Exosomes derived from dendritic cells

Dendritic cells (DC) are potent antigen presenting cells and the only ones capable of inducing primary cytotoxic immune responses both in vivo and vitro. DCs secrete a 60-80 nm membrane vesicle population of endocytic origin, called exosomes. The protein composition of exosomes was analyzed using a systematic proteomic approach. Besides MHC and costimulatory molecules, exosomes bear several adhesion proteins, probably involved in their specific targeting. Exosomes also accumulate several cytosolic factors, most likely involved in exoxome's biogenesis in late endosomes. Like DCs, exosomes induce potent anti tumor immune responses in vivo. Indeed, a single injection of DC-derived exosomes sensitized with tumor peptides induced the eradication of established mouse tumors. Tumor-specific cytotoxic T lymphocytes were found in the spleen of exosome treated mice, and depletion of CD8+ T cells in vivo inhibited the anti tumor effect of exosomes. These results strongly support the implementation of human DC-derived exosomes for cancer immunotherapy.     

Production of l-Methionine-enriched cells of a mutant derived from a methylotrophic yeast,Candida boidinii

l-Methionine-enriched cells production of an ethionine-resistant mutant of Candida boidinii no. 2201 was greatly improved by the control of pH and by feeding of methanol and other medium components during cultivation in a jar fermentor. Under the optimal conditions, 38.5 g (as dry weight)_of cells abd 282 mg of pool methionine (intracellular pool of free l-methionine) per l of culture broth were obtained after 11 d of cultivation.The culture conditions for production of l-methionine-enriched cells in continuous culture were investigated. With limited methanol in continuous cultivation, pool methionine productivity reached a maximum value of 1.14 mg·l⁻¹·h⁻¹ at a dilution rate of 0.05·h⁻¹. During methanol-limited growth in continuous cultivation, the pool methionine content of the mutant was about 20–35% higher than that in batch cultivation.     

Pluripotential stem cells derived from migrating primordial germ cells

Pluripotent stem cells termed embryonic germ cells (EGCs) have earlier been derived from pre- and post-migrating mouse primordial germ cells (PGCs). We have recently obtained four EGC lines from migrating PGCs of 9.5 days post coitum (dpc) embryos. All lines were male with normal karyotype and showed properties that are similar to previously established EGC lines, including colony morphology, expression of alkaline phosphatase (AP), and expression of SSEA-1 antigen. The developmental potency of two of these lines was tested in vivo. They contributed to a range of tissues in fetal chimeras including heart, lung, kidney, intestine, muscle, brain and skin. We also examined the methylation status of the imprinted genes: Igf2r, p57Kip2, Lit1, H19 and Igf2. Igf2r, p57Kip2 and Lit1 were unmethylated in all analysed EGC lines, whereas H19 and Igf2 showed significant hypo-methylation in the 9.5 dpc EGC-1 line when compared to previously derived 11.5 dpc male EGC lines. This suggests that imprint erasure in the male germ line occurs prior to 9.5 dpc for all imprinted genes examined.     

Tumorigenesis in cells derived from induced pluripotent stem cells

Induced pluripotent stem (iPS) cells are an attractive source for potential cell-replacement therapy. However, transplantation of differentiated products harbors the risk of teratoma formation, presenting a serious health risk. Thus, we characterized Nanog-expressing (undifferentiated) cells remaining after induction of differentiation by cytological examination. To induce differentiation of iPS cells, we generated embryoid bodies (EBs) derived from iPS cells carrying a Nanog–green fluorescent protein (GFP) reporter and then injected GFP-positive and GFP-negative EBs into nude mice. GFP-positive EB transplantation resulted in the formation of immature teratoma grade 3, but no tumors were induced by GFP-negative EB. GFP-positive cells revealed significantly lower cytoplasmic area and higher nucleus/cytoplasm ratio than those of GFP-negative cells. Our results suggest that morphological analysis might be a useful method for distinguishing between tumorigenic and nontumorigenic iPS cells.     

Chondrocytes derived from mouse embryonic stem cells

Our knowledge of cellular differentiation processes during chondro- and osteogenesis, in particular the complex interaction of differentiation factors, is still limited. We used the model system of embryonic stem (ES) cell differentiation in vitro via cellular aggregates, so called embryoid bodies (EBs), to analyze chondrogenic and osteogenic differentiation. ES cells differentiated into chondrocytes and osteocytes throughout a series of developmental stages resembling cellular differentiation events during skeletal development in vivo. A lineage from pluripotent ES cells via mesenchymal, prechondrogenic cells, chondrocytes and hypertrophicchondrocytes up to osteogenic cells was characterized. Furthermore, we found evidence for another osteogenic lineage, bypassing the chondrogenic stage. Together our results suggest that this in vitro system will be helpful to answer so far unacknowledged questions regarding chondrogenic and osteogenic differentiation. For example, we isolated an as yet unknown cDNA fragment from ES cell-derived chondrocytes, which showed a developmentally regulated expression pattern during EB differentiation. Considering ES cell differentiation as an alternative approach for cellular therapy, we used two different methods to obtain pure chondrocyte cultures from the heterogenous EBs. First, members of the transforming growth factor (TGF)-β family were applied and found to modulate chondrogenic differentiation but were not effective enough to produce sufficient amounts of chondrocytes. Second, chondrocytes were isolated from EBs by micro-manipulation. These cells initially showed dedifferentiation into fiboblastoid cells in culture, but later redifferentiated into mature chondrocytes. However, a small amount of chondrocytes isolated from EBs transdifferentiated into other mesenchymal cell types, indicating that chondrocytes derived from ES cells posses a distinct differentiation plasticity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lineage-switching by pluripotent cells derived from adults

When proceeding normally, embryonic morphogenesis begins with germ layer formation through the process of gastrulation. Each primordial germ layer gives rise to a particular set of lineages. Until recently, it was considered that fate switches between germ layers were impossible. In the last two or three years however, a fair number of such switches have been described (Table I), the most spectacular of which entails the differentiation of neural stem cells into various derivatives. This unexpected plasticity opens important prospects for cell therapy. Stem cells, which are the cells that display this plasticity, are defined by the two properties of self renewal and pluripotency. They are set apart during ontogeny and are responsible for maintaining the homeostasis of a tissue. This notion, first established in the case of hematopoietic stem cells was later extended to other fast renewing cells, such as those in the intestinal epithelium or epidermis, and more recently to cells reputedly non-renewable, i.e. neurons. A new strategy has been described, which has the interesting feature that it can be applied to the isolation of stem cells from various lineages. It consists in sorting out cells on the basis of the efflux of Hoechst 33342 dye (Goodell et al., 1996). When a cell suspension stained with this dye is examined under two distinct wave lengths, a "side population" (SP), characterized by weak fluorescence, can be identified and sorted out. The dye efflux property of these cells is due to the activity of the mdr (multidrug resistance) gene, which encodes a protein responsible for the building of a canal which serves to extrude toxins from the cells. A means of distinguishing a truly multipotent stem cell from a progenitor committed to a specific lineage has been reported. This consists in the expression of the Pax7 gene. Pax7-/- mouse muscles have no satellite cells, i.e. they miss the cells normally responsible for the regeneration of muscle. In contrast they do have an SP population. These SP cells are incapable of differentiating into muscle, but give rise to 10 times more hematopoietic colonies, when cloned in vitro, than SP cells from wild type muscle do. Thus Pax7 appears to be a commitment gene, in the absence of which stem cells cannot become specified to the muscle lineage. As a conclusion, this review emphasizes various features of the recent findings: 1) the unexpected plasticity uncovered in recent years is restricted to the stem cells of each tissue; 2) the switch in phenotype has to be "forced" on these stem cells by drastic experimental conditions enforced in the host: often sublethal irradiation is superimposed on a genetic deficiency. Progress in this field, concerning both conceptual and applied aspects, will require the identification of the factors characterizing the niches which promote integration and fate switches of stem cells, probably a combination of growth factors and intercellular interactions. Finally a key issue, before any therapeutical applications can be considered, is how to control the proliferation of transplanted stem cells in their new environment.     

Immunosuppressive lymphokine derived from natural suppressor cells

Cloned natural suppressor (NS) cells derived from spleens of total lymphoid irradiated BALB/c mice were incubated with the phorbol ester, PMA, and calcimycin for 4 h. After thorough washing, the induced NS cells were incubated in serum-free medium for 24 h and the supernatants were collected. The supernatants suppressed the MLR between normal adult responder and stimulator spleen cells. There was no Ag specificity or H-2 haplotype restriction of the MLR suppression. The supernatants did not inhibit [3H]thymidine incorporation per se, because they did not suppress mitogen stimulation of spleen cells. Protease digestion of the supernatants removed the suppressive activity, and dialysis studies indicated that the molecular size of the suppressive factor was larger than 50,000 Da and smaller than 100,000 Da. The suppressive activity was stable at 56 degrees C, pH 2, for 1 h. Thus, NS cell clones can be induced to secrete an immunosuppressive lymphokine, NS factor.     

Immunofluorescence in cells derived from Burkitt's lymphoma

Henle, Gertrude (Children's Hospital of Philadelphia, Philadelphia, Pa.), and Werner Henle. Immunofluorescence in cells derived from Burkitt's lymphoma. J. Bacteriol. 91:1248-1256. 1966.-Indirect immunofluorescence tests led to the brilliant staining of a small proportion of the cells in five different cultures derived from Burkitt's (African) lymphomas. The reaction was not restricted to the 17 sera from cases of this disease but extended to many sera from American individuals, whether healthy donors or patients suffering from a variety of illnesses. The incidence of positive sera increased with age from about 30% in childhood to > 90% in adults. Fluorescein-isothiocyanate-conjugated human gamma-globulins were suitable for direct staining of the same proportion of cells. The stained cells appeared to be in varying stages of degeneration, but cultural conditions leading to an increase in the cellular death rates failed to result in a rise in fluorescent cells. Several observations suggest that the stainable cells might be those which are seen to harbor virus particles under the electron microscope. Two cell lines derived from leukemic patients in this country also contained a small fraction of stainable cells but two others, and numerous primary human leukocyte cultures, gave consistently negative results. Attempts to relate the staining to known viral antigens have failed to implicate herpes simplex, varicella, cytomegalo, and reo viruses types 1, 2, and 3. The nature of the virus carried by the lymphoma cells as well as of the staining reactions remains to be determined.     

Eupatilin, a pharmacologically active flavone derived from Artemisia plants, induces apoptosis in human promyelocytic leukemia cells

Extracts of the whole herb of Artemisia asiatica Nakai (Asteraceae) have been used in traditional oriental medicine for the treatment of inflammation, cancer and other disorders. In the present work, we have evaluated the apoptosis-inducing capability of eupatilin (5,7-dihydroxy-3,4,6-trimethoxyflavone), a pharmacologically active ingredient of A. asiatica, in cultured human promyelocytic leukemia (HL-60) cells. Thus, eupatilin exhibited concentration-dependent inhibitory effects on viability and DNA synthesis capability of HL-60 cells. The anti-proliferative effect of eupatilin was attributable to its apoptosis-inducing activity as determined by characteristic nuclear condensation, in situ terminal end-labeling of fragmented DNA (TUNEL), release of mitochondrial cytochrome c into cytoplasm, proteolytic activation of caspases-9, -3, and -7, and cleavage of poly(ADP-ribose)polymerase. Eupatilin-induced HL-60 cell apoptosis does not appear to be mediated via alteration in Bcl-2/Bax-2. Taken together, the above findings suggest that eupatilin has chemopreventive and cytotoxic effects.     

Clinical,hormonal, and hematologic characteristics of bovine calves derived from nuclei from somatic cells

Although healthy animals are born after nuclear transfer with somatic cells nuclei, the success of this procedure is generally poor (2%-10%) with high perinatal losses. Apparently normal surviving animals may have undiagnosed pathologies that could develop later in life. The gross pathology of 16 abnormal bovine fetuses produced by nuclear transfer (NT) and the clinical, endocrinologic (insulin-like growth factors I and II [IGF-I and IGF-II], IGF binding proteins, post-ACTH stimulation cortisol, leptin, glucose, and insulin levels), and biochemical characteristics of a group of 21 apparently normal cloned calves were compared with those of in vitro-produced (IVP) controls and controls resulting from artificial insemination. Oocytes used for NT or IVP were matured in vitro. NT to enucleated oocytes was performed using cultured adult or fetal skin cells. After culture, Day 7, grade 1-2 embryos were transferred (one per recipient). All placentas and fetuses from clones undergoing an abnormal pregnancy showed some degree of edema due to hydrops. Mean placentome number was lower and mean placentome weight was higher in clones than in controls (69.9 +/- 9.2 placentomes with a mean weight of 144.3 +/- 21.4 g in clones vs. 99 and 137 placentomes with a mean individual weight of 34.8 and 32.4 g in two IVP controls). Erythrocyte mean cell volume was higher at birth (P < 0.01), and body temperature and plasma leptin concentrations were higher and T4 levels were lower during the first 50 days and the first week (P < 0.05), respectively, in clones. Plasma IGF-II concentrations were higher at birth and lower at Day 15 in clones (P < 0.05). Therefore, apparently healthy cloned calves cannot be considered as physiologically normal animals until at least 50 days of age.     

Integrin-like substrate adhesion in RTG-2 cells, a fibroblastic cell line derived from rainbow trout

Fluorometric cell attachment assays together with competitive inhibitors of adhesion were used to probe for the presence of integrins, a diverse family of heterodimeric cell-surface glycoproteins involved in cell-cell and cell-extracellular matrix adhesion, in the fibroblastic rainbow trout cell line, RTG-2. The adhesive properties of this cell line were evaluated. RTG-2 cells adhered poorly to TC plastic in the absence of serum but as little as 2.5% fetal bovine serum allowed over 75% of the cells to attach after 5 h. Surfaces coated with the extracellular matrix proteins collagen I, collagen IV, fibrin, fibrinogen, or fibronectin were able to support attachment of RTG-2 cells. Adhesion of RTG-2 cells to fibronectin varied linearly with fibronectin coating densities in the range 0 to 65 ng/mm(2). Oligopeptides containing the sequence Arg-Gly-Asp (RGD) caused dose-dependent inhibition of adhesion to microtiter plates coated with fibrin, fibrinogen, and fibronectin, whereas attachment to collagen I and collagen IV was less severely affected. In all cases, peptides containing Arg-Gly-Glu (RGE) or Asp-Gly-Arg (DGR) sequences caused no reduction of cell attachment. Since many integrins mediate adhesion by binding to RGD sequences in their target ligands, these results suggest the presence of integrin-like adhesion molecules on the surface of RTG-2 cells.     

Comparison of human post-embryonic,multipotent stem cells derived from various tissues

Multipotent stem cells were isolated from human fetal heart, liver, muscle, lung, derma, kidney, and adipose tissue, and then analyzed for their characteristics and function. Cells with characteristics similar to bone marrow-derived post-embryonic multipotent stem cells can be selected and cultured from tissues other than bone marrow. This may then help explain the “stem cell plasticity” found in multiple human tissues. Baijun Fang and Ning Li contributed equally to this study.     

Characteristics of a clone of endothelial cells derived from a line of normal adult rat lung cells

Summary A strain of endothelial cells derived from a single cell cloned from a line of normal adult rat lung parenchyma has been maintained in tissue culture for more than 3 years. These cells have been identified as endothelial cells based on the combination of their growth characteristics, cell morphology as observed with both light and electron microscopy, and their physiological properties. They have continued to produce granules, which stain specifically for glycosaminoglycans with Alcian blue, for over 2 1/2 years. During the same period of time, glycosaminoglycans were identified biochemically in both cells and medium. They have maintained the ability to degrade bradykinin over this period as well. This work was supported in part by NIH Program Project HL 15832.     

Characteristics of a clone of endothelial cells derived from a line of normal adult rat lung cells

A strain of endothelial cells derived from a single cell cloned from a line of normal adult rat lung parenchyma has been maintained in tissue culture for more than 3 years. These cells have been identified as endothelial cells based on the combination of their growth characteristics, cell morphology as observed with both light and electron microscopy, and their physiological properties. They have continued to produce granules, which stain specifically for glycosaminoglycans with Alcian blue, for over 2 1/2 years. During the same period of time, glycosaminoglycans were identified biochemically in both cells and medium. They have maintained the ability to degrade bradykinin over this period as well.     

Recloned dogs derived from adipose stem cells of a transgenic cloned beagle

A number of studies have postulated that efficiency in mammalian cloning is inversely correlated with donor cell differentiation status and may be increased by using undifferentiated cells as nuclear donors. Here, we attempted the recloning of dogs by nuclear transfer of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) from a transgenic cloned beagle to determine if cAd-MSCs can be a suitable donor cell type. In order to isolate cAd-MSCs, adipose tissues were collected from a transgenic cloned beagle produced by somatic cell nuclear transfer (SCNT) of canine fetal fibroblasts modified genetically with a red fluorescent protein (RFP) gene. The cAd-MSCs expressed the RFP gene and cell-surface marker characteristics of MSCs including CD29, CD44 and thy1.1. Furthermore, cAd-MSCs underwent osteogenic, adipogenic, myogenic, neurogenic and chondrogenic differentiation when exposed to specific differentiation-inducing conditions. In order to investigate the developmental potential of cAd-MSCs, we carried out SCNT. Fused-couplets (82/109, 75.2%) were chemically activated and transferred into the uterine tube of five naturally estrus-synchronized surrogates. One of them (20%) maintained pregnancy and subsequently gave birth to two healthy cloned pups. The present study demonstrated for the first time the successful production of cloned beagles by nuclear transfer of cAd-MSCs. Another important outcome of the present study is the successful recloning of RFP-expressing transgenic cloned beagle pups by nuclear transfer of cells derived from a transgenic cloned beagle. In conclusion, the present study demonstrates that adipose stem cells can be a good nuclear donor source for dog cloning.     

Ascopyrone P,a novel antibacterial derived from fungi

AIMS: To assess the antimicrobial efficacy of ascopyrone P (APP), a secondary metabolite formed by the fungi Anthracobia melaloma, Plicaria anthracina, Plic. leiocarpa and Peziza petersi belonging to the order Pezizales. METHODS AND RESULTS: In vitro testing using a well diffusion procedure showed that APP at a high concentration (approximately 5%) inhibited the growth of Gram-positive and Gram-negative bacteria. Using an automated microbiology reader, growth curve analysis showed that 2000-4000 mg l(-1) APP caused total or significant bacterial inhibition after incubation for 24 h at 30 degrees C. Against certain yeast strains, 1000- 2000 mg l(-1) APP enhanced growth, although at higher concentrations inhibition of some yeasts was observed. Clostridium and fungal strains were not sensitive to 2000 mg l(-1) APP. No significant cidal effect was observed after 2 h against Listeria monocytogenes or Escherichia coli. Results were identical whether the APP samples tested had been produced enzymatically or chemically. CONCLUSIONS: At a level of 2000 mg l(-1), APP demonstrated growth inhibitory activity against a broad range of bacteria, but not yeasts or moulds. SIGNIFICANCE AND IMPACT OF THE STUDY: A possible application for this novel natural antimicrobial is in food preservation, to control the growth of Gram-negative and Gram-positive bacteria in raw and cooked foods. Effective dosage levels would be 500-4000 mg kg(-1), depending on food type. The efficacy, organoleptic and safety aspects of this compound in food still need to be assessed.     

Neural precursors derived from human embryonic stem cells

Human embryonic stem (hES) cells provide a promising supply of specific cell types for transplantation therapy. We presented here the method to induce differentiation of purified neural precursors from hES cells. hES cells (Line PKU-1 and Line PKU-2) were cultured in suspension in bacteriological Petri dishes, which differentiated into cystic embryoid bodies (EBs). The EBs were then cultured in N₂ medium containing bFGF in poly-L-lysine-coated tissue culture dishes for two weeks. The central, small cells with 2–3 short processes of the spreading outgrowth were isolated mechanically and replated. The resulting neurospheres were cultured in suspension for 10 days, then dissociated into single cell suspension with a Pasteur pipette and plated. Cells grew vigorously in an attached way and were passed every 4–5 days. Almost all the cells were proved nestin positive by immunostaining. Following withdrawal of bFGF, they differentiated into neurons expressing β-tubulin isotype_III, GABA, serotonin and synaptophysin. Through induction of PDGF-AA, they differentiated into astrocytes expressing GFAP and oligodendrocytes expressing O₄. The results showed that hES cells can differentiate into typical neural precursors expressing the specific marker nestin and capable of generating all three cell types of the central nervous system (CNS)in vitro.