A protoplast-to-plant system for the methionine resistant variant of Astragalus cicer L. has been developed. The friable calli induced from stem segments of variant plants were used as materials for protoplast isolation through enzyme digestion. The effects of different media and plating densities on protoplast divisions and plant regeneration were studied. Sustained cell divisions and colony formation from the protoplasts of the methionine resistant cell line of Astragalus cicer L. were obtained by a DPD medium containing 2.0 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D), 0.2 mg/L 6 -benzylaminopurine(6-BA), 0.3 mol/L mannitol, 200 mg/L casein hydrolysate and 2% (W/V) sucrose at a plating density of 2x10(5) /ml. The division frequency was 38.3%. At the same time, different dividing types of protoplasts were found. Organogenesis and shoot formation from the protoplast-derived calli were induced on MS medium supplemented with 0.5 mg/L NAA, 10 mg/L KT and 2% (W/V) sucrose. The protoplast-derived calli still expressed resistance to methionine. The protoplast to plant regeneration protocol developed in this study might provide the foundation for the resistant cell line as a parent for somatic hybridization.

Adipose were obtained from patients underwent liposuction treatment (total 19 female donors, 31.5 +/- 5.8 years old). Liposuction tissues were digested with type I collagenase, cells were isolated and cultured up to passage 10. To evaluate the proliferation potential of ADCs, growth curve and cumulative population doubling were achieved by cell counting. CD29,CD105, CD106, CD166, CD49d, CD34, CD31, 3G5 were analyzed by flow cytometry and immunocytochemistry to characterize the cell population. The multi-lineage potential of ADCs was testified by differentiating cells with osteogenic,chondrogenic and adipogenic inducer. A total of 5x10(7) nucleared cells could be obtained from 300ml liposuction tissues. After in vitro cultivation,cumulative population doubling number reached 15.53 at passage 10 (average 1.59 +/- 0.224 /passage). Flow cytometry and immunocytochemistry showed that ADCs expressed high level (>60%) of stem cell-related antigen (CD29, CD105, CD106, CD166), while cells expressed hematopoiesis-related antigen CD34 and CD31 around 7.3% and 29.2% respectively. Collagen II (both in mRNA and protein level) was detected in chondrogenic differentiation. The calcified nodules were observed by von Kossa and Alizarn Red staining and the expressions of AKP and Osteonectin were detected by RT-PCR in osteogenic differentiation. PPARr2, GLU-4, and Leptin genes were detected in adipogenic differentiation and intracellular lipid droplets could be observed by Oil Red staining. ADCs can be abundantly harvested and have high proliferative and multi-lineage differentiation potential.

After allogeneic hematopoietic stem cell transplantation (allo-HSCT), the donor cells present a profound immunization therapy efficiency. Among these effector cells, allo-reactivity natural killer (NK) cell activation are concerned with the graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) effect. As known, GVHD is primarily a T-cell-mediated event but not initiated by NK cells. NK cells may significantly enhance GVL immune response by using an integration of activating and inhibitory receptors. Allo-reactivity NK cell infusion after allo-HSCT already transits from experiments to clinic. In this review the background on NK cells, and their clinical roles in Allo-HSCT were summarized.

Application of ex vivo expanded megakaryocytic progenitor cells (MKPC) is a strategy for the treatment of thrombocytopenia after hematopoietic stem cell transplantation. Some growth factors including thrombopoietin (TPO), megakaryocyte growth and development factor (MGDF), interleukin (IL)-1, IL-3, IL-6, IL-11, platelet-derived growth factor (PDGF), and serotonin (5-HT) have been demonstrated to play an important role on the regulation of megakaryocyte/platelet development, the efficient conditions for the expansion of the megakaryocyte (MK) progenitors from hematopoietic stem/progenitor cells were discussed in this review article. TPO alone produced a high proportion of MK progenitors but a low total cell count. The addition of IL-1 beta, IL-3, IL-6 and Flt-3L improved the expansion outcome. The combination of three to five cytokines produced more efficient expansions of hematopoietic stem and MK progenitors. PDGF also enhanced the ex vivo expansion of CD61+ CD41+ cells and CD34+ cells in combination with TPO, IL-1 beta, IL-3, IL-6 and Flt-3L. PDGF is a suitable growth factor to improve the ex vivo expansion of MKPC without promoting their in vitro maturation. More importantly, PDGF also enhanced the engraftment of human stem and progenitor cells in NOD/SCID mice. It has been reported that MKPC can be safely administered to autologous peripheral blood progenitor cell transplant recipients. In short, MKPC can be expanded ex vivo and safely applied to autologous transplant.

To evaluate the clinical efficacy of unrelated umbilical cord blood transplantation (UCBT) on the treatment of children with hematologic malignancies and nonmalignancies, between August 2001 and June 2004, 32 patients were transplanted by using unrelated umbilical cord blood supplied by Shandong Umbilical Cord Blood Bank. Out of them, 13 patients suffered from ALL, 9 from AML, 3 from AA, 3 from HAL, 2 from CML and 2 from NHL. The median age was 8 years (range 2-15), the median weight was 31.5 kg (range 14-55). All patients received ablative conditioning regiment according to the disease and the disease status. Conditioning regiments Cy/TBI were used for 4 patients and Bu/Cy for 21 patients, other for 7 patients. All patients received cyclosporin A and/or methotrexate for GVHD prophylaxis. The mean number of infused nuclear cells were 5.57 (2.16-12.3) x 10(7)/kg, CD34+ cells 1.78 (0.85-5.59) x 10(5). All of UCB units were tested for HLA-A, -B, and DRB1 using low and high resolution techniques. There were HLA-matched in 10, 5/6 in 16 and 4/6 in 6. The results showed that 20 out of 32 patients achieved complete engraftment. Median time of neutrophil > or = 0.5 x 10(9)/L, and platelet > or = 20 x 10(9)/L were 17 (9-38) and 42 (18-102) days respectively. The incidence of aGVHD II-IV and aGVHD III-IV were 35% and 15% respectively. After a median follow-up of 18 months (1.5-28.5), overall survival rate at one year was 59.4%, overall survival rate at two years was 40.6%. It is suggested that UCBT is promising for children patients who is lack of matched bone marrow donors.

This study was aimed to investigate the difference of immunological properties between recombination human granulocyte colony-stimulating factor (rhG-CSF) mobilized peripheral blood grafts (G-PB) and rhG-CSF primed bone marrow grafts (G-BM). The lymphocyte proliferation ability and the quantities of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) secreted by T cells were determined by using MTT assays and sandwich ELISA; T cell subgroups, dendritic cells (DC), monocytes and the expression of CD28 costimulatory molecules on T cells were determined by multicolor flow cytometry. The results showed that the absolute numbers of lymphocytes, monocytes, CD3+, CD4+ and CD8+ T cells as well as DC1 and DC2, the ratios of CD4/CD8 in G-PB were significantly higher than those in G-BM, respectively (P < 0.001). T cell proliferation ability was significantly higher in G-PB than that in G-BM (P < 0.05). The quantities of IFN-gamma and IL-4 secreted by T cells per microliter of G-PB was significantly higher than those of G-BM, the ratios of IL-4/IFN-gamma were significantly lower in G-PB than that in G-BM (P < 0.001). As compared with G-BM, the ratio between DC2 and T-lymphocyte was significantly low in G-PB (P < 0.01), whereas the percentage and overall expression of CD28 on CD4+ and CD8+ cells were significantly high in G-PB (P < 0.05). It is concluded that T cell hyporesponsiveness of G-PB and G-BM induced by rhG-CSF in vivo were confirmed to be different, and the difference of immunological properties between G-PB and G-BM may explain the lower incidence of GVHD and lower relapse after G-BM and G-PB transplantation respectively.

Sphingosine 1-phosphate (S1P), which can be impacted by different growth factors through sphingosine kinase (SphK), is a bioactive lipid produced by metabolism of sphingolipid with various biological responses. Recombinant human granulocyte-colony-stimulating factor (rhG-CSF) have been used widely in peripheral blood stem/progenitor cell mobilization. This study was aimed to investigate the effects of rhG-CSF on S1P concentration in plasma of donors. The peripheral blood mononuclear cells and blood plasma were collected from 8 peripheral blood progenitor cell donors before mobilization and on the fifth day of mobilization with rhG-CSF. The SphK mRNA expression of blood mononuclear cells were detected by RT-PCR. The changes of S1P concentration were assayed with SphK enzyme catalyzed reaction. The results showed that both kinds blood mononuclear cells collected before and after rhG-CSF mobilization expressed SphK mRNA. The S1P concentration is low in donor's plasma both before and after mobilization with rhG-CSF, and there was no markedly change of S1P concentration in plasma before and after mobilization (P > 0.05). In conclusion, mobilization with rhG-CSF does not impact S1P concentration in donors' plasma.

The aim of this study was to evaluate the effect of all-trans retinoic acid (ATRA) on cell adhesion molecule expression and adhesion capacity of bone marrow stromal cells (BMSC) in patients after conditioning treatment for peripheral blood stem cell transplantation (PBSCT). BMSC of 27 patients before and after conditioning treatment for PBSCT were cultured in vitro. After treated with ATRA at 0.01, 0.1, or 1 micromol/L, expression of intercellular adhesion molecule-1 (ICAM-1) protein and vascular adhesion molecule-1 (VCAM-1) protein were detected by flow cytometry, and soluble ICAM-1 (sICAM-1) protein was determined by using radioimmunoassay. Then BMSC was co-cultured with CD34+ cells, and adhesion rate of BMSC to CD34+ cells was measured. The results showed that after pretreatment with conditioning regimen for PBSCT, the expressions of ICAM-1 and VCAM-1 proteins in BMSC and the expression level of sICAM-1 protein in supernatant of BMSC culture were down-regulated, and the adhesion rate of BMSC to CD34+ cells was decreased, after administration of ATRA, the expression of ICAM-1 protein in BMSC, sICAM-1 protein in culture medium and adhesion rate of BMSC to CD34+ cells all increased significantly, but expression of VCAM-1 protein changed no significantly. It is concluded that the ATRA can partly restore adhesion function of BMSC injured by pretreatment for PBSCT and contribute to hematopoietic reconstitution.

This study was aimed to investigate the effect of some culture system composed of various cytokine combinations (TPO, SCF, FL, IL-1, IL-3, IL-6) on ex vivo expansion of megakaryocytic progenitors induced from CD34+ cells of peripheral blood and to seek a optimal cytokine combination and culture time. Mononuclear cells were isolated from mobilized peripheral blood (MPB) by density gradient centrifugation over Ficoll. CD34+ cells were purified by using an immunomagnetic bead separation system. The selected CD34+ cells were seeded in Iscove's modified Kulbecco's medium (IMDM) supplemented with fetal calf serum (FCS) and various kinds of cytokines. After 15 days of culture, the content of CD41+ cells in culture system were determined by flow cytometry, and the number of megakaryocyte colony-forming unit (CFU-MK) was measured simultaneously. The results showed that after definited days of culture, the cytokine combination TPO/FL/IL-6/IL-3 was the most suitable for MPB to obtain high number of MK, and better than any other three groups (P < 0.05). The increase multiple of CD41+ cells was 93.97 +/- 17.27 on day 5 and 131.23 +/- 18.26 on day 10. On day 15, the proportion and the increase multiple of CD41+ cells decreased obviously. The expansion multiples of CFU-MK were 93.33 +/- 10.02 on day 5 and 120.67 +/- 13.01 on day 10, higher than any other groups. It is concluded that TPO/FL/IL-6/IL-3 combination was the best optimal for expansion ex vivo of megakaryocytic progenitors from MPB, and its suitable duration of culture was 10 days; a culture system for expansion ex vivo of megakaryocytic progenitors have been established in this study.

This study was aimed to investigate the effect of angiotensin II on differentiation of cord blood CD34+ cells into megakaryocytes in vitro. The CD34+ cells from eight fresh umbilical cord blood samples sorted by a high-gradient magnetic cell sorting system (MACS) were cultured in serum-free culture medium containing thrombopoietin (TPO) 50 ng/ml, IL-3 10 ng/ml, stem cell factor (SCF) 50 ng/ml and different concentrations of angiotensin II (0, 50, 100, 1000 microg/ml) for 14 days. Mononuclear cells (MNC) were counted by automatic cell analyzer. Cultured CD41+ cell and platelet counts in cultured system, and cell cycle were analyzed by flow cytometry. CD41 specific monoclonal antibody staining was observed by immunofluorescence microscopy. The results showed that as compared with the control group, the number of MNC not increased significantly (P > 0.05), but the number of CD41+ cells and platelets increased significantly in treatment group (P < 0.05). Cell cycle analysis revealed that the amounts of 4N cells increased and apoptosis cells obviously existed in treatment group (P < 0.05). After fluorescence staining, more CD41+ cells of different sizes were observed by means of fluorescence microscopy in both groups. It is concluded that angiotensin II can induce the cord blood CD34+ cells to differentiate towards megakaryocyte, and enhance the function of megakaryocyte to produce platelet.

To compare the growth characteristics of non-hematopoietic adult stem cells (NASC) derived from rat fetal blood and rat bone marrow in vitro, and to study the differentiation of these stem cells into neuron-like cells in vitro, the fetal blood of pregnant rats and bone marrow of adult rats were sterilely collected; mononuclear cells (MNC) were isolated by using standard Ficoll-hypague techniques and then cultured in DMEM/LG containing 10% fetal bovine serum (FBS). The acquired NASCs were subcultured for passage. The immunophenotype of NASCs was detected by flow cytometry. The expanded NASCs were induced to differentiate into neurons-like cells by beta-mercaptoethanol (beta-ME), dimethylsulfoxide (DMSO), butylated hydroxyanisole (BHA). The specific markers of these neuron-like cells were detected by immunocytochemistry. The results showed that two kinds of subcultured NASCs showed homogeneous spindle-shaped and expressed antigens CD44 and CD54, but did not expressed CD11b and CD45. The both induced cells were similar to neuron in morphology and were positive for nestin and neuron-specific enolase (NSE), but negative for glial fibrillary acidic protein (GFAP). It is concluded that no significant difference of NASCs derived from pregnant rat fetal blood and adult rat bone marrow found in cell morphology and biological characteristics. NASCs of both origins can be induced to differentiate into neuron-like cells, so fetal blood can be regarded as another resource of NASC.

To investigate the related factors affecting the isolation of multipotent non-hematopoietic adult stem cells (MNASCs) from human umbilical cord blood in low serum (2%) condition, the isolation conditions were optimized and the yield of MNASCs was improved. MNASCs from human umbilical cord blood samples were isolated, and the effects of medium component, medium exchange time and initial plating density for isolation of MNASCs were studied. Then, the MNASCs were isolated and cultured in optimal condition, the surface antigen expression and differentiation potential of MNASCs were detected. The result showed that the medium of DMEM/F12 was better than IMDM and DMEM-LG for MNASCs culture in low serum condition. The optimal yield of MNASCs was obtained when mononuclear cells were cultured at a initial plating density of 1 x 10(6) cells/cm2 and the medium was exchanged to remove the nonadherent cells after 72 hours of inoculation. MNASCs isolated and cultured under the above-mentioned conditions maintained a homogenous morphology, high potential ability of expansion and differentiation. It is concluded that culture conditions with low serum defined in this study is optimal for the successful isolation and expansion of umbilical cord blood MNASCs with high numbers for subsequent cellular therapeutic approaches.

To screen and separate the genes differentially expressed in human embryonic aorta-gonad-mesonephros (AGM)-derived stromal cells, a subtracted library was generated through the suppression subtractive hybridization using the cDNA of human embryonic AGM-derived stromal cells as target and human fetal liver (FL)-derived stromal cells as drivers. Then a high though screening technique, gene chip, was used to screen the differentially expressed genes in the established subtractive library. Approximately 18 of the resulting subtracted cDNA clones were partially sequenced and analyzed by blastn in the GenBank database. The results showed that 211 Clones were selected and identified from the established subtractive library, the positive ratio was amount to 76.4%. 18 over-expressed genes were screened by gene chip with more than a 5-fold difference expression levels between AGM and FL-derived stromal cells, and were selected to sequence, results of sequencing indicated that the 18 sequences was compared to known sequences in the GenBank database, and among the sequenced clones, 14 sequences were considered as part of the known genes, and 4 sequences representing previously unknown genes. The known genes were reported to involve the regulation of cell migration, cell differentiation, cell proliferation, cell cycle, signal transduction, and angiogenesis. Most of these genes have not been reported to relate to the haematogenesis in ontogeny. It is concluded that many genes both known and unknown are differentially expressed in human embryonic aorta-gonad-mesonephros-derived stromal cells. Discovery of these genes provides a solid foundation to elucidate the mechanism of haematogenesis in ontogeny.

To induce DNA oxidative damage in colorectal crypt cells by hydrogen peroxide in vitro.

To investigate the fluences on the proliferation and undifferentiation of spermatogonial stem cells in vitro.

To investigate the effect of vasoactive intestinal polypeptide (VIP) on differentiation of hematopoietic stem cells (HSC) into hepatic related cells and probe into the possibility that VIP affects HSC transdifferentiation.

Neural stem cells are a potential therapeutic source for cellular transplantation therapy in neurological diseases. The present paper was aimed to investigate whether neural stem cells could be obtained from the spinal cords of low temperature preserved abortuses. Fourteen weeks old abortuses were stored in a refrigerator at 4 degrees C without any additional treatments for 2, 6 and 12 h before use. The spinal cords were anatomized out and divided into cervical cords, thoracic cords and lumbar/sacral cords. Then the spinal cord segments were used for cell culture separately. Neural stem cells were isolated from the segments and cultured in bFGF, EGF and N2 supplement containing free-serum DMEM/F12 (1:1) medium. In order to examine the differentiation potential, the stem cells were induced to differentiate with 5% fetal bovine serum on poly-l-lysine substrate. Clonal culture was carried out to demonstrate that the isolated cells met the standard of stem cells. Indirect fluorescent immunocytochemistry was used to examine the expressions of neural stem cell marker (nestin), neuron marker (MAP2), astrocyte marker (GFAP) and cholinergic marker (ChAT). The stem cells in different cultures were compared. One-way analysis of variance and Kruskal-Wallis test were used for the statistical comparison. As a result, neural stem cells were obtained from all the spinal cord segments with different postmortem intervals. Both the cells on the surface and inside the neurospheres showed nestin immunoreactivity. Therefore, nearly all the cells that composed the neurospheres were nestin-positive undifferentiated cells. When the spheres were induced to differentiate, they could yield GFAP-positive astrocytes and MAP2-positive neurons including ChAT-positive cholinergic neurons. Primary neurospheres could be dissociated mechanically, expand in subcultures and maintain the differentiation potential. In clonal cultures, single cells from a single primary sphere could give rise to new neurospheres, which had the same differentiation potential as the primary spheres. The lumbar/sacral cord cultures gave rise to the most abundant primary neurospheres. When the preservation time of the fetus was prolonged to 12 h, the number of primary neurospheres decreased sharply. The clonal formation and phenotype capacity were similar in all cultures. In conclusion, spinal neural stem cells can be isolated from low temperature preserved abortuses and represent an alternative source for experimental and potential therapeutic purposes.

To screen differentially expressed genes involved in osteogenic differentiation of human bone marrow stromal cells (BMSCs) at defined stages, subtractive cDNA library was established by means of suppression subtractive hybridization. The BMSCs cultured for 12 and 21 d were used as driver and tester, respectively. A subtract library was successfully constructed and five positive clones were selected from the library. Sequencing analysis and homology comparison showed that the five clones differentially expressed in BMSCs cultured for 21 d were at least 90% homologous with the known genes in human GenBank. It was interestingly found that the osteogenic BMSCs cultured for 21 d differentially expressed decorin and Bax inhibitor 1. RT-PCR was performed to confirm the differentially expressed genes. The results showed that the expression of Bax inhibitor 1 was significantly higher in the cells of 21-day than that of 12-day, while the expression of decorin was only detected in the cells of 21-day.

The immunoregulatory effects of mescenchymal stem cell (MSC) and its application have become a hot research topic in recent years. This article reviews the up-to-dated research advances in the features and mechanisms of immune regulation of MSC and its application.