To observe dystrophin and utrophin expression in muscle tissues of Duchenne muscular dystrophy (DMD) mouse model (dko mouse) after having been treated with bone marrow mesenchymal stem cells (MSC) transplantation.

To study the hematopoiesis controlled by mesenchymal stem cell (MSC) in the absence of exogenous hematopoietic growth factors in vitro. MSCs were isolated and cultured in vitro, and the hematopoietic growth factors secreted by MSCs were detected with RT-PCR. Mononuclear cells from bone marrow were seeded in 6-well plates in which MSCs had been seeded as feeder cells. After culture for 3 weeks, cells in coculture were observed with wright's staining, immunocytochemisty and flow cytometry analysis to identify the lineage of hematopoietic cells. The results showed that SCF, Flt3 Ligand and M-CSF were detected in MSCs, but G-CSF and GM-CSF were not detected. In the coculture system, a number of round shape cells grew adherently to the spindle shape MSCs after coculture for 2 weeks. Wright's staining displayed that the round shape cells had abundant plasma and kidney-shaped or oval nucleus, some cells had typical monocytic morphology, and Flow cytometry analysis displayed that the CD45+ round shape cells were positive for CD14 and were negative for CD15, CD7, CD19, CD41 and glycoporin A. Our data suggested that hematopoietic precursors from bone marrow were able to differentiate into monocyte controlled by mesenchymal stem cells in vitro in absence of exogenous hematopoitic growth factors, the committed differentiation might be regulated by the hematopoietic growth factors secreted by MSCs, and the cell-cell contact might also contribute to the differentiation.

To investigate the potential of adult mesenchymal stem cells (hMSCs) derived from human bone marrow to undergo cardiomyogenic differentiation after exposure of 5-azacytidine in vitro. A small bone marrow aspirate was taken from the iliac crest of human volunteers, and hMSCs were isolated by 1.073 g/mL Percoll and cultured in the right cell culturing medium as previously described. The phonotypes of hMSCs were identified by flow cytometry. The stem cells were cultured in cell culture medium (as control) and medium mixed with 5-azacytidine (5-aza, 3, 5, 10 micromol/L) (n=5, respectively) for cellular differentiation. We examined respectively with immunohischemistry at 21 days of inducement on desmin, cardiac-specific cardiac troponin I (cTnI), GATA4 & connexin43. The ultrastructures of induced cells were examined by transmission electron microscope. The results indicated that the hMSCs showed a fibroblast-like morphology with vortex distribution in their peak propagation, and express high level of CD44 but negative for CD34 and CD45. 20%-30% cells grown after 5, 10 microl/L 5-aza treatment connected with adjoining cells and coalesced into myotube structures after 14 days. After 21 days of culturing, immunohistochemistry revealed expression of desmin, GATA4, cTnI and connexin43 in 5, 10 micromol/L showed positive, but no cardiac specific protein were found in neither 3 micromol/L nor in control group. The ratio of cTnI positive stained cells in 10 micromol/L group were higher than that in 5 micromol/L group (65.3+/-4.7% vs 48.2+/-5.4%, p<0.05). Electron microscopy revealed myofilaments were formed. The results indicated that purified hMSCs from adult bone marrow can be differentiated into cardiac-like muscle cells with 5-aza inducement in vitro and the differentiation is in line with the 5-aza concentration.

To subclone human neurotrophin-3 gene (NT3) and transfer this gene into human bone marrow mesenchymal stem cells (BM-MSCs) to construct genetically engineered cells that produce NT3 in vitro.

Dedifferentiation is an important event in developmental biology and morphology but frequently neglected by the biologists. In the past, attention was given predominantly to its relationship with tumorigenesis. In our series of researches, we found important and inherent relations between dedifferentiation and regeneration of damaged cells and tissues in the emergence of large numbers of Schwann cells that digested their own myelin sheath components and senescent organelles by autophagic mechanism during regeneration of the peripheral nerves following injuries, when some apocytosomes (apo+cyto+sis), by means of apocytosis (apo+cyto+sis), budded from the cells containing such primitive organelles as free ribosomes and polyribosomes and retaining a very small quantity of active mitochondria and Golgi apparatus, all characterizing an immature state of the cells after dedifferentiation. Many autophagic bodies were found enwrapped by membranes L02 cells (a liver cell line) after vincristine-induced damage, with numerous apocytosomes dissociated following budding around the cells. The cells survived the treatment presented immature appearance, a phenomenon we termed as dedifferentiation, after which the metabolic burden of the cells was relieved, the senescent and damaged organelles were cleared, and the cells resumed their viability and proliferated vigorously to repair and reconstruct the damaged tissues. As most researchers have currently give their full attention to the important role of stem cells in the regeneration of damaged cells and tissues, we, after long-term observations and experiments, propose that mature cells also take part in the regeneration and reconstruction process after dedifferentiation.

To study the effect of hydroquinone on apoptosis of bone marrow mononuclear cells, and to evaluate the toxic effect of benzene on stem cells.

Dendritic cell (DC), the most powerful antigen presenting cell, has been paid close attention to tumor immunotherapy. The tumor antigens were extracted by different ways to pulse DC, and then the mature DC were infused to patients to accelerate anti-tumor immuno responses. Its clinical use would provide a new therapeutic pathway other than surgical operation, irradiation, chemotherapy. In this paper, biological characteristics of DC, tumor-vaccine preparation and its clinical use were reviewed.

Recombinant adenoviral vectors have been widely applied for the basic research and clinical trials of gene therapy. However, the inability of adenovirus to infect hematopoietic cells which lack the specific adenovirus receptors-coxsackie virus and adenovirus receptor (CAR) represents an important limitation in therapeutic applications. This limitation may be overcome by several approaches including modification of adenovirus vector and improvement of the susceptibility of hematopoietic cells. The current progresses in this field were summarized.

To study the effects of short-term cryopreservation of cord blood hematopoietic cells in liquid nitrogen, the viability and function of cord blood hematopoietic cells were investigated by using each of 8 samples cryopreserved for six months, one and two years after thawing respectively. Nucleated cells (NC) were detected by blood cell analyzer. CD34+ cells were analyzed by flow cytometry, CFU-GM were cultured and detected in vitro, the survival rate was determined by trypan blue staining. The results showed that the differences of recovery rate of NC, CD34+, CFU-GM were nonsignificant at three different cryopreserved times. In conclusion, the short-term storage in liquid nitrogen showed a good effect on cord blood hematopoietic cell without any significant change of activities and number of the cryopreserved hematopoietic cells.

To explore the efficacy of the different combinations of stem cell factor (SCF), FLT3 ligand (FL) and interleukin (IL) 2, 7, 15 on the induction and expansion of cord blood (CB) derived CIK/ NK cells in vitro, according to the different combinations of cytokines, combinations of cytokines were divided into 3 groups: group A (SCF + IL2 + IL7 + IL15), group B (SCF + FL + IL2 + IL7 + IL15) and group C (IL2 + IL7 + IL15 as control group). At 21 days of culture, the proportion and the expansion rate of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells were measured. The results showed that at 21 days in culture, the percentages of CD3(+)CD56(+) CIK cells derived from CB-MNC in group A, B and C were (19.84 +/- 2.93)%, (26.20 +/- 4.05)%, (24.03 +/- 4.99%) and that of NK cells were (49.60 +/- 1.40)%, (51.16 +/- 6.45)% and (30.85 +/- 8.12)%, respectively. The proliferation of CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells was the most effective in group B (SCF + FL + IL2 + IL7 + IL15). Expansion multiple of CIK cell number increased from 575.81 +/- 221.72 (control) to 796.09 +/- 278.47 with peak value of 1 313, and that of NK cells increased from 30.39 +/- 14.47 (control) to 65.85 +/- 30.83 with peak value of 121.06. In conclusion, a proper cytokines (SCF + FL + IL2 + IL7 + IL15) culture system can effectively induce and expand CB CD3(+)CD56(+) CIK cells and CD3(-)CD56(+) NK cells simultaneously.

In order to study the feasibility of multi-umbilical cord blood transplantation (multi-UCBT) in adult, 13 cases of unrelated allogeneic multi-UCBT performed from June 1998 to July 2003 were analyzed retrospectively. All cord blood units were obtained from full term normal vaginal and cesarean deliveries in Guangzhou Maternity and Neonatal Hospital. The fractionation, cryopreservation and thawing of cord blood were done according to the regulation of New York Umbilical Cord Blood Bank and pertinent literatures. Donors of HLA 1/6-2/6 mismatch were accepted at registry search. The results showed that from June 1998 to July 2003, 28 umbilical cord blood units were selected by 7 transplantation centers for 13 cases. The median age of recipients was 22 (8 - 41) years, and the median weight was 50 (21 - 75) kg, the median infused dose of total nuclear cells was 2.91 x 10(7)/kg. Six out of thirteen cases were engrafted after cord blood infusion with absolute neutrophil count of > 5.0 x 10(8)/L at 19 days post-infusion. Only one case suffered from graft versus host disease, the total survival of multi-UCBT was 46.2% (6/13). It is concluded that good prospects in the field of multi-umbilical cord blood transplantation is likely to be realized.

This study was aimed to investigate the clinical outcome of ricin-immunotoxin mediated T cell partially depleted HLA/MLC mismatched allogeneic hematopoietic stem cell transplantation. 13 patients with hematological malignancies were treated by ricin-immunotoxin mediated T cell partially depleted allogeneic hematopoietic stem cell transplantations from HLA/MLC mismatched donors, including 6 cases of CML in CP(1), 1 case of ALL in CR(1), 1 case of ALL in CR(2), 1 case of ALL in relapse, 2 cases of AML in CR(1), 1 case of AML in CR(2), 1 case of MDS-RAEBT-AML (M(4)) in CR(1). The results showed that 8 cases were engrafted successfully, 2 cases of them developed grade II acute GVHD and 2 cases developed grade III-IV acute GVHD. Within following-up of 8 - 90 months, 2 patients who experienced grade III-IV acute GVHD died early after transplantation; 1 patient died of late onset of infection; the other 5 patients survived free from diseases. After failure at first infusion, 4 patients were given reinfusion of peripheral blood hematopoietic stem cells from the same donor. 3 out of 4 cases failed to engraft and only one patient got engraftment but died of related complications of transplantation. One patient was performed a second transplantation from a syngeneic donor and survive free of disease until now. In conclusion, T cell partially depleted HLA/MLC mismatched allogeneic hematopoietic stem cell transplantation by ricin-immunotoxin decreases the occurrence of severe acute GVHD but with high risk of rejection, which clinical outcome still needs further evaluation.

To explore the effects of bone marrow cells expanded under different conditions on hematopoietic reconstitution, in the liquid expanded cultural system with several cytokines and/or bone marrow stromal cell layers, the BMMNC of mice were expanded for 5 days. Then the expanded cells were transplanted into the lethal-dose irradiated mice via the caudal vein. The hematopoietic reconstitution of transplanted mice were evaluated by detecting the number of bone marrow nuclear cells and various colony forming cells. The results showed that ex vivo expansion of bone marrow mononuclear cells mediated with cytokines under cultural conditions could not improve the hematopoietic engraftment in post-irradiated mice, but the expansion supported by bone marrow stromal cells could benefit the reconstruction significantly regardless of addition with cytokines. In conclusion, the ex vivo hematopoietic cell expansion supported by bone marrow stromal cells can maintain the properties of the hematopoietic stem/progenitor cells for hematopoietic reconstitution.

To study and compare the immunomodulatory functions of human bone marrow mesenchymal stem cell (MSC) and cord blood mononuclear cell (CBMNC) in vitro, human bone marrow MSC were separated with Percoll (1.073 g/L) and cultured in low-glucose DMEM, and cord blood mononuclear cells were isolated with Ficoll (1.077 g/L). These two kinds of cells were added to mixed lymphocyte cultures and PHA induction transformation cultures with various concentrations. The proliferation of lymphocytes was measured by MTT method, effects of MSC and CBMNC on mixed lymphocyte response and PHA-induced lymphocyte transformation were investigated. The results showed that both 5 x 10(4), 1 x 10(4) MSC and 2 x 10(5) CBMNC could inhibit the mixed lymphocyte response (MLR) and PHA induced transformation. But with lower concentrations (MSC < or = 1 x 10(3), CBMNC < or = 1 x 10(5)), the inhibition effects of MSC and CBMNC were less consistent. 1 x 10(2) MSC and 1 x 10(4) CBMNC mainly increased the lymphocyte activation. In addition, the inhibition ratio of 5 x 10(4) MSC (MLC 65.3%, PHA 79.1%) was higher than that of 2 x 10(5) CBMNC (MLC 8.6%, PHA 37.3%). It is concluded that larger numbers of both MSC and CBMNC showed negative immunomodulatory functions and inhibited the mixed lymphocyte response and induction of transformation in vitro. Moreover, the inhibitory effect of MSC was much stronger than that of CBMNC.

In the present study, the effects of murine bone marrow endothelial cell conditioned medium (mBMEC-CM) on the growth of yolk sac and bone marrow hematopoietic stem/progenitor cells (HSPC) were investigated. Nonadherent cells of yolk sac and bone marrow were collected for semisolid culture assay of CFU-GM and HPP-CFC after being cultured in DMEM with 10% FBS, 10% mBMEC-CM and/or FL (5 ng/ml), TPO (2 ng/ml) for 24 hours. The number of CFU-GM and HPP-CFC was counted by day 7 and 14 respectively. Atlas cDNA Expression Array was used for analysis of cytokine receptor expression of yolk sac and bone marrow HSPC. The results showed that mBMEC-CM could support the expansion of CFU-GM and HPP-CFC in liquid culture system. The expansion effects of mBMEC-CM were enhanced by combination with FL and TPO. mBMEC-CM was more effective on expansion of bone marrow CFU-GM and HPP-CFC than that of yolk sac CFU-GM and HPP-CFC. The differential expression of cytokine receptors were detected between yolk sac and bone marrow HSPC. PDGF-Rbeta, PDGF-Ralpha and corticotropin releasing factor receptor (CRFR) were only expressed in yolk sac hematopoietic cells while IFN-gammaR, GM-CSFR, Dopamine D2R and follicle-stimulating hormone receptor were only expressed in bone marrow hematopoietic cells. In conclusion, mBMEC-CM could support the growth and proliferation of yolk sac and bone marrow HSPC, and this effect was further enhanced by addition of FL and TPO. mBMEC-CM was more effective on expansion of bone marrow HSPC than on expansion of yolk sac HSPC. The comparative study indicated that the different expressions of cytokine receptors existed between yolk sac and bone marrow hematopoietic cells, which might lead to the difference in expansion in vitro between embryonic and adult HSPC.

Ex vivo maintainance of human stem cells is crucial for many clinical applications. Current culture conditions provide some level support but cytokines induce most quiescent stem cells to proliferate and differentiate. Better control of primitive cells is needed to extend the time and range of manipulation of such cells. A recently identified plant lectin Flt3 receptor-interacting lectin (FRIL) present may a special ability to preserve primitive CB progenitors for extended periods in culture without exogenous cytokines. But the mechanisms of FRIL preserving quiescent primitive cells are still unknown. Recently a novel protein HTm4 and its alternatively spliced variant HTm4S, which serve as hematopoietic cell cycle regulators, have been identified. In this report we studied the effect of FRIL on the in vitro maintenance of quiescent human cord blood stem cells and the expression of the novel hematopoietic cell cycle regulator HTm4 and HTm4S in progenitor cells cultured in FRIL. We analyzed the proliferation and the HPP-CFC proportion of CD34(+) cells treated with FRIL. The human HTm4 and HTm4S mRNA expression was detected by semi-quantitative RT-PCR, and the cell cycle status of CB CD34(+) cells was analyzed by FACS. The results showed that incubation of CD34(+) cells in FRIL resulted in a low proliferation of progenitor cells and fewer cycling cells, but FRIL selectively maintained a higher number of primitive cells with proliferative potential in suspension culture. CB CD34(+) cells cultured in FRIL showed significant diversity in the expression of HTm4 and HTm4S during 0~14 d. On d 0, HTm4 was detected at high level, downregulated on d 1, but upregulated during d 3 to d 14, and reaching the highest level on d 7. But the expression levels of HTm4S changed little in the cells cultured in FRIL except the obviously increased expression on d 7. Exogenous expression showed that HTm4 was localized around the karyon while HTm4S scatted in the cytoplasm, respectively, which may be responsible for their difference in function. Thus, FRIL can preserve quiescent primitive CD34(+), and FRIL's ability to preserve quiescent primitive cells in a reversible manner may significantly expand the time and range of ex vivo manipulations of human stem cells for clinical applications. In other words, HTm4 and HTm4S may play a crucial role in the cell cycle modulation of CD34(+) progenitor cells maintained with FRIL in vitro.

To improve the method of hard tissue slicing for research of bone tissue engineering.

To study the effect of allogenic different cells injected into denervated muscles on nerve regeneration.

To optimize the culture conditions of the human bone marrow mesenchymal stem cells (hMSCs).

To study the feasibility of using mice marrow stromal stem cells(MSCs) as seed cells for tissue engineering cartilage to embed the seed cells in acellular cartilage matrix of human auricle.