Application of perfluorocarbon emulsions for the administration of photosensitizing preparations into bone marrow stem cells

It has been shown that, upon incubation of mouse bone marrow stem cells (BMSC) in vitro with the nanoparticles of perfluorocarbon (PFC) emulsion stabilized by proxanol 268, these nanoparticles penetrate into cells and stay there for a long time (up to 20 days of observation). It has been found that, under in vitro conditions, mouse BMSC loaded with the nanoparticles of both the original emulsion and the emulsion preliminarily incubated with radachlorine do not differ from control stem cells in the rate of division, stretching on a plastic support, and the formation of a monolayer. It has been shown that the exposure to laser radiation of BMSC incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine under in vitro conditions leads to the death of these cells due to the destruction of the cell membrane. The treatment with laser radiation of BMSC incubated with the nanoparticles of the starting PFC emulsion (without preliminarily incubation with radachlorine) causes no death of these cells. It has been shown in in vivo experiments that, when transplanted to the organism of a recipient mouse, BMSC of a donor mouse incubated with the nanoparticles of a PFC emulsion preliminarily incubated with radachlorine retain their functional activity, in particular the ability to migrate in the animal body. In this case, radachlorine contained in these stem cells retains its major function, to induce the death of stem cells by the action of laser radiation due to the destruction of the cell membrane. The observation period after the transplantation was 5–7 days.     

In vitro imaging of embryonic stem cells using multiphoton luminescence of gold nanoparticles

Recent advances in nonlinear optical techniques and materials such as quantum wells, nanowires and noble-metal nanoparticles have led to advances in cellular imaging wherein various nanoparticles have been shown to improve both in vitro and in vivo visualization. In this paper, we demonstrate in vitro imaging using multi-photon photoluminescence of gold nanoparticles from two different cell types Dictyostelium discoideum and mouse embryonic stem cells. By observing nanoparticles we show that embryonic stem cells maintained their ability to proliferate for several passages while grown in the presence of gold nanoparticles. The advantages of multi-photon luminescence using gold nanoparticles have important implications for use in stem cell proliferation experiments and in vitro experiments to monitor differentiation.     

Gene expression profile of mouse bone marrow stromal cells determined by cDNA microarray analysis

Bone marrow stromal cells (BMSC) have gained increased attention because of their multipotency and adult stem cell character. They have been shown to differentiate into other cell types of the mesenchymal lineage and also into non-mesenchymal cells. The exact identity of the original cells, which are isolated from bone marrow by their selective adherence to plastic, remains unknown to date. We have established and characterized mouse BMSC cultures and analyzed three independent samples by cDNA microarrays. The expression profile was compared with two previous expression studies of human BMSC and revealed a high degree of concordance between different techniques and species. To gain clues about the positional context and biology of the isolated cells within the bone marrow stroma, we searched our data for genes that encode proteins of the extracellular matrix, cell adhesion proteins, cytoskeletal proteins and cytokines/cytokine receptors. This analysis revealed a close association of BMSC with vascular cells and indicated that BMSC resemble pericytes.     

Temperature comparisons for antibody production in vitro by plaque-forming cells from trout, Salmo gairdneri (Richardson), and mice

Anterior kidney and splenic cells were taken from rainbow trout and splenic cells from BALB/c mice immunized with a T-dependent (sheep red blood cells) or T-independent (DNP-Ficoll) antigen. The cells were incubated at different temperatures in Jerne plaque assays (direct or passive haemolytic plaque assays). The optimum numbers of in vitro plaque-forming cells (PFC) after incubation with homologous complement were directly correlated with normal body temperatures of the respective species. The optimum incubation temperature was 37°C for mouse cells and 10°C for fish cells. Incubation of mouse cells at lower temperatures of 30, 20, 10, 4 or 0°C appeared to yield a direct line reduction in numbers of PFC. Trout cells developed significantly fewer PFC at 4 and 20°C and none at 30°C or above; however, significant numbers still appeared at 0°C. More PFC per million white blood cells were obtained from the anterior kidney; however, related to temperatures, no differences in development of numbers of PFC could be seen between the spleen and anterior kidney cells of trout. When the incubation time was lengthened for both trout and mouse cells held at low temperatures, the numbers of PFC approached those of the cells incubated at the optimum temperatures for 10 h.     

Immune competence of splenic lymphocytes following graft-vs-host disease in mouse allogeneic radiation chimeras.

The abnormal immune response of long-term mouse allogeneic chimeras is reflected by qualitative deficiencies in either T or B lymphocytes. The present study was undertaken to determine if a relationship existed between the severity of graft-vs-host disease (GVHD) that these animals had experienced and a functional defect in either the T or B cell population. The in vitro PFC response of chimera spleen cells to sheep red blood cells (SRBC) was evaluated in the presence of normal T or B lymphocytes 4 to 8 months after marrow transplantation and well beyond the GVHD period. In an analysis of several different allogeneic radiation chimeras, our results showed no relationship between the severity of GVHD experienced and the immunologic capacity of either T or B cells. Thus, different chimera combinations showing similar degrees of GVHD were functionally deficient in one or the other of these two cells types or both with no apparent predilection for abnormality in either population. In examining the quantitative in vitro PFC response to sheep RBC by spleen cells from individual chimeras, we found that the number of PFC formed was related to the severity of GVHD experienced by that animal. A general relationship between severity of GVHD and PFC capacity may also exist between chimeras of different genetic combinations. However, this relationship is not precise since gross exceptions occur. Our results, although documenting further the qualitative abnormalities in T and/or B lymphocytes of radiation chimeras, do not reveal the factor or mechanisms by which these cells are made unresponsive. It is suggested that the tolerance-inducing mechanism of these animals, whether it be humoral blocking factors or suppressor cells, is in some way interfering with the collaboration of T and B cells for antibody production.     

A comprehensive study on optimization of proliferation and differentiation potency of bone marrow derived mesenchymal stem cells under prolonged culture condition

Bone marrow derived stem cells (BMSC) have paved way to clinical approaches for its utilization in a variety of diseases due to its ease of isolation combined with its multilineage differentiation capacity. However, the applicability of BMSC is not successful due to the lesser number of nucleated cells obtained from large samples. Hence, culture expansion of BMSC is a prerequisite, as high numbers of stem cells are needed to meet the standards of clinical advancement. There are attempts on optimizing culture condition for large scale production of BMSC. It was believed that, prolonged culture of BMSC is difficult since they tend to lose their characteristics and differentiation potential. Hence, our study aims to determine whether BMSCs could retain its proliferative and differentiation capacity in prolonged in vitro culture by a comparative study on extensive culturing of BMSC with the following four media, DMEM LG (DMEM-Low Glucose), DMEM KO (DMEM-Knock Out), Alpha MEM (Alpha Minimal Essential Medium), DMEM F 12. We found that two samples among the three cultured tend to lose their property in long term culturing. Besides, we also found that DMEM LG and Alpha MEM were the optimal media for in vitro culturing of BMSC. Overall, it was concluded that BMSC can be cultured until passage 15 without losing its characteristics. However, its potency beyond passage 15 has to be further elucidated for utilization of the ex vivo expanded BMSC for subsequent cellular therapies.     

Selective photothermal efficiency of citrate capped gold nanoparticles for destruction of cancer cells

Gold nanoparticles are recently having much attention because of their increased applications in biomedical fields. In this paper, we demonstrated the photothermal efficacy of citrate capped gold nanoparticles (AuNPs) for the destruction of A431 cancer cells. Citrate capped AuNPs were synthesized successfully and characterized by UV–visible–NIR spectrophotometry and High Resolution Transmission Electron Microscopy (HR-TEM). Further, AuNPs were conjugated with epidermal growth factor receptor antibody (anti-EGFR) and applied for the selective photothermal therapy (PTT) of human epithelial cancer cells, A431. PTT experiments were conducted in four groups, Group I—control cells, Group II—cells treated with laser light alone, Group III—cells treated with unconjugated AuNP and further laser irradiation and Group IV—anti-EGFR conjugated AuNP treated cells irradiated by laser light. After laser irradiation, cell morphology changes that were examined using phase contrast microscopy along with the relevant biochemical parameters like lactate dehydrogenase activity, reactive oxygen species generation and caspase-3 activity were studied for all the groups to determine whether cell death occurs due to necrosis or apoptosis. From these results we concluded that, these immunotargeted nanoparticles could selectively induce cell death via ROS mediated apoptosis when cells were exposed to a low power laser light.     

A study of the functional activity of macrophages of peritoneal exudate of mice exposed to low-intensity laser radiation in vitro and in vivo

The effect of low-intensity laser radiation generated by semiconductor devices in the red (650 nm) and infrared (850 nm) regions of the spectrum in vitro and in vivo on the phagocytic activity and synthesis of proinflammatory cytokines by peritoneal macrophages during the phagocytosis of bacterial cells has been studied. A culture of the clinical strain of the enteropathogenic bacterium Escherichia coli was used as an object. The radiation dose was varied by changing the power and duration of exposure. The results obtained indicate that infrared low-intensity laser radiation has a stimulating effect on the phagocytic activity of macrophages. It was shown that the effect of low-intensity laser radiation on the activity of the phagocytic process, the enhancement of the adhesion of bacteria by macrophages, killing of bacteria, and the production of proinflammatory cytokines is dose-dependent. The exposure to the rays of the red region of the spectrum on phagocytizing macrophages induced a decrease in their activity; as the dose was increased, the destruction of cells was registered.     

Cellular uptake of Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles synthesized through solvent emulsion evaporation and nanoprecipitation method

Poly-(D,L-lactide-co-glycolide) (PLGA) nanoparticles have been widely studied for drug delivery. The aim of this study is to determine how cellular uptake of these nanoparticles is influenced by different surface properties, incubation time, particle concentration and cell types. Spherical coumarin-6 loaded PLGA nanoparticles with a size of about 100 nm were synthesized through solvent emulsion evaporation and nanoprecipitation methods. In vitro cellular uptake efficiency was determined using human bronchial epithelial cells (BEAS-2B) and murine monocyte-derived macrophage (RAW264.7) cells. PLGA nanoparticles were incubated with these cells in a concentration range of 10-300 μg/ml for different time periods. The results show that cellular uptake decreased for nanoparticles surface coated with PVA surfactant and was especially limited for severely aggregated particles. At higher particle concentration, the total amount of particles taken up by cells increased while the uptake efficiency decreased. In addition, cells could take up more particles with longer incubation time, although the uptake rate decreased gradually with time. Finally, RAW264.7 cells show increased uptake compared to BEAS-2B cells. The information drawn from this study would provide important clues on how nanomaterials interact with cells and how these interactions can influence biocompatibility or toxicity.     

转化生长因β1基因真核表达质粒的构建及其在小鼠骨髓间充质干细胞中的表达

目的：构建转化生长因β1（TGF-β1）表达载体,在骨髓间充质干细胞（BMSC）中表达。方法：以小鼠肺组织cDNA为模板,PCR扩增TGF-β1基因,并将其插入pCDH1-MCS1-EF1-copGFP载体质粒,转化感受态大肠杆菌DH5α,抽提质粒,经PCR和测序鉴定后转染BMSC,利用激光共聚焦显微镜和Western印迹对其表达进行检测。结果：经PCR及测序鉴定,构建入载体质粒的基因为TGF-β1基因,pCDH1-TGFβ1-EF1-copGFP重组质粒能在BMSC中表达。结论：构建了pCDH1-TGFβ1-EF1-copGFP重组质粒,且能表达于BMSC,为进一步研究TGF-β1影响间充质干细胞的生理功能奠定了基础。     

增强型绿色荧光蛋白标记兔间充质干细胞体外复合小肠黏膜下层的生长特性

目的利用绿色荧光蛋白（EGFP）标记兔来源的骨髓间充质干细胞（BMSC）,观察其作为种子细胞复合SIS基质体外培养的生长特性,为复合物回植体内时间提供参考。方法通过物理和化学方法制备小肠黏膜下层（SIS）;取新西兰大白兔分离BMSC,体外培养增殖至P3代,行转染增强型EGFP,转染前后根据增殖速度,绘制生长曲线。然后将转染后的第3代间BMSC,以30×106的浓度接种于1 cm×1 cm大小的SIS支架材料上培养。观察细胞转染后的生长特性,检测复合后的细胞总数,荧光显微镜观察复合后细胞形态。结果 BMSC经转染后生长增殖速度未受明显影响。细胞DNA检测显示复合物培养第7天培养结果显示细胞增殖状态最佳差异有统计学意义（P〈0.05）。荧光显微镜观察显示复合物培养7天细胞增殖良好差异有统计学意义（P〈0.05）,第10天后呈现老化状态。结论增强型的EGFP转染不影响间充质干细胞传代以及增殖。间充质干细胞-SIS基质复合物体外培养7 d回植较为合适。     

Studies on immune responses to parasite antigens in mice: VII. Cells secreting antibodies to modified mouse erythrocytes in Babesia rodhaini-infected mice

Increased numbers of plaque-forming cells (PFC) secreting antibodies to bromelain-treated mouse erythrocytes were detected in the spleens of BALB/c·nu/+ mice heavily infected with Babesia rodhaini but not in the spleens of infected hypothymic BALB/ c·nu/nu mice. Whether the products of these cells, antierythrocyte autoantibodies, play any role in the destruction of intact erythrocytes in parasitized mice is unknown, but it is noteworthy that only low numbers of PFC were detected, and the modification of mouse erythrocytes was obligatory for detection of PFC.     

Conjugating folic acid to gold nanoparticles through glutathione for targeting and detecting cancer cells

Gold nanoparticles (GNPs) were modified with glutathione (GSH) to form GSH-capped GNPs, which have carboxyl groups on the surface of these nanoparticles. Then folic acid (FA) was conjugated with GNPs through the reaction between amino group of FA and carboxyl group of GSH. These folic acid-conjugated nanoparticles (FA-GSH-GNPs) were stable in aqueous solution over a broad range of pH and ionic strength values. The targeting of FA-GSH-GNPs in human cervices carcinoma cells (HeLa cells) with high-level folate receptor expression was confirmed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). No cellular uptake of these nanoparticles was observed in A549 cells lack of folate receptor. HeLa cells and mouse fibroblasts incubated with FA-GSH-GNPs were assayed by measuring the relative absorbance of the supernatant collected at low-speed centrifugation. Based on this simple spectroscopic method, HeLa cells have been detected with a detection limit of 10² cells/mL.     

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

EphrinA I-targeted nanoshells for photothermal ablation of prostate cancer cells

Gold-coated silica nanoshells are a class of nanoparticles that can be designed to possess strong absorption of light in the near infrared (NIR) wavelength region. When injected intravenously, these nanoshells have been shown to accumulate in tumors and subsequently mediate photothermal treatment, leading to tumor regression. In this work, we sought to improve their specificity by targeting them to prostate tumor cells. We report selective targeting of PC-3 cells with nanoshells conjugated to ephrinA I, a ligand for EphA2 receptor that is overexpressed on PC-3 cells. We demonstrate selective photo-thermal destruction of these cells upon application of the NIR laser.     

Replacement of mouse embryonic fibroblasts with bone marrow stromal cells for use in establishing and maintaining embryonic stem cells in mice

We have investigated the use of BMSC (bone marrow stromal cell) as a feeder cell for improving culture efficiency of ESC (embryonic stem cell). B6CBAF1 blastocysts or ESC stored after their establishment were seeded on to a feeder layer of either SCA-1+/CD45-/CD11b- BMSC or MEF (mouse embryonic fibroblast). Feeder cell activity in promoting ESC establishment from the blastocysts and in supporting ESC maintenance did not differ significantly between BMSC and MEF feeders. However, the highest efficiency of colony formation after culturing of inner cell mass cells of blastocysts was observed with the BMSC line that secreted the largest amount of LIF (leukaemia inhibitory factor). Exogenous LIF was essential for the ESC establishment on BMSC feeder, but not for ESC maintenance. Neither change in stem cell-specific gene expression nor increase in stem cell aneuploidy was detected after the use of BMSC feeder. We conclude that BMSC can be utilized as the feeder of ESC, which improves culture efficiency.     

Primary in vitro immunogenicity of liposomal model membranes in mouse spleen cell cultures.

Neither 2,4-dinitrophenyl-6-N-aminocaproylphosphatidylethanolamine (DNP-Cap-PE) nor fluoresceinthiocarbamylphosphatidylethanolamine (F1-PE) induces hapten-specific plaque-forming cells (PFC) when incubated with suspensions of spleen cells from unimmunized C57BL/6J mice. However, PFC are produced after incorporation of these synthetic lipid antigens into liposomal model membranes. The in vitro response is characterized by the following: a) it is time and dose dependent; b) the frequency of IgM PFC exceeds IgG PFC; c) both nonadherent and adherent cells are required (2-mercaptoethanol can replace the requirement for adherent cells in some experiments); d) depletion of thymus-derived cells by treatment with anti-theta antiserum plus complement does not diminish the response; e) spleen cells from nude BALB/c mice also produce PFC. Thus, the essential features of the in vivo immunogenicity of DNP-Cap-PE and F1-PE sensitized liposomes, which have been previously described, can be replicated in an in vitro cell culture system.     

Lysophosphatidic acid accelerates lung fibrosis by inducing differentiation of mesenchymal stem cells into myofibroblasts

Lung fibrosis is characterized by vascular leakage and myofibroblast recruitment, and both phenomena are mediated by lysophosphatidic acid (LPA) via its type‐1 receptor (LPA1). Following lung damage, the accumulated myofibroblasts activate and secrete excessive extracellular matrix (ECM), and form fibrotic foci. Studies have shown that bone marrow‐derived cells are an important source of myofibroblasts in the fibrotic organ. However, the type of cells in the bone marrow contributing predominantly to the myofibroblasts and the involvement of LPA‐LPA1 signalling in this is yet unclear. Using a bleomycin‐induced mouse lung‐fibrosis model with an enhanced green fluorescent protein (EGFP) transgenic mouse bone marrow replacement, we first demonstrated that bone marrow derived‐mesenchymal stem cells (BMSCs) migrated markedly to the bleomycin‐injured lung. The migrated BMSC contributed significantly to α‐smooth muscle actin (α‐SMA)‐positive myofibroblasts. By transplantation of GFP‐labelled human BMSC (hBMSC) or EGFP transgenic mouse BMSC (mBMSC), we further showed that BMSC might be involved in lung fibrosis in severe combined immune deficiency (SCID)/Beige mice induced by bleomycin. In addition, using quantitative‐RT‐PCR, western blot, Sircol collagen assay and migration assay, we determined the underlying mechanism was LPA‐induced BMSC differentiation into myofibroblast and the secretion of ECM via LPA1. By employing a novel LPA1 antagonist, Antalpa1, we then showed that Antalpa1 could attenuate lung fibrosis by inhibiting both BMSC differentiation into myofibroblast and the secretion of ECM. Collectively, the above findings not only further validate LPA1 as a drug target in the treatment of pulmonary fibrosis but also elucidate a novel pathway in which BMSCs contribute to the pathologic process.     

Extracellular nicotinamide adenine dinucleotide induces t cell apoptosis in vivo and in vitro.

Incubation of mouse T cells expressing the cell surface enzyme ADP ribosyltransferase with nicotinamide adenine dinucleotide (NAD) had been reported to cause ADP ribosylation of cell surface molecules, inhibition of transmembrane signaling, and suppression of immune responses. In this study, we analyze the reasons for these effects and report that contact of T cells with NAD causes cell death. Naive T cells when incubated with NAD and adoptively transferred into semiallogeneic mice fail to cause graft-vs-host disease, and when injected into syngeneic, T cell-deficient recipients do not reconstitute these mice. Rather, they accumulate in the liver, leading to an increase of apoptotic lymphocytes in this organ. Similar effects are induced by injection of NAD, shown to cause a dramatic increase of apoptotic CD3(+), CD4(+), and CD8(+) cells in the liver. Consistent with this, in vitro incubation of naive T cells with NAD is shown to induce apoptosis. In contrast, no cell death is demonstrable when T cells are activated before incubation with NAD. It is concluded that ecto-NAD, as substrate of ADP ribosyltransferase, acts on naive, but not on activated CD69(+) T cells.