To investigate the differentiation status of autologous bone marrow mononuclear cells (BM-MNC) and peripheral endothelial progenitor cells (EPC) transplanted into myocardial ischemia reperfusion injury region in swine.

To explore the feasibility of magnetic resonance imaging (MR) on detecting transplanted nanometer small superparamagnetic iron oxides (SPIO) labeled mesenchymal stem cells (MSCs) in swine model with acute myocardial infarction (MI).

To investigate the possibility of transdifferentiation of adipose mesenchymal stem cells (AMSCs) into hepatocytes.

To establish a method through which murine bone marrow mesenchymal stem cells (MSCs) can be induced into hepatocytes in vitro.

To study the effects of rat endothelial progenitor cell (EPC) transplantation on hepatic fibrosis in carbon tetrachloride (CCl4) induced hepatic fibrosis rats.

To explore the expression of T cell factor 4(Tcf-4) in the process of severe abdominal infection in rats.

Mesenchymal stem cells are a kind of non-hematopoietic adult stem cells with selfrenewal and multilineage differentiation potential, which have special biological characteristics, such as secreting hematopoietic growth factors, reconstructing hematopoietic microenvironment, low immunogenicity, and can be transfected and expressed by exogenous gene. This article summarizes the biological characteristics of MSCs and their models of application to acute radiation disease in animals.

With the capacities of multiple differentiation, immunoregulatory activities and easy handling for isolation and culture expansion, human bone marrow mesenchymal stem cells (MSCs) have been utilized in clinical trials for the prevention and treatment of graft-versus-host disease in allogeneic bone marrow transplantation, repair of bone and cartilage defects and treatment of cardiac infarction and liver injury. However, increasingly experimental data indicate that a great deal of issues, such as intra-neutralization of calf serum proteins into cultured MSCs, survival of engrafted cells and subsequent cell differentiation tendency, should be in stringent consideration before clinical trials are designed. In this paper, these issues that should be raised and solved in clinical trials with MSCs were reviewed.

In order to explore the security and feasibility of double autologous peripheral blood stem cell transplantation (APBSCT) for treatment of multiple myeloma, a 49 years old female patient with multiple myeloma was therapied with double APBSCT. The first peripheral blood stem cell (PBSC) mobilization regimen included CTX 2 g/m(2) x 1d and G-CSF [10 microg/(kgxd)] x 5 d. The conditioning regimen was given melphalan 200 mg/m(2). The transplanted number of mononuclear cells was 6.1 x 10(8)/kg and that of CD34(+) cells was 4.7 x 10(6)/kg. The second APBSCT was performed six months later. PBSC mobilization regimen was G-CSF [10 microg/(kgxd)] x 5 d. The conditioning regimen was melphalan 200 mg/m(2). The transplanted number of mononuclear cells was 10.2 x 10(8)/kg and that of CD34(+) cells was 5.9 x 10(6)/kg. The results showed that the absolute neutrophil count (ANC) rose to above 0.5 x 10(9)/L on day 17 and platelet count exceeded 20 x 10(9)/L on day 15 after first transplantation. After second transplantation ANC rose to above 0.5 x 10(9)/L on day 22 and platelet count exceeded 20 x 10(9)/L on day 13. There were neither obvious adverse reaction nor severe complication during the double transplantations. The patient's ostealgia and anemia were healed through above therapy. In the follow-up of 7 months, the patient's general status was good and she remained in complete remission phase. It is concluded that double APBSCT is safe, effective and feasible for the treatment of multiple myeloma.

This study was aimed to investigate the therapeutic effect of growth factor-primed donor peripheral mononuclear stem cell infusion (DMNCI) for patients with relapsed leukemia after haploidentical bone marrow transplantation (BMT). The donor was the same individual for both BMT and DMNCI. All the three patients described here were Philadelphia chromosome positive leukemia before haploidentical BMT; one case was newly diagnosed as acute lymhoblastic leukemia (ALL) and the others were chronic myeloid leukemia (CML). Two cases (one with ALL and one with CML) manifested with clinical relapse and the third case was in the stage of molecular relapse. The former 2 patients received a single bulk dose of DMNCI, the inoculums of which contained mononuclear cells of 8.25 x 10(8)/kg or 5.24 x 10(8)/kg and CD3-positive cells of 1.87 x 10(8)/kg or 1.14 x 10(8)/kg respectively. The third case received initial dose of DMNCI which was 2.0 x 10(7)/kg, and received CD3 positive cells of 1.1 x 10(7)/kg. The results indicated that the different therapeutic responses were found in all three patients. Two patients with clinical relapse received temporal remission, and died of severe graft versus host disease (GVHD), relapse and failure at day 41 and 49 after DMNCI. The third patient with molecular relapse received molecular remission after 2 infusions of DMNCI. All three patients developed acute GVHD, but two patients among them developed GVHD of grad IV, other one developed GVHD of grad I and has survived in disease-free state during half a year follow-up. It is concluded that the DMNCI may be effective for the treatment of relapsed leukemia after haploidentical BMT and this treatment can be safe if the initial dose of DMNCI is 10(7)/kg and subsequent single dose of DMNCI gradually increases.

This study was purposed to investigate the change of Th cell subsets in the patients with acute graft-versus host disease (aGVHD) and to explore its role in the pathogenesis of aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT). 23 patients underwent allo-HSCT were selected for analysis. The aGVHD in patients was diagnosed according to clinical features, and was confirmed by skin biopsy in some patients. The peripheral blood from 23 patients was collected before and after allo-HSCT. The quantitative chenges of Th1 and Th2 cells in peripheral blood samples were detected by using flow cytometry. The results showed that out of 23 patieats the aGVHD occured in 8 patients including 1 case of grade I, 2 case of grade II, 3 cases of grade III; no aGVHD occured in 15 patients. The flow cytometry analysis revealed that the amount of Th1 cells in patients with aGVHD was much higher than that in patients without aGVHD (p < 0.01), the IFN-gamma expression of Th1 cells in patients with aGVHD of grad II - III significantly was higher than that in patients without aGVHD (p < 0.01), meanwhie the IL-4 expression of Th2 cells in patients with aGVHD of grade II - III was significantly lower than that in patients without aGVHD (p < 0.05). Dynamical detection indicated that the Th1 obviously increased before occurrence of aGVHD and before treatment of aGVHD, while the Th1 cells obviously decreased after treatment of aGVHD. The Th1 cells not changed significantly in patients without aGVHD before and after allo-HSCT. It is concluded that Th1 cells obviously increase in patients with aGVHD, this increase is related to aGVHD pathogenesis. Detecting the changes of Th cell subsets in the early stage after allo-HSCT may be contributed to early diagnosis and therapy of aGVHD.

The objective of this study was to investigate the curative effect of combined sibling umbilical cord blood and bone marrow transplantation in treatment of beta-thalassemia major. Combined umbilical cord blood and bone marrow transplantation from an HLA-identical sibling were performed for 3 patients with beta-thalassemia major. The nucleated cells infused into 3 recipients were 19.5 x 10(7)/kg, 20.8 x 10(7)/kg and 23.3 x 10(7)/kg respectively. They accepted the conditioning regimen consisting of busulfan, cyclophosphamide, antithymocyteglobulin. The results showed that three patients gained protracted and stable engraftment. The time to achieve more than 0.5 x 10(9)/L neutrophils in three patients was 16, 18, and 17 days respectively; the time to achieve more than 50 x 10(9)/L platelet in three patients was 48, 50, and 49 days respectively. The speed of hematopoietic recovery was faster than that of umbilical cord blood transplantation (UCBT) only. Three patients all suffered from acute graft-versus-host disease (aGVHD) of I grade. They had lived with free-thalassemia for 1.5, 2.0 and 2.1 years respectively. Their Hb had been maintained at normal level without transfusion. It is concluded that combined UCBT and BMT may be an effective and safe way to treat pediatric beta-thalassemia major.

This study was purposed to investigate the dynamic change of clonal proliferation of T cell receptor V subfamilies in peripheral blood of patients received allo-hematopoietic stem cell transplantation (allo-HSCT) and to analyze the relationship between T cell clonal proliferative changes and GVHD. The peripheral blood mononuclear cell samples from 70 cases (17 GVHD patients) undergoing allo-PBCST patients were detected for CDR3 (complementarity determining region 3 repertoire analysis of T cell receptor Vbetagene) using reverse-transcriptase-polymerase chain reaction (RT-PCR). The products were further analyzed by genescan to identify T cell clonality. The results showed that the patients of HSCT generally passed through a transformation from monoclone to polyclone. At day 60 - 90 after HSCT, half of the cases were monoclonal and the remainders were polyclonal. After 120 days, most of patients without GVHD transferred into polyclones, however, patients with GVHD remained monoclonal after one year because of immunosuppressive agents and GVHD itself. The peripheral blood of GVHD patients mainly expressed monoclone/biclone at the time of target organ damage conspicuously, after medication intervention, partial monoclone or bioclone expressed TCR Vbeta subfamilies were diverted to polyclonal expression. It is concluded that the T cells present clonal proliferation and T cell receptors are prone to be used when patients are in earlier period of transplantation or with GVHD especially. The expression of TCR Vbeta subfamilies can return to normal polycloning along with the recovery of hematopoiesis and immunity in patients.

To evaluate the effects of rhG-CSF on mobilization of mesenchymal stem cells (MSCs) of mouse bone marrow at different time point, thirty mice were randomly divided into rhG-CSF treatment group and control group. The mice were subcutaneously injected with rhG-CSF in a dose of 80 microg/kg or saline for 5 days. The bone marrow and peripheral blood were obtained at time points of 6, 12, 168 hours after final injection of rhG-CSF or saline. Bone marrow mononuclear cells (BMMNCs) were seeded at density of 1 x 10(6) MNCs onto 12-well plate for culture expansion in DMEM supplemented with 10% FBS, and the number of colony forming unit - fibroblast (CFU-F) was counted after 14 days. The cells were collected by trypsinization and the surface antigens CD34, CD133, CD90 and CD105 were analyzed by flow cytometry. The multi-differentiation of MSCs were done in the culture condition of induced-adipocyte and osteocyte. Peripheral blood MNCs examination was same as the bone marrow. The results indicated that the number of CFU-F of bone marrow in rhG-CSF group was more than that in control group (p < 0.01), the number of CFU-F in rhG-CSF group at 6 hours was more than that at 12 hours and 168 hours, respectively (p < 0.01). There was no obvious difference between CFU-F at 12 hours and at 168 hours (p > 0.05). MSCs were positive for CD90, CD105 and negative for CD34 and CD133. MSCs were found to differentiate into adipocyte and osteocyte in vitro. The CFU-F of PBMNCs obtained and cultured in vitro in the same culture conditions could be observed after the rhG-CSF injection at 6 hours, but cloning efficiency was (0.50 +/- 0.11) x 10(-6) MNCs and showed statistical difference as compared with control. It is concluded that rhG-CSF to mobilize hemopoietic stem cells can be used to induce mouse MSCs in vivo expansion, which showed the peak value within 6 hours after final injection of rhG-CSF. rhG-CSF have the mini-mobilization effect on murine MSCs derived from bone marrow.

The study was purposed to investigate the immune regulatory effects of human bone marrow mesenchymal stem cells (hMSCs) on Foxp3 expressing CD4(+)CD25(+) regulatory T cells and to explore the mechanism of immune modulation by hMSCs. Human MSCs were isolated and expanded from bone marrow cells, and identified with cell morphology, and the phenotypes were assessed by immunohistochemistry. Human peripheral blood mononuclear cells (hPBMNCs) were prepared by centrifugation on a Ficoll Hypaque density gradient. The hMSCs (1 x 10(3), 1 x 10(4), 1 x 10(5)) were added into wells containing hPBMNCs (1 x 10(6)) from an unrelated donor in the presence of rhIL-2. After 5 days of co-culture, the percentage of CD4(+)CD25(+) T cells was detected by flow cytometry. T cell proliferation was assessed by [(3)H] thymidine incorporation using a liquid scintillation counter. The expression of Foxp3 in CD4(+)CD25(+) T cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Cytokines (TGF-beta, IL-12, IFN-gamma, IL-10) concertrations of cultured supernatants were measured with ELISA. The results indicated that in all the experiments, the presence of hMSCs with hPBMNCs resulted in a statistically significant decrease in T cell proliferation, in dose-dependent manner. The increase of percentage of CD4(+)CD25(+) T cells in the peripheral CD4(+) T cell was observed after coculturing lymphocytes with hMSCs (p < 0.01). The expression of Foxp3-mRNA (Foxp3/beta-actin) in hMSCs groups was significantly higher than that in the control and was negatively associated with the value of CPM representing T proliferation. The levels of TGF-beta and IL-10 were higher in hMSCs groups than that in the control, and the levels of TGF-beta and IL-10 correlated positively with Foxp3-mRNA expression and the percentage of CD4(+)CD25(+) T cells. However, the secretion of IL-12 and IFN-gamma was significantly attenuated by hMSCs coculture, and there was no correlation with Foxp3-mRNA expression and the percentage of CD4(+)CD25(+) T cells. It is concluded that the Foxp3 expressing regulatory T cells may play an important role in the immune regulatory by hMSCs. Its mechanism is related to that the hMSCs-mediated TGF-beta and IL-10 convert CD4(+)CD25(-) T cells into CD4(+)CD25(+) T regulatory T cells, which specifically inhibits the proliferation of T cells.

This study was purposed to investigate the colony formation of high-proliferative potential colony-forming units (CFU-HPP) from bone marrow-derived hematopoietic cells of psoriatic patients and p16 gene promotor methylation in CFU-HPP cells, and to explore the relationship between the colony formation and the methylation status of p16 gene promoter. Bone marrow-derived mononuclear cells from psoriatic patients and normal controls were separated by density gradient centrifugation, and were cultured in methycellulose semi-solid culture medium with SCF, GM-CSF, IL-3 and IL-6 for 14 days to measure the colonies of CFU-HPP. The CFU-HPP colony cells were collected and methylation status of p16 gene promoter of CFU-HPP cell DNA modified with sodium bisulfite was detected by the methylation-specific polymerase chain reaction (MSP). The results showed that in methycellulose semi-solid culture system, the number and the size of CFU-HPP colonies of bone marrow of psoriatic patients were all significantly less than that of normal controls, the positive frequency of p16 gene promoter methylation in CFU-HPP cells was lower than that in CFU-HPP colony cells of normal controls. It is concluded that the colony formation capability of CFU-HPP from bone marrow hematopoietic progenitor cells in psoriatic patients is lower than that in normal controls, and the lower positive frequency of P16 gene promoter methylation in CFU-HPP cells perhaps closely correlated with lower CFU-HPP colony-forming capability.

The study was purposed to investigate the effects of the panaxadiol saponin (PDS) from Ginseng on proliferation and differentiation of human CD34(+) cells from human bone marrow. Highly purified CD34(+) cells were isolated from human bone marrow by using the Dynal CD34 Cell Selection System (Dynal, Norway). The cells were exposed to PDS at various concentrations in both agar semi-solid culture of CFU-Mix and suspension culture of myeloid and erythroid cells in order to observe the effects of PDS on proliferation of CD34(+) cells. The cells were marked with 4 kinds of monoclonal antibody in related with their differentiation toward to myeloid and erythroid lineages, then examined by flow cytometry (FACS) after being incubated with PDS for 14 days. The results showed that the number of CD34(+) cells was 1.0 +/- 0.15% out of marrow nuclear cells after being purified by Dynal beads system. The enrichment of CD34(+) cells reached to 86.8 +/- 2.8%. The best efficiency in promoting proliferation of CD34(+) cells in vitro was obtained when the concentration of PDS was 25 mg/L, the formation of CFU-Mix colonies significantly increased by 56.3 +/- 3.5% over those of no-PDS control (p < 0.01). The results from suspension culture revealed that myeloid cells elevated in a dose-dependent manner with a peak increasing rate of 35.6 +/- 3.2%, and erythroid cells significantly increased by 22.3 +/- 2.1% over those of no-PDS control (all p < 0.01). After incubation with PDS for 14 days, number of CD33(+) cells increased in a dose-dependent manner with a peak increasing rate at 50 mg/L. CD71(+) cells reaching the peak were at 25 mg/L, while G-A(+) cells were increased by 7.2 +/- 1.3% (p < 0.01) at 10 mg/L, but the number of CD15(+) cells was not found to be changed before and after treating with PDS. It is concluded that PDS not only enhance the proliferation of CD34(+) cells, but also induce differentiation of CD34(+) cells toward to myeloid and erythroid lineages. PDS may play the roles as like hematopoietic growth factor, or provide synergistic effects on growth factor in the regulation of hematopoiesis.

The objective of this study was to investigate the expression of BMP-4 in stromal cells in vitro derived from human aorta-gonad-mesonephros (AGM) region. Stromal cells derived from human AGM region (hAGM S1-S5) and fibroblasts derived from human fetal trunk (hFT) were cultured in vitro. RT-PCR was used to analyze the expression of BMP-4 in hAGM S1-S5 stromal cells at mRNA level. And BMP-4 level was detected in the supernatant of hAGM S1-S5 stromal cells by ELISA assay. hFT cells were used as control group. The results showed that the heterogenous hAGM S1-S5 stromal cells displyed shapes of fibroblast-like and endothelial-like cells. hAGM S1-S5 stromal cells expressed BMP-4 mRNA, but fetal trunk fibroblasts (hFT) did not express BMP-4 mRNA. In the supernatant of hAGM S1-S5 cells, BMP-4 could be detected by ELISA assay ana its levels were statistically higher than that in hFT group (p < 0.05), while there was no significant difference between groups of hAGM S1-S5 (p > 0.05). It is concluded that human AGM-derived stromal cells in vitro express BMP-4, and the establishment of a new culture system based on the feeder cells of AGM stroma would promote the differention of embryonic stem cells into hematopoietic stem cells at a high proportion.

To investigate the effect of a novel p21-modulating protein WISp39 on proliferation, apoptosis and cell cycle of leukemia cells, the plasmid pLenti6/V5-WISp39 was constructed and transfected into the human myelocytic leukemia cell line-U937 cells. The expression of WISp39 was detected by real-time PCR at 48 hours after transfection, proliferation of U937 cells assayed by CCK-8, apoptosis and cell cycle were determined by flow cytometry. The results showed that plasmid pLenti6/V5-WISp39 could readily enhance the expression of WISp39 in U937 cells. A significant growth inhibition (37.6%) was observed in cells tranfected with pLenti6/V5-WISp39, while the control plasmid pLenti6/V5-lacZ showed little effect on U937 growth. Further analysis revealed that pLenti6/V5-WISp39 did not show obvious apoptosis induction effect, but it could really regulate U937 proliferation via cell cycle modulation. Compared with pLenti6/V5-lacZ, pLenti6/V5-WISp39 resulted in increase of cells in G(0)/G(1) phase by 10% at 48 hours after transfection. It is concluded that the WISp39 gene has no significant apoptosis induction effect on leukemic cells, but it can increase cells at G(0)/G(1) phase via effect on cell cycle, thus inhibiting the U937 proliferation. This result means WISp39 gene can act as a negative modulator on tumour cells.

To investigate the neuroprotective effect of basic fibroblast growth factor (bFGF) on neurological function after hypoxic-ischemic brain damage (HIBD) in neonatal rats.