To evaluate the effect of mobilization of peripheral blood stem cells (PBSC) with high dose cyclophosphamide combination chemotherapy and G-CSF in breast cancer patients, a new mobilization protocol was designed on the basis of standard combination chemotherapy regimen, in which the dose of cyclophosphamide was raised to 2 to 4 times, and G-CSF began to be used at the dose of 150 micro g twice everyday when white blood cell (WBC) decreased below 1.0 x 10(9)/L. PBSC collection was performed while WBC increased over 5.0 x 10(9)/L during bone marrow recovering. The PBSC mobilization protocol was completed in 10 patients, the median nadir of WBC was 0.8 (0.4 - 1.0) x 10(9)/L, the median time of PBSC collection was 2 (2 - 4), the median number of collected CD34(+) cells was 6.43 (1.99 - 8.75) x 10(6)/kg. The results showed that the protocol, high dose cyclophosphamide combination chemotherapy, was an optimal PBSC mobilization regimen in breast cancer patients.

Recent reports have clearly demonstrated that bone marrow cells can be differentiated into neurons, suggesting the existence of cells with the differentiation capacity in the bone marrow cell population. It is well known that hematopoietic stem cells as well as mesenchymal stem cells (MSCs) can be transplanted and therefore, alternative of them might contribute to the process. In the present study it was addressed whether marrow MSCs could be coaxed into neuron-specific antigen bearing cells and if so, whether the differentiated cells possess the cytochemical features seen in neurons. The report here showed that high concentration of 2-mercaptoethanol (2-ME) could induce some of the MSCs into neuron-like cells expressing neurofilament (NF) and neuron specific enolase (NSE). The neuron-like cells were alkaline phosphotase positive while the others MSCs were kept negative. Cells treated with 2-ME were positive for alpha-naphthylacetate esterase and glycogen and negative for acetylchonlinesterase, which were similar with the results seen in untreated cells. Furthermore, Nissel body was not observed in treated cells shown by toluidine blue staining. Therefore, it is likely that the cells described here seem not belong to the neuronal lineage. These findings, however, reveal that human MSCs could alter their committed fates under some circumstances.

Recent clinical reports have demonstrated that the use of umbilical cord blood (UCB) opened a new source of stem cell for hematopoietic stem cell transplantation, leading to the development of cord blood banks world-wide. Prior to the large scale construction of UCB banks, quality control must be performed for health care providers and manufactures. With increasingly stringent regulatory requirement in blood industry, quality control is playing an important role in the operation of blood centers and stem cell laboratories. Reviewed the lectures in the biology of UCB and UCB banks published in recent years, our experiences were discussed in setting up Shandong blood bank to define process variables associated with the collection of UCB, to determine and optimize the procedures and materials used, to ascertain how UCB can be processed in clean room as mononucleated cell preparations, and to analyze using of long-term storage of UCB in research and clinic in the future. Our conclusions are: (1) the establishment of UCB banks for use in transplantation appears to be easy, effective and particularly suitable approach in China under cGMP conditions; (2) the procedures for volume reduction by closed and semi-automated blood processing system, SSP HLA typing, biocode and local computer net, microbiological tests and the 50 ml cryobags for storage constitute a cost efficient system for large-scale UCB banking; (3) the average of 60 ml UCB collection may contain sufficent marrow repopulating cells for children and most of adult recipients; and (4) hematopoietic stem and progenitor cells in cord blood have a more potent proliferative ability than those derived from bone marrow in cell expansion potentials.

Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective and proven treatment for malignant and nonmalignant diseases. Most of the natural HSC allografts hardly show overall advantages of high engraftment, slight GVHD and rare relapse. The graft engineering including stem cell engineering to make a tailor-made graft ex vivo is promising to conquer all the risks of low engraftment, lethal GVHD and high relapse, which becomes the key program in current HSC research. To combine HSC allotransplant with gene therapy and immune therapy is the novel therapeutic strategy for malignancies, the real meaning of "cytotherapy" or "cell therapy" updated. The rapid expansion of umbilical cord blood banks (CBBs) makes the substantial increase of cord blood transplants (CBT) both possible and likely world-widely. In China, however, owing to lack of hematological pediatricians specified in HSCT and pediatric laminar-flow wards, clinical application rate of cord blood is extremely low despite of the high collection rate in the CBBs under the GMP standards. In evaluating a CBB, the release rate and the clinical efficacy of the released cord blood should be most emphasized as well as the banking quality control. In all CBBs worldwide, it is unworthy of mentioning the banking of umbilical cord blood for autologous transplant. Only one or two commercial companies in the world run it for profitable purpose to charge the donor parents regularly. It is because no autologous CBT can cure the inherited diseases and its efficacy of treating malignancies is doubtable since the cord blood is of weak immune competence against tumor and may be contaminated with autologous malignant or premalignant cells. Moreover, there is no report so far about how long the repopulating activity of cryopreserved hematopoietic stem/progenitor cells of cord blood can keep. No honest guarantee can be made about the effective quality and adequate amount of stem cells to meet the therapeutic requirement when used after a long storage.

Glycophorin A (GPA) is one of the important molecular markers in studies of somatic cell mutations. To investigate the relationship between the gamma-irradiation and the frequency of GPA variation, the frequency of variant erythrocytes at the GPA locus was determined in peripheral blood of 3 subjects with accidental whole-body gamma-irradiation. The biological dose of individuals was 2.5, 2.9 and 1.9 Gy estimated by the chromosome aberration assay, respectively, and the frequency of GPA variation was 3.9, 4.3 and 4.1 times greater than that from normal controls, respectively. Our results suggest that the variant frequency of erythrocyte GPA was increased. On account of the GPA gene mutations are preserved in hematopoietic stem cells during all irradiated individual's life, the frequency of GPA variation could be used as a permanent marker for mutagenesis of radiation.

The experience with the umbilical cord blood (UCB) stem cells for unrelated transplantation from our 3 000 UCB storage was described. UCB, collected from closed blood bags, were mixed with hydroxyethyl starch for nucleated cell (NC) enrichment. After finishing CD34 analysis, culture of hematopoietic progenitors (CFU-GM and CFU-GEMM) assays, microbial culture, HLA Class I (A, B) serology and class II (DR) low resolution SSP typing, cord blood units are stored in the liquid nitrogen for clinical applicatoin. Cord blood contained an average of nuclear cell (NC) (1.2 +/- 0.6) x 10(9), CD34(+) cells (3.0 +/- 3.7) x 10(6), CFU-GM (1.1 +/- 0.7) x 10(6) and CFU-GEMM (1.1 +/- 1.2) x 10(6) for storage and the recovery rates were 91%, 88%, 85% and 82%, respectively. The recovery rates for red blood cell and Hb were (39 +/- 9)% and (40 +/- 8)%, respectively. The storage volume was (35.1 +/- 7.1) ml in a 50 ml storage bags. The mean time from collection to processing of 15 hours (range 4 - 24 hours) had no influence on cell viability. The cell viability before processing is more than 95% and 92% after UCB thawing. The recovery rates of NC, CD34(+) cells and CFU-GM post-thawing were 96%, 90% and 91%, respectively. There were no HIV antibody (HIVAb) positive in all of UCB units. For an incidence of processed samples, infection with syphilis, HBsAg, HBcAb, HCVAb, CMV, bacterial contamination and abnormal hemoglobin were 0.1%, 0.8%, 3.2%, 0.2%, 87.1%, 1.2% and 0.1%, respectively. More than 3 HLA loci matched can be found for random patients in our cord blood bank and 6 HLA loci matched have 5%. For transplantation with nucleated cell counts of > 2.7 x 10(7) cells/kg, our cord blood bank will be able to provide all of the umbilical cord blood stem cell samples for children and 50% of units can be used for some of adult recipients transplantation in the country. It is concluded that: (1) The large cord blood banking for 20 000 UCB storage is feasible in China. (2) Our system of whole procedure and methods is functionable for supplying qualified cord blood units in transplantation. (3) The volume for collection is critical to the yield of CD34(+) cells or hematopoietic progenitor cells, however cord blood NC is also important and proportional with CD34(+) cells. Only the units containing more than 8 x 10(8) cells and more than 60 ml of cord blood can be in the procession for storage.

Expression of AML1/ETO mRNA was observed in bone marrow cells from 49 untreated leukemic patients, and continuously detected during different periods after chemotherapy (12 cases) or bone marrow transplantation (8 cases). The results showed that AML1/ETO mRNA could be expressed in cells from AML-M(2), AML-M(4) and MDS-RAEB-T patients. The positive expression changed into negative at different duration in patients who achieved complete remission either by chemotherapy (9 cases), allogeneic bone marrow transplantation (5 cases) and autologous peripheral blood stem cell transplantation (1 case), and they were sustained in complete remission status. In chemotherapeutic group, patients whose AML1/ETO expression turning from negative (2 cases) or faint positive (1 case) to positive relapsed later. Two patients treated with Allo-BMT showed continuously positive results and died of GVHD and relapse, respectively. These observations suggest that AML1/ETO chimeric mRNA could disappeared after chemotherapy or bone marrow transplantation. The patients have a great probability to relapse if the results of RT-PCR are continuously positive or change from negative to positive. Regular detection is necessary for leukemic patients.

Long-tem culture initiating cells(LTC-IC), and in vitro assay of hematopoietic stem/progenitor cells, still represent a heterogeneous population in terms of proliferative capacity and sensitivity to different growth factors. Human umbilical cord (CB) is rich of hematopoietic progenitor cells measured by clonogenic assays and stem cells capable of reconstituting the marrow after transplantation. The influence of culture conditions on the in vitro behavior of LTC-IC from CB was evaluated. First, by using IRES sequence, FL and TPO cDNA were recombined with retroviral vector pLXSN by gene recombination technology. The recombinant plasmid pLFSN, pLTSN, pLFTSN were transfected into human stromal cell line HFCL. Then, LTC-IC were evaluated in long term cultures, comparing five types of stromal feeder layers: human bone marrow stromal cell, human stromal cell line HFCL, and stromal cell lines HDF tranfected with FL gene, HLT transfected with TPO gene or HFT co-transfected with FL and TPO genes. The results were demonstrated that after 8 weeks of coculture, three types of stromal cell lines that supported the maintenance of CFU-C for up to 3 weeks in vitro were identified. However, cocultivation of human bone marrow stromal cell and CB CD34(+) cells on HFT, CFU-C production continued up to 6 weeks or longer on these stroma. The absolute LTC-IC frequency in CD34(+) cells on human bone marrow stromal cell (2.65 +/- 0.76/1 000 cells) was no significant difference with on HFT (3.65 +/- 0.58/1 000 cells). Thus, HFT acts by direct contact to maintain the phenotype and function of the most primitive and quiescent human progenitors. Furthermore, HFT cell line was selected as the optimal one for supporting long-term culture feeder. It was concluded that LTC-IC progenitors from cord blood maintain growing upon the FL/TPO gene-modified stromal feeder layers in vitro.

The objectve of this investigation was to explore the use of nonmyeloablative allogeneic peripheral blood stem cell tranplantation for treatment of hematologic diseases. Six patients were included: 3 cases with acute leukemia in first complete remission (2 AML and 1 ALL), 2 severe aplastic anemias and 1 chronic myeloid leukemia in chronic phase. All of the 6 cases were received HLA-identical siblings donor peripheral blood stem cell transplantation after a nonmyeloablative conditioning. The donor cells were engrafted in all patients (3 cases were full engrafment of donor cells and 3 were mixed chimerism). Hematopoietic recovery was appeared in all of the cases (ANC recovered to more than 0.5 x 10(9)/L and platelet count to more than 30 x 10(9)/L on day 9 to day 21 and day 14 to day 28 after transplantation, respectively). Two patients developed III or IV degree GVHD. Our preliminary results suggest that the procedure is more safe, efficient and less complications than myeloablative conditioning regimens and represents another new approach in the management of patients with hematologic diseases.

It has been well-known that intravenously infused hematopoietic stem and progenitor cells can home to the bone marrow and reconstitute hematopoiesis. However, little is understood about the homing efficiency or percentage of infused stem and progenitor cells. In order to examine distribution pattern of infused hematopoietic cells in the organs and tissues, a direct assay system to trace transplanted cells in vivo by employing PKH-26, a red fluorescent membrane dye, to label hematopoietic cells in inbred strain of mice transplanted cells (stem cell antigen-1 positive subpopulation cell, Sca-1(+) cells) was introduced. The numbers of labeled cells was measured by means of flow cytometry and fluorescence microscopy. The early fate of infused Sca-1(+) donor bone marrow cells after intravenous administration in a allogeneic mouse model was examined. The presence of infused donor cells with the fluorescent dye PKH-26 was evaluated within 60 hours in hematopoietic organ (bone marrow and spleen) and non-hematopoietic organ (lungs and liver) of recipients. The data showed that (1) Following intravenous infusion, Sca-1(+) donor bone marrow cells were detained in lungs shortly. (2) Sca-1(+) donor bone marrow cells localized to both hematopoietic organ (bone marrow and spleen) and non-hematopoietic organ (lungs and liver) for periods of up to 60 hours following infusion, however, the number of donor hematopoietic cells localized to bone marrow was more than that localized to non-hematopoietic organ (P < 0.05). These results indicated that there were also donor early hematopoietic cells in non-hematopoietic organ of recipients at the homing phase in allo-BMT mice.

The purpose of this study was to designed to analyze and explore the diagnosis and treatment on a patient with beta-thalassemia major accompanied with intestinal acute graft-versus-host disease (aGVHD) grade IV after allo-peripheral blood stem cell transplantation (allo-PBSCT). The conditioning regimen was consisted of busulfan (18 mg/kg), cyclophosphamide (200 mg/kg), thiotepa (6 mg/kg) and horse anti-thymocyte globulin (110 mg/kg). The nucleated cells (10.6 x 10(9)/kg) and CD34(+) CD38(-) cells (11.7 x 10(6)/kg) were infused. At +14 days PBSCT, ANC and WBC achieved 0.66 x 10(9)/L and 1.4 x 10(9)/L respectively. The engraftment was successful but the patient depended on transfusion. The range of blood WBC was from 1.4 x 10(9)/L to 2.4 x 10(9)/L. The myelogram showed aplastic anemia. Hematopoiesis was restored continually after the second transfusion of the donor's cells. At +11 days the patient was suffered from cutaneous aGVHD grade II. Diarrhea occurred at +33 day when cutaneous aGVHD had been improved, and exaggerated obviously after the second transfusion at +39 days. The serious diarrhea continued for 2 months and intestinal aGVHD grade IV was finally diagnosed. Our conclusion was that intestinal aGVHD possibly occurred even when cutaneous aGVHD was improved. For a patient with refractory diarrhea after allo-PBSCT, intestinal aGVHD was considered first when the engraftment evidence was definited but peripheral blood cells and bone marrow were abnormal.

Bone marrow cells in cultures were divided into four groups and cultured with various cytokines in vitro. These four groups are: control, IL-2 group, CD3-AK group, and CIK group. The morphological (cell volume, nucleus/plasm) changes of bone marrow cells in culture were observed. Immunophenotype analysis (CD34, CD38, CD3, CD56) were done before and after culture in all groups. Cytotoxicity against fresh acute leukemia cells were detected by modified MTT methods. The cell volume became larger with increased nucleus/plasm ratio in IL-2 group, CD3-AK group and CIK group. The plasm filled with PAS positive granules in most of cells in CD3-AK group and CIK group. The positive ratio of CD3, CD56, CD38 in CD3-AK or CIK group increased markedly after culture (P < 0.05), but no significant difference between the two groups. The CD56(+) cell increased in IL-2 group. CD34(+) cells decreased in all groups and there were no significant differences among those four groups. The cytotoxicity to fresh leukemia cells: CD3-AK group and CIK group > IL-2 group > control group. There was no significant difference between CD3-AK group and CIK group. This experiment showed different effect on bone marrow cells by different cytokine combination. The cytokine combination of CD3-AK group or CIK group can make immunocytes of bone marrow proliferating and retained certain amount of stem/progenitor cells.

To explore methods of maintaining the self-renewal capacity of hematopoietic stem cells, inhibiting their overdue differentiation and expanding hematopoietic cells massively, the murine bone marrow stromal cells were coated on microcarriers, then co-cultured with hematopoietic cells from murine bone marrow as group 2 (G2). The G2 contents were wrapped by sodium alginate, then cultivated as group 1 (G1). The only microcarriers coated with stromal cells as group 3 (G3) and the only bone marrow cells as group 4 (G4) were cultivated as control groups. Contrasting observation and microphotograph were performed; the number of total marrow cells, the colony efficiency of CFU-GM and the percentages of CD34(+) cells were determined. Three repeated experiments indicated that the colony efficiency of CFU-GM before culture (G0) were 118.8 +/- 38.1/10(5) marrow cells, and the total outputs of CFU-GM (G0) were 9 501.3 +/- 3 049.0. After culture for two weeks, hematopoietic cells were adhered to or embedded in stromal cells coating the microcarriers, and had formed hematopoietic focus. The colony efficiency of CFU-GM per 10(5) mononuclear cells in group G1, G2, G3 and G4 averaged 30.9 +/- 13.7, 147.3 +/- 66.0, 23.4 +/- 23.1 and 15.9 +/- 8.1, respectively; the total outputs of CFU-GM in group G1, G2, G3 and G4 averaged 273.8 +/- 75.3, 9 424.8 +/- 7 933.7, 419.1 +/- 305.6 and 140.7 +/- 20.7, respectively; the measured CFU-GM output in group G2 was significantly higher than that in group G4, and still significantly higher than the sum of groups G3 and G4 (t = 6.553, t = 5.494; P < 0.05). The percentage of CD34 cells before culture was 10.0 +/- 1.0; after cultuer for two weeks, the percentages of CD34(+) cells in G1, G2, G3 and G4 averaged 4.0 +/- 1.0, 11.0 +/- 1.0, 3.3 +/- 1.5 and 2.2 +/- 0.8, respectively. The percentage of CD34 positive control (3T3 cells) was 17.0 +/- 1.0. This result was consistent with the result of CFU-GM outputs measured. These data suggest that microcarriers coated with stromal cells can perfectly support the ex vivo hematopoiesis at least to four weeks, while hematopoietic cells fixed by alginate are not significantly different from control groups. The hematopoiesis-simulating model of microcarriers is successful, whereas the hematopoiesis-simulating model of alginate macrocarriers can not support the ex vivo hematopoiesis.

Embryonic stem cells are continuously growing stem cell lines of embryonic origin. The distinguishing features of ES cells are their capacity to be maintained in an undifferentiated state indefinitely in culture and their potential to develop into various kinds of cells in the body. At molecular level, in vivo and in vitro differentiation of ES cells are similar in many ways. To date, ES cells can differentiate in vitro into myocytes and myocardial cells, neural precursor cells, hematopoietic cells and endothelial cells. Recent evidence suggests that hematopoietic stem cells (HSCs) originate from a region within the embryo proper. It is likely that the genesis of HSCs is regulated by embryonic growth factors, which have not been discovered yet. ES cells have provided a convenient and effective experimental approach to define early hematopoietic process within embryos. Research on ES cells has broad application prospect.

To simplify the traditional method for cryopreservation of peripheral blood stem cells(PBSCs) at -196 degrees C with rate-controlled freezing with 10% dimethyl sulfoxide(DMSO), the simplified method was carried out by freezing the cells to -80 degrees C in low-temperature freezer with the combination of 5% DMSO, 3% hydroxyethyl starch(HES) and 4% human serum albumin(HSA) as cryoprotectant. PBSCs were cryopreserved by different methods. Cell viability and recovery rate of mononuclear cells (MNC), CFU-GM and CD34(+) cells were compared. It was observed that the higher MNC and CFU-GM recovery rates were achieved and without agglutination with the simplified method. It was also found with this simplified method, satisfactory recovery rates of CFU-GM and CD34(+) cells could be obtained when PBSCs were preserved at -80 degrees C as long as 24 months. There was no difference observed in parameters of cryopreserved PBSCs thawed at 37 degrees C and 20 degrees C. After the cells being exposed to 5% DMSO at room temperature for 1 hour, the cell viability decreased from 93.2% to 84.5%, however, the CFU-GM recovery rate was not decreased. It is concluded that the simplified cryopreservation technique is better and simpler than the traditional crypreservation method, will be useful for institutions without rate-controlled freezing facility. Moreover, this method diminishes the amount of DMSO infused into patients, thus decreasing its toxicity.

To analyze the relation of early immune reconstitution with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (all-HSCT), the changes of CD3(+), CD4(+), CD8(+), CD25(+) and CD69(+) cells in peripheral blood from 26 patients with hematologic malignancies were assayed by flow cytometry within 2 months after allo-HSCT. All patients achieved hematopoietic reconstitution, and grade I and II - IV GVHD were developed in 9 and 5 patients, respectively. CD25(+) cells were increased in patients aGVHD at week 2 after transplantation and the peak value was appeared at week 3. The increase of CD25(+) cells was preceded the occurence of clinical signs of aGVHD. The maximal levels of CD25(+) cells increase correlated significantly with the severity of aGVHD. The increase of CD25(+) cells was declined along with remission of aGVHD signs. Our results suggest that analyzing immune reconstitution after allo-HSCT could predict occurence of aGVHD, and CD25(+) cell increase prior occurence of aGVHD is predictive marker for aGVHD.

There has been an increasing interest in recent years in the stromal cell system. The stroma is a complex tissue, composed of a number vascular and connective tissue cell types including endothelial cells, adipocytes, smooth muscle cells, osteogenic cells, and stromal cells. The marrow mesenchymal stem cells are capable of self-renewal and differentiate into various connective tissue lineages including bone, cartilage, tendon, muscle, fat, and marrow stroma. Recently, techniques for the isolation of adult bone marrow-derived human and animal mesenchymal stem cells have been described, as well as the methods to directing their differentiation into osteogenic, chondrogenic, tendogenic, myogenic, adipogenic, and marrow stromal lineages. But there is no report about the fetal bone marrow- and liver-derived mesenchymal stem cells. Are they the same or not? In our assay, human fetal mesenchymal stem cells from 4 - 5 months old human fetal bone marrow and liver low-density mononuclear cells were cultured, and the cell cycle, immunophenotype and ex vivo expansion properties were studied. Results showed that the mesenchymal stem cells from fetal liver and fetal bone marrow were similar in morphology, growth character and immuno-phenotypes, but the liver mesenchymal cells manifested higher ability to support hematopoiesis than the marrow-derived mesenchymal cells. This study demonstrates that adherent fetal marrow- and liver-derived cells cultured in the absence of differentiation stimulus gave rise to a population of cells with phenotypical features of mesenchymal stem cells, and should be enough to sustain a steady supply of low differentiated cells upon proliferation.

To observe the effect of Quanjia Yangshen capsule (QJYS) on cyclophosphamide (Cy) induced teratogenesis and bone marrow in mice and to explore the mechanism of its clinical therapeutic effect.

To observe the leukocyte increasing effects of Shaunghuang Shengbai Granule (SHSBG) in tumor patients treated by chemotherapy (CT) and its function on bone marrow hematopoietic microenvironment in mice.

To investigate the action of Ginsenosides (GS) in inducing transcription factor c-fos and GATA-1 to explore the mechanism of GS in hematopoietic cells.