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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.

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.

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.

Human mesenchymal stem cells (hMSC) are a population of multipotent cells that can differentiate into osteoblasts, chondrocytes, adipocytes, and other cells. The exact mechanism governing the differentiation of hMSC into osteoblasts remains largely unknown. Here, we analyzed protein expression profiles of undifferentiated as well as osteogenic induced hMSC using 2-D gel electrophoresis (2-DE), mass spectrometry (MS), and peptide mass fingerprinting (PMF) to investigate the early gene expression in osteoblast differentiation. We have generated proteome maps of undifferentiated hMSC and osteogenic induced hMSC on day 3 and day 7. 2-DE revealed 102 spots with at least 2.0-fold changes in expression and 52 differently expressed proteins were successfully identified by MALDI-TOF-MS. These proteins were classified into 7 functional categories: metabolism, signal transduction, transcription, calcium-binding protein, protein degradation, protein folding and others. The expression of some identified proteins was confirmed by further RT-PCR analyses. This study clarifies the global proteome during osteoblast differentiation. Our results will play an important role in better elucidating the underlying molecular mechanism in hMSC differentiation into osteoblasts.

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.

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.