of these parameters was observed among islets from the same mouse. In contrast, the levels of proinsulin-1 transcripts in islets of different ages did not change significantly. Moreover, mitotic activities correlated significantly with telomere lengths of individual islets. Lastly, after 5.5 d pancreatic duct labeling, a few EGFP-positive islets could be identified in neonatal but not from adult pancreases. Compared with unlabeled control islets, EGFP-positive islets had higher mitotic activities and longer telomeres. The results suggest that islets are born at different time points during the embryonic and neonatal stages and imply that young islets might play an important role in the maintenance of islet mass in the adult pancreas.

Heterogeneity in mitotic activity and telomere length implies an important role of young islets in the maintenance of islet mass in the adult pancreas.

Biopolyesters of polyhydroxyalkanoates (PHAs), including poly-3-hydroxybutyrate (PHB), co-polyester of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), and co-polyester of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) have been well investigated for their biocompatibility. For in vivo application, it is very important that the degradation products of PHAs, especially the oligomers, are not harmful to the cells and surrounding tissues. In this study, in vitro effects of oligo(3-hydroxybutyrate) (OHB), oligo(3-hydroxybutyrate-co-4-hydroxybutyrate) (O3HB4HB) and oligo(3-hydroxybutyrate-co-3-hydroxyhexanoate) (OHBHHx) on growth and differentiation of the murine beta cell line NIT-1 were investigated. Among the three oligo-hydroxyalkanoates (Oligo-HAs), cells treated with OHBHHx displayed higher viability, as measured by CCK-8 assay. Flow cytometric analysis of NIT-1 cells indicated that Oligo-HAs had an inhibitory effect on cell apoptosis. The cytosolic Ca(2+) transient of NIT-1 cells increased when fed with 0.04 g/l Oligo-HAs. For gap junction intercellular communication of cells, the effect of OHBHHx was the best among all materials tested. More importantly, extracellular insulin secretion was up-regulated after growing in OHBHHx for 48 h. The results demonstrated that the degradation products of PHAs, especially OHBHHx from PHBHHx, were not harmful to the beta cells. Therefore, PHBHHx warrant further study for application as a pancreatic tissue engineering material.

Effects of oligo(3-hydroxyalkanoates) on the viability and insulin secretion of murine beta cells.

Gene therapy provides a promising approach to curing diabetes. However, an effective route for islet-specific targeting has yet to be established. Toward this end, the pancreatic blood circulation system in Balb/c mice was determined by the injection of rhodamine-containing beads. The efficiency of islet targeting was then measured by the injection of adenoviral vectors carrying a green fluorescence gene via the celiac trunk (C.T.). The results showed that >95% of islets and about 60% of β cells within the pancreatic body and tail could be labeled 3 days after surgery. α-Cell labeling was not as efficient, whereas labeling of nonendocrine tissues was barely detectable. For proof of principle, adenoviral vectors carrying a Sirtuin transgene were injected similarly to test the islet protection effect in the streptozotocin (STZ)-induced type 1 diabetic model. The results demonstrated that overexpression of Sirtuin in STZ-treated mice reduced the level of β-cell death and extent of glucose intolerance. This study reports on efficient islet-specific targeting by using adenoviral injection. This procedure could be invaluable to the treatment of diabetes and the study of islet biology.

Intra-arterial targeted islet-specific expression of Sirt1 protects β cells from streptozotocin-induced apoptosis in mice.

Chronic administration of nicotinic acid (NA), a potent antilipidemic compound, aggravates glycemic control in diabetic patients. It is not known if NA has direct effects on islet β cells.

Nicotinic acid inhibits glucose-stimulated insulin secretion via the G protein-coupled receptor PUMA-G in murine islet β cells.

Our previous study showed an efficient targeting of islets of Langerhans by adenoviral injection via the celiac trunk. Unexpectedly, none of the endothelial cells was infected given the direct contact between adenoviruses and the capillary wall. The present study intended to provide an efficient approach for adenoviral targeting of the microcapillary endothelial cells in the pancreas.

Transient embolization with microspheres of polyhydroxyalkanoate renders efficient adenoviral transduction of pancreatic capillary in vivo.

Ectopically expressed Cre recombinase in extrapancreatic tissues in RIP-Cre mice has been well documented. The objective of this study was to find a simple solution that allows for improved beta-cell specific targeting. To this end, the RIP-Cre and reporter CMV-loxP-DsRed-loxP-EGFP expression cassettes were configurated into a one-plasmid and two-plasmid systems, which labeled approximately 80% insulin-positive INS-1 cells after 48 h transfection. However, off-target labeling was robustly found in more than 15% insulin-negative Ad293 cells. When an IRES element was inserted in front of Cre to reduce the translation efficiency, the ratio of recombination between INS-1 and Ad293 cells increased 3-4-fold. Further, a series of Cre mutants were generated by site-directed mutagenesis. When one of the mutants, Cre(H289P) in both configurations, was used in the experiment, the percentage of recombination dropped to background levels in a number of insulin-negative cell lines, but decreased only slightly in INS-1 cells. Consistently, DNA substrate digestion assay showed that the enzymatic activity of Cre(H289P) was reduced by 30-fold as compared to that of wild-type. In this study, we reported the generation of constructs containing RIP and Cre mutants, which enabled enhanced beta-cell specific labeling in vitro. These tools could be invaluable for beta-cell targeting and to the study of islet development.

Increased Specific Labeling of INS-1 Pancreatic Beta-Cell by Using RIP-Driven Cre Mutants with Reduced Activity.

The objective of this study was to determine if plasma membrane vesicles (PMVs) could be exploited for efficient transfer of macro-biomolecules and mitochondria. PMVs were derived from mechanical extrusion, and made fusogenic (fPMVs) by incorporating the glycoprotein G of vesicular stomatitis virus (VSV-G). Confocal microscopy examination revealed that cytoplasmic proteins and mitochondria were enclosed in PMVs as evidenced by tracing with cytoplasmically localized and mitochondria-targeted EGFP, respectively. However, no fluorescence signal was detected in PMVs from cells whose nucleus was labeled with an EGFP-tagged histone H2B. Consistently, qRT-PCR measurement showed that mRNA, miRNA and mitochondrial DNA decreased slightly; while nuclear DNA was not measureable. Further, Western blot analysis revealed that cytoplasmic and membrane-bound proteins fell inconspicuously while nuclear proteins were barely detecsle. In addition, fPMVs carrying cytoplamic DsRed proteins transduced about ~40 % of recipient cells. The transfer of protein was further confirmed by using the inducible Cre/loxP system. Mitochondria transfer was found in about 20 % recipient cells after incubation with fPMVs for 5 h. To verify the functionalities of transferred mitochondria, mitochodria-deficient HeLa cells (Rho0) were generated and cultivated with fPMVs. Cell enumeration demonstrated that adding fPMVs into culture media stimulated Rho0 cell growth by 100 % as compared to the control. Lastly, MitoTracker and JC-1 staining showed that transferred mitochondria maintained normal shape and membrane potential in Rho0 cells. This study established a time-saving and efficient approach to delivering proteins and mitochondria by using fPMVs, which would be helpful for finding a cure to mitochondria-associated diseases. Graphical abstract Schematic of the delivery of macro-biomolecules and organelles by fPMVs. VSV-G-expressing cells were extruded through a 3 μm polycarbonate membrane filter to generate fusogenic plasma membrane vesicles (fPMVs), which contain bioactive molecules and organelles but not the nucleus. fPMVs can be endocytosed by target cells, while the cargo is released due to low-pH induced membrane fusion. These nucleus-free fPMVs are efficient at delivery of cytoplasmic proteins and mitochondria, leading to recovery of mitochondrial biogenesis and proliferative ability in mitochondria-deficient cells.

Incorporation of VSV-G produces fusogenic plasma membrane vesicles capable of efficient transfer of bioactive macromolecules and mitochondria.

We previously found niacin receptor GPR109A was expressed in murine islet beta-cells, and signaling through GPR109A inhibited glucose stimulated insulin secretion (GSIS). However, the expression of GPR109A in human islets and its functional relevance is still not known.

Niacin receptor GPR109A inhibits insulin secretion and is down-regulated in type 2 diabetic islet beta-cells.

The objective of this study was to formulate a novel gene delivery system based on the erythrocyte ghost (EG) integrated with fusogenic viral glycoprotein vesicular stomatitis virus glycoprotein G (VSV-G). VSV-G proteins were harvested as condition medium of Ad293 cells carrying a VSV-G transgene and then incorporated into EG. Plasmid DNA was condensed by various transfection reagents. A luciferase expression construct (pGL3-control) and a DsRed expression cassette (pCMV-DsRed) were used to evaluate the delivery efficiency of DNA/EG/VSV-G complexes. VSV-G proteins could be incorporated into EG in static incubation under acidic conditions as evidenced by the Western blot analysis. Condensed plasmid DNA was bound mostly to the outer surface of EG, which could be detected by electromicroscopy and measured by electrophoresis. EG/VSV-G complexes stimulated the delivery of pGL3-control into Ad293 cells significantly with the luciferase activity increased about 4-fold as compared to that of the control. The delivery of pCMV-DsRed was also enhanced with the percentage of DsRed-positive Ad293 cells increased from 55 % to about 80 %. Moreover, the transfection efficiency in 3T3, HeLa, INS-1, and bone marrow stem cell (BMSC) cells increased about 2-3-fold. Finally, confocal microscopy analysis showed that incorporation of VSV-G significantly enhanced the endocytosis of EG into target cells. In the present study, a novel type of non-viral DNA delivery vehicle consisting of EG and fusogenic VSV-G proteins was formulated, which showed superior transfection efficiency even in cells resistant to classical transfection.

Incorporation of Viral Glycoprotein VSV-G Improves the Delivery of DNA by Erythrocyte Ghost into Cells Refractory to Conventional Transfection.

To investigate whether selected cytokines are detectable in the embryo culture medium (EM) of human preimplantation embryos (HPE) and what the relationship is of the cytokines with clinical outcomes.

Evaluation of in vitro fertilization outcomes using interleukin-8 in culture medium of human preimplantation embryos.

Vesicular stomatitis virus G glycoprotein (VSV-G) has been widely used for pseudotyping retroviral, lentiviral and artificial viral vectors. The objective of this study was to establish a potential approach for large scale production of VSV-G. To this end, VSV-G was cloned with a N-terminal His-tag into Pichia pastoris expression vector pPIC3.5K. Three clones (Mut(s)) containing the VSV-G expression cassette were identified by PCR. All clones proliferated normally in expansion medium, while the proliferation was reduced significantly under induction conditions. VSV-G protein was detected in cell lysates by Western Blot, and the highest expression level was observed at 96 h post induction. VSV-G could also be obtained from the condition medium of yeast protoplasts. Further, VSV-G could be incorporated into Ad293 cells and was able to induce cell fusion, leading to the transfer of cytoplasmic protein. Finally, VSV-G mediated DNA transfection was assayed by flow cytometry and luciferase measurement. Incubation of VSV-G lysate with pGL3-control DNA complex increased luciferase activity in Ad293 and HeLa cells by about 3-fold. Likewise, incubation of VSV-G lysate with pCMV-DsRed DNA complex improved transfection efficiency into Ad293 by 10% and HeLa cells by about 1-fold. In conclusions, these results demonstrated that VSV-G could be produced from Pichia pastoris with bio-functionalities, demonstrating large scale production of the viral glycoprotein is feasible.

Chi-ju Wei

Multidisciplinary Research Center

Shantou University

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

Chi-ju Wei

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Multidisciplinary Research Center

Shantou University

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

Multidisciplinary Research Center