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We explored the potential therapeutic value of transplanting bone marrow (BM)-derived mesenchymal stromal cells (MSC) into utrophin/dystrophin-deficient double knock-out (dko) mice, a murine model of Duchenne muscular dystrophy.MSC from male rats were isolated and transplanted into female dko mice via the caudal vein. Behavior and locomotor function were later evaluated, along with the expression of dystrophin and utrophin in the sarcolemma of myofiber tissues. The presence of grafted cells was confirmed via polymerase chain reaction for the sex-determining region of the Y-chromosome.Locomotor activity improved significantly (P0.05) from 5 to 15 weeks after cell transplantation, as measured by traction, rotating rod and running wheel tests. We also found that the expression of dystrophin and utrophin increased significantly (P0.05) and progressively in the sarcolemma from 5 to 15 weeks after transplantation. The median lifespan of mice in the normal group (74.1 weeks) was significantly (P0.001) higher than those in the control (22.0 weeks) and transplantation (35.0 weeks) groups, and the median lifespan of mice in the transplantation group was significantly (P0.001) higher than that in the control group.Results of this study demonstrate that BM MSC have potential value in xenogeneic transplantation therapy for muscular dystrophy.

The full potential of embryonic stem (ES) cells to generate precise cell lineages and complex tissues can be best realized when they are differentiated in vivo-i.e. in developing blastocysts. Owing to various practical and ethical constraints, however, it is impossible to introduce ES cells of certain species into blastocysts of the same species. One solution is to introduce ES cells into blastocysts of a different species. However, it is not known whether ES cells can contribute extensively to chimerism when placed into blastocysts of a distantly related species. Here, we address this question using two divergent species, Apodemus sylvaticus and Mus musculus, whose genome sequence differs by approximately 18% from each other. Despite this considerable evolutionary distance, injection of Apodemus ES cells into Mus blastocysts led to viable chimeras bearing extensive Apodemus contributions to all major organs, including the germline, with Apodemus contribution reaching approximately 40% in some tissues. Immunostaining showed that Apodemus ES cells have differentiated into a wide range of cell types in the chimeras. Our results thus provide a proof of principle for the feasibility of differentiating ES cells into a wide range of cell types and perhaps even complex tissues by allowing them to develop in vivo in an evolutionarily divergent host-a strategy that may have important applications in research and therapy. Furthermore, our study demonstrates that mammalian evolution can proceed at two starkly contrasting levels: significant divergence in genome and proteome sequence, yet striking conservation in developmental programs.

Embryonic stem cells can proliferate indefinitely and are capable of differentiating into derivatives of all three embryonic germ layers in vitro, including the neural lineage. The main objective of this study is to test the effects of neural stem cell conditioned medium on the neural differentiation of mouse embryonic stem cells. When cultured in neural stem cell conditioned medium, mouse embryonic stem cells can form floating cell spheres composed of many nestin-positive cells. After trypsinization and growth on gelatin, these embryonic stem cell-derived neural progenitor cells can be expanded for more than 3 months without loss of neural progenitor characteristics. Both neuronal and glial cells can be readily generated from these cells under differentiation conditions. Thus, neural stem cell conditioned medium is a highly potent reagent for inducing the development of mouse embryonic stem cells into the neural lineage, especially neural progenitor cells.

The prospects of using immature CD8a(+) dendritic cells (DC2) to establish transplant immunologic tolerance and treatments for autoimmune diseases in the future are promising. However, the methods for inducing DC2 are still being explored. The present study was aimed to investigate the optimal in vitro conditions for preparing large numbers of predominant DC2 from murine bone marrow cells.Three groups of bone marrow cells cultured under different conditions were examined, namely a cytokine-induced experimental group (cytokine group), a control group with a low concentration of granulocyte-macrophage colony stimulating factor (GM-CSF, low GM-CSF group) and a control group without endogenous cytokines. The cytokine group was cultured with 5 ng/ml GM-CSF, 25 ng/ml Flt3 ligand (Flt3L), 20 ng/ml interleukin 4 (IL-4) and 100 ng/ml stem cell factor (SCF). The low GM-CSF control group was cultured with 0.4 ng/ml GM-CSF, 25 ng/ml Flt3L and 100 ng/ml SCF, without IL-4. The control group without exogenous cytokines was cultured without additional cytokines. All cells were cultured at 37 degrees C under 5% CO2. On days 3, 7 and 16, 4-color flow cytometry was carried out to analyze the cell phenotypes, and the total cell numbers were counted to analyze the cell yields. Phase-contrast microscopy was used to observe the cell morphologies.The cytokine group exhibited higher proportions of typical immature CD8a(+) DC, especially on day 3, but the total cell number and DC2 proportion decreased during prolonged culture. The low GM-CSF control group showed the same tendencies as the cytokine group on days 16 and 22, but produced higher total cell numbers (P0.05) with lower DC2 proportions and cell numbers. The control group without exogenous cytokines spontaneously generated a certain proportion of DC2, but with low total cell and DC2 numbers that decreased rapidly, especially during prolonged culture (days 7 and 16, P0.05).Culture in the presence of 5 ng/ml GM-CSF, 25 ng/ml Flt3L, 20 ng/ml IL-4 and 100 ng/ml SCF can rapidly induce large quantities of predominant immature CD8a(+) DC from murine bone marrow cells. Therefore, these represent optimal culture conditions for preparing murine immature DC2 in vitro.

Neurospheres are free-floating heterogeneous spheroid structures maintained in culture and have been used for in vitro expansion of neural stem/progenitor cells. But the growth characteristics and proliferation kinetics of cell population in neurospheres are poorly understood. Using clonogenic culture and immunocytochemistry, we observed the growth dynamics of cell populations in neurospheres and identified key parameters relating to the growth dynamics of cells in neurospheres, including the fraction of dividing cells, cell cycle duration, time delay of first division, and survival rate of cells. Based on these parameters, we established two mathematical models that describe kinetic features of neural stem/progenitor cells under clonogenic conditions. These models provide a powerful tool to explain and predict the experimental outcome and clarify the cell growth characteristics of neural stem/progenitor cells.

The thymus provides a unique cellular and hormonic microenvironment for the development of immunocompetent T cells. Thymic polypeptides have been widely used clinically for the treatment of tumors, infectious diseases and immune deficiency diseases. They have already shown the ability to stimulate the maturation of hematopoietic stem cells towards the CD3+CD4+ T cell lineage. However, their effects on the thymopoiesis of embryonic stem cells are still unexplored. In this paper, we compared the effects of three thymic polypeptides, thymopentin (TP5), thymosin alpha-1 (Talpha-1) and thymopeptides on the in vitro thymopoiesis of mouse embryonic stem (ES) cells. Using the embryoid body induction system, we found that both Talpha-1 and thymopeptides effectively induced ES cells to differentiate sequentially into the CD3+ and CD4+/CD8+ T cells. These T cells had T cell receptor (TCR) Vbeta gene rearrangement and most were TCRalphabeta T cells. We also found that the expression of the Notch receptor and its ligands Delta-like-1 and Delta-like-4 gradually increased during the induction. However, TP5 failed to induce the T cell differentiation of the ES cells. In summary, this is the first report to demonstrate that Talpha-1 can stimulate the T cell early stage differentiation from ES cells using the embryoid body protocol. These findings provide a powerful model for studying T cell development and may open new venues for the clinical application of Talpha-1.

Neural stem cells are multipotent and self-renewing cells with important potential application in cell replacement therapy in brain damage. Many studies have shown that nestin-positive cells represent neural stem and progenitor cells in the central neural system. Here, we derived neural stem cells from the subventricular zone of a newborn nestin-promoter-driven green fluorescent protein mouse, and found that the percentage of nestin-positive cells decreased continuously at each passage in neurosphere culture. Using the relative proliferation ratio and relative division ratio analysis, we concluded that the slower cycling of nestin-positive cells was responsible for this decrease.

To investigate effects of rat bone marrow mesenchymal stem cells (rBMMSC) on hematopoiesis after allo-hematopoietic stem cell transplantation (HSCT), allogeneic BMT model from Fischer 344 rats (RT-1Al) to Wistar rats (RT-1Au) was established; effects of MSCs on hematopoietic reconstitution were studied by survival rate, peripheral blood counts, histological analysis and FACS at day 30 after transplantation. The results showed that (1) MSCs from donor Fisher344 could survive in recipient irradiated by lethal dose and could be found in the thymus, spleen and bone marrow of the recipient at 30 days after cotransplantation with BM by measuring EGFP gene. (2) Cotransplanation of MSCs and BM improved hematopoietic reconstitution. Lymphocyte and platelet counts of peripheral blood in cotransplantation group were higher than those in the control group. Active hematopoiesis and increase of bone marrow nucleated cells were observed in cotransplantation group. MSCs significantly enhanced hematopoiesis of B lymphocyte and megakaryocytopoietic lineages by FACS analysis. It is concluded that (1) MSCs of Fisher344 can be found in the thymus, spleen, bone marrow of the recipients at 30 days after cotransplantion by measuring EGFP gene. (2) hematopoietic reconstitution is significantly enhanced by MSCs cotransplanted with BM.

Embryonic stem cells (ESCs) are derived from totipotent cells of early embryo and they are potential to differentiate to any kind of cells of tissues in the body. Some reports showed that ESCs had broad capabilities of differentiating to variety of hematopotietic cells, such as erythroid, granulocyte/macrophage, megakaryocyte, mast and lymphocyte precursors. However, it is very difficult to control the phase of differentiation for ESCs in vitro. There is few report about hematopotietic stem cells (HSCs) from ESCs. Therefore, this research was designed to establish a culture system for generation of CD(34)(+)/Sca-1(+) HSC from ESC in vitro.Single mouse E 14.1 cells were suspended in methylcellulose medium, containing 40 ng/ml stem cell factor (SCF) and 20 ng/ml vascular endothelial growth factor (VEGF) and incubated at 37 degrees C with 5% CO2. In order to ensure the viability of the primary differentiation cultures over an extended period of time, the cultures were fed on day 7 with a dilute methylcellulose medium containing VEGF, SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (EPO), which promoted their primary differentiation into embryoid bodies (EBs) with more CD(34)(+)/Sca-1(+) cells. Then, EBs with peak level of CD(34)(+)/Sca-1(+) cells were dispersed into single cells and replanted either in methylcellulose medium or in bone marrow stromal cells differentiation system containing 15% fetal bovine serum (FBS), 160 ng/ml SCF, 20 ng/ml VEGF, 30 ng/ml IL-3, 30 ng/ml IL-6, 3 U/ml EPO and 20% BIT for HSC into second-step differentiation. The HSCs were characterized by flow cytometric analysis, colonogenic cell assay and Wright-Giemsa stains.VEGF had the strongest stimulatory effect on the enhancement of the CD(34)(+)/Sca-1(+) cells population when combined with SCF, IL-3, IL-6 and EPO. It could markedly accelerate mouse E14.1 cells to differentiate into EB with more CD(34)(+)/Sca-1(+) cells. Cell cytometric analysis showed CD(34)(+)/Sca-1(+) cells were up to (1.91 +/- 0.40)% by day 5 and (8.11 +/- 1.17)% by day 8, and the peak level of CD(34)(+)/Sca-1(+) cells was (13.72 +/- 1.92)% by day 12. However, CD(34)(+)/Sca-1(+) cells could not increase in number with the prolongation of differentiation. So renewal single cells suspension from EB by day 12 was dispersed into the second step differentiation. The results showed that HSC was slowly generated with a few hematopoietic colony formations in methylcellulose medium differentiation system. CD(34)(+)/Sca-1(+) cells got (2.74 +/- 0.80)% by day 5 and (11.37 +/- 1.84)% by day 10, and apex percentage of CD(34)(+)/Sca-1(+) cells was about (20.52 +/- 2.78)% by day 14. However, EBs generated quickly for HSC with increased hematopoietic cell population by co-culture on bone marrow stromal cells feeder. Flow cytometric analysis showed that the percentages of CD(34)(+)/Sca-1(+) cells was (7.33 +/- 1.61)% by day 5, (13.28 +/- 2.59)% by day 8, and (20.81 +/- 3.19)% by day 10. EB cells were induced after 12 days to reach the peak level of (34.60 +/- 3.71)%. Hematopoietic colony formation unit (CFU) analysis showed that CFU was sufficient from cells on bone marrow stromal cells differentiation system in the second step compared to that in methylcellulose medium differentiation system, and Wright-Giemsa stain could confirm its characteristics of hematopoietic progenitors.Using two-step differentiation, the investigators got a good way to control the phase of differentiation from ESC to HSC. The bone marrow stromal cell differentiation system combining with VEGF, SCF, IL-3, IL-6 and EPO was an optimal system for the generation of HSC with CD(34)(+)/Sca-1(+) surface marker from ESC differentiated in vitro. This study demonstrated that these cells could form more hemopoietic colonies.

To explore in-vivo regulation of T cells in pulmonary tissues of ovalbumin(OVA)-sensitized mice by BCG and its effect on airway inflammatory reaction.Thirty BALB/c mice (male and female, 6 to 8 weeks of age) were divided into 3 groups. For the mice of first group, aerosol OVA was inhaled daily for 20 minutes once for 10 days running, so as to establish a OVA-sensitized mouse model. In the mice of second group, aerosol normal solution was inhaled according to the time and frequency of the first group. In the third group, the mice were intradermally injected with BCG of 0.04 to 0.06 mg on 10 and 14 days before OVA sensitization, respectively, and then aerosol purified protein derivative (PPD) was inhaled to the mice on 6 days after OVA sensitization. The changes in the number of CD4(+), CD8(+) and IFN-gamma(+) T cells in the inflammatory tissues and changes in the inflammatory cells in the inflammatory tissues and broncho-alveolar lavage fluid (BALF) were detected by SABC immuno-histochemical staining.The number of CD4(+) T cells significantly increased in the pulmonary tissues of OVA-sensitized mice, which mainly was the increased number of CD4(+)/IFN-gamma(-) T cells, While the number of CD8(+) T cells had no notable variation, thus the ratio of CD4(+)/IFN-gamma(+) T cells descended. After BCG treatment, the number of CD8(+) T cells markedly increased, so the ratio of CD4(+)/ IFN-gamma(+) T cells variously rose, whereas the number of inflammatory cells decreased in BALF.BCG can upregulate the number of Th1 cells and lighten the airway inflammatory reaction of experimental asthma mice.

This study was undertaken to establish a murine model for unrelated allogeneic umbilical cord blood transplantation (UCBT). The characteristics and percentage of hematopoietic stem/progenitor cells between near-term fetal and neonatal murine peripheral blood (FNPB) and bone marrow (BM) were evaluated by flow cytometry and semisolid methylcellulose culture. BABL/c (H-2(d)) recipient mice conditioned with high dose CTX were transplanted with FNPB form C57BL/6 (H-2(b)) mice and the survival rate, hematopoietic and immunological reconstruction, graft versus host disease (GVHD) and engraftment level were observed. The results showed that the numbers of day 14 CFU-GM and CFU-GEMM in FNPB (176.40 +/- 78.39)% and (141.40 +/- 56.57)%, respectively were much higher than those in BM (75.20 +/- 26.41)% and (68.80 +/- 23.95)%, respectively. Moreover the percentage of Sca-1(+) CD34(+) cell subsets in FNPB (3.63 +/- 1.13)% was also higher than that in BM (1.41 +/- 0.8 7)%. FNPB transplantation improved survival rate and reconstituted hematopoietic and immune function in recipients. There was no evidence of GVHD. Chimeric analysis showed that the proportion of donor cells in BM of recipients was 27.94% at 21 days after transplantation. It was concluded that FNPB contains more hematopoietic stem and progenitor cells with high expansion ability and weak allogeneic immunity, which was similar to human UCB. The murine model for allogeneic UCBT (C57BL/6--BALB/c) was established successfully.

AIM: Recent reports have shown the capacity of mesenchymal stem cells (MSCs) to differentiate into hepatocytes in vitro and in vivo. MSCs administration could repair injured liver, lung, or heart through reducing inflammation, collagen deposition, and remodeling. These results provide a clue to treatment of liver fibrosis. The aim of this study was to investigate the effect of infusion of bone marrow (BM)-derived MSCs on the experimental liver fibrosis in rats. METHODS: MSCs isolated from BM in male Fischer 344 rats were infused to female Wistar rats induced with carbon tetrachloride (CCl4) or dimethylnitrosamine (DMN). There were two random groups on the 42nd d of CCl4:CCl4/MSCs, to infuse a dose of MSCs alone; CCl4/saline, to infuse the same volume of saline as control. There were another three random groups after exposure to DMN: DMN10/MSCs, to infuse the same dose of MSCs on d 10; DMN10/saline, to infuse the same volume of saline on d 10; DMN20/MSCs, to infuse the same dose of MSCs on d 20. The morphological and behavioral changes of rats were monitored everyday. After 4-6 wk of MSCs administration, all rats were killed and fibrosis index were assessed by histopathology and radioimmunoassay. Smooth muscle alpha-actin (alpha-SMA) of liver were tested by immunohistochemistry and quantified by IBAS 2.5 software. Male rats sex determination region on the Y chromosome (sry) gene were explored by PCR. RESULTS: Compared to controls, infusion of MSCs reduced the mortality rates of incidence in CCl4-induced model (10% vs 20%) and in DMN-induced model (20-40% vs 90%).The amount of collagen deposition and alpha-SMA staining was about 40-50% lower in liver of rats with MSCs than that of rats without MSCs. The similar results were observed in fibrosis index. And the effect of the inhibition of fibrogenesis was greater in DMN10/MSCs than in DMN20/MSCs. The sry gene was positive in the liver of rats with MSCs treatment by PCR. CONCLUSION: MSCs treatment can protect against experimental liver fibrosis in CCl4-induced or DMN-induced rats and the mechanisms of the anti-fibrosis by MSCs will be studied further.

Previously we reported that a uni-axial cyclic stretch treatment of rat 3Y1 fibroblasts induced focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation (Wang et al., 2001) [Wang, J.G., Miyazu, M., Matsushita, E., Sokabe, M., Naruse, K., 2001. Uni-axial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Comm. 288, 356-361]. In the present study, we investigated whether stretch-induced MAPK activation leads to proliferation of fibroblasts. 3Y1 fibroblasts were subjected to a uni-axial cyclic stretch treatment (1 Hz, 120% in length) and the bromodeoxyuridine (BrdU) incorporation was measured to access cell proliferation. BrdU incorporation increased in a time-dependent manner and became significant within 6 hours. To investigate the involvement of FAK, we transiently expressed FAK mutants that lacked tyrosine phosphorylation site (s) (F397Y, F925Y, F397/925Y). Transient expression of wild-type FAK or mock vector did not inhibit the stretch-induced BrdU incorporation, however, the FAK mutants significantly blocked BrdU incorporation. Treatment of the cells with MAPK inhibitors, PD98059 or SB203580, blocked extracellular signal-regulated kinase (ERK) phosphorylation and p38 MAPK phosphorylation, respectively, and also blocked stretch-induced BrdU incorporation. These results suggest that the stretch-induced FAK activation followed by MAPK activation plays an important role in the stretch-induced proliferation of 3Y1 fibroblasts.