The effects of > 10 kD component of serum-free conditioned medium from murine bone marrow-derived endothelial cell line on the formation of CFU-GM and HPP-CFC were investigated. We found that > 10 kD component alone could stimulate CFU-GM colony formation in dose-dependent. Above- 10 kD component supplemented with GM-CSF not only resulted in significant increase of CFU-GM as compared with > 10 kD or GM-CSF alone, but also could stimulate HPP-CFC formation. The effect of > 10 kD component on the formation of HPP-CFC was also in dose-dependent. The component above- 10 kD combined with IL-3, IL-6, EPO, SCF or GM-CSF further enhanced HPP-CFC colony formation. The results suggest that > 10 kD component may be used as the substitutes of hematopoietic growth factors to stimulate the formation of CFU-GM and HPP-CFC.

This paper deals with a new cell line established from 5 carcinoma specimens of hysterectomy and designated as HCE1. This cell line has been passaged more than 80 times(more than 18 months). It has the characteristics: 1. The pathological features of the tumor transplanted in nude mice were similar to that of the original inoculated lesion; 2. Electromicroscopically, there were rich microvilli on the surface of the cells. Abundant ribosomes were also observed; 3. The growth curves of the 25th passage were determined, the population doubline time calculated in the log phase of growth was 45.8 h; 4. The cloning efficiency in soft agar averaged 14.8% after counting 6 plates; 5. Karyo-type analysis showed aneuploidy with the modal chromosomal number 64-97.

To investigate the effects of sera on the growth of single CD34(+) cells from patients with paroxysmal nocturnal hematoglobinuria (PNH), sera from both PNH patients and normal individuals were added separately to the single cell culture system and semi-solid colony formation system. The growth of the normal CD34(+) and PNH CD34(+) CD59(+) and CD34(+) CD59(-) cells was evaluated. No growth difference was found for growth of the normal CD34(+) and PNH CD34(+) CD59(+) cells when PNH or normal sera were added to the culture media in either single cell culture or colony formation culture. While no difference was detected for single PNH CD34(+) CD59(-) cells growth when PNH or normal sera were added, more colonies were observed in semi-solid culture with PNH sera. A conclusion was reached that compared with those from normal controls, the sera from PNH patients had no significant influence on single hematopoietic stem/progenitor cells derived from normal subjects and from PNH patients, but the PNH sera might promote the colony formation of the CD34(+) CD59(+) cells in semi-solid culture

The cell-surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood were studied. The CD34(+) cells freshly isolated from cord blood displayed low CD95 expression, combinations of cytokines such as SCF+FL upregulated the expression of CD95 in CD34(+) cells. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) further increased the CD95 expression induced by positive cytokines. The functional status of CD95-mediated apoptosis was analysed by incubation of CD34(+) cells in the presence of anti-CD95 monocloned antibody (McAb). The effect of anti-CD95 McAb was measured by viable cells counting, flow cytometry, LTC-IC and CFU-C assays. Viable cells and CFU-C numbers were 31.9 +/- 11.2 and 43 +/- 2.0 respectively, the rate of apoptosis was 42.9 +/- 12.4 in the presence of anti-CD95 McAb and TNF-alpha or IFN-gamma. However, growth factor deprivation or the early-acting cytokine such as SCF and FL cross-linking of CD95 lead to low apoptosis of CD34(+) cells. The correlation of increased intracytoplasmic levels of Bcl-2 and the presence of CD95 on surface of CD34(+) cells suggests that Bcl-2 may be involved in protecting against CD95-mediated apoptosis of cord blood CD34(+) cell.

Bone marrow transplantation (BMT) is an effective therapy for a variety of hematologic and immunodeficient diseases. Two possible situations of chimerism can be encountered when assessing the hematological status in recipient post-allo-BMT: 1. complete chimerism, where the marrow and circulating blood cells all are donor origin; 2. mixed chimerism, the cell components in recipient are both donor and recipient origins. Formation of the different kinds of chimerism are influenced by some factors, included the amount of infused hematopoietic stem cells, the ratio of T cells from donor and recipient, conditioning regimen and patient's condition. The clinical significance was different for the forms and development of chimerism. There are several detecting methods for chimerism, such as microsatellite assay, karyotyping of chromosome, conversion of blood group, red blood cell antigen, restriction fragment length polymorphism, variable number of tandem repeat, and PCR. Above-mentioned aspects of chimerism research were reviewed.

Stem cells in the individual life are the cell population with high self-renewal capacity and multiple differentiation potential. At present, embryonic stem cells and tissue stem cells are the major objects for study in the field of stem cell engineering. At the same time, with the development of tissue engineering, cell replacement therapy became a new approach to treat some diseases. Tissue stem cells were tried to expand and committedly induce in vitro to some cells that are needed, then implanted them into patients to repair damage, replace regressive tissue and improve the function of hereditarily defect tissue. Based on recent progress of research on stem cells, this paper reviewed the biological characters and clinic application prospects of tissue stem cells.

Clinical transplantation evidence has indication that umbilical cord blood (UCB) can be useful in the hematopoietic reconstitution in the children, but not well in the adult patients because of the low cell count. The purpose of our study was to evaluate a new method for collection of blood cells from human placenta and umbilical cord. We have simultaneously harvested blood cells from umbilical cord (UCB), placenta blood (UPB) and placenta tissue (UPT) for their content of nucleated cells, CD34 (hematopoietic stem progenitor marker) positive cells. Result showed that the nuclear cell (NC) from UPB and UPT has three to four times than that from umbilical cord blood only, (8.3 +/- 1.04) x 10(8) (UCB), (16.33 +/- 5.54) x 10(8) (UPB), and (8.01 +/- 2.64) x 10(8) (UPT). CD34(+) cells are (0.77 +/- 0.01) x 10(6), (1.25 +/- 0.55) x 10(6) and (4.21 +/- 1.90) x 10(6) respectively. The cells from UPB and UPT have more survival ability than the cells from UCB in the long-term cell culture condition. It is clear that the blood stored in the liquid nitrogen did not show large loss of total nucleated cell count and CD34(+) cells. It was observed that UPT and UPB contained more suppressor lymphocytes, which may be important in prevention of graft-versus-host disease. In conclusion, our data may have implications for the development of placental blood collection together with umbilical cord blood banking for the stem cell transplantation.

In this study, to investigate the effect on expression of IL-2 in lymphocytes from bone marrow and peripheral blood of normal donors after they were mobilized by G-CSF in allo-BMT, 7 normal donors bone marrow and peripheral blood were harvested before and after G-CSF administration. The separated lymphocytes were measured by FCM after they were stained intracellularly by anti-IL-2, and their expressions of IL-2 were compared. The degree of aGVHD in patients after bone marrow transplantation was evaluated clinically, and it was compared with the status of aGVHD of 15 patients whose donors didn't receive G-CSF administration in our department, and 2 groups of patients are comparable in age, types of diseases and status of donors. The results showed that the expression of IL-2 in lymphocytes in 7 G-CSF mobilized donors decreased significantly after G-CSF administration and more severe aGVHD than grade II didn't develop in these recipient patients, and comparing with 15 patients received the bone marrow from donors who didn't receive G-CSF, the incidence of aGVHD decreased. It is suggested that the expression of IL-2 in lymphocytes was influenced by donors' G-CSF administration, and it is likely that thereby reduces the incidence of aGVHD in patients after BMT.

Adhesion to extracellular matrix plays important roles in the regulation of survival, proliferation, differentiation and homing of hematopoietic cells and is regulated by a wide variety of growth factors, adhesion receptors and other ligands that mediate the cell to matrix and cell to cell interaction. Stem cell factor (SCF) plays important roles in the regulating growth and self-renewal of hematopoietic stem/progenitor cells. In the report, the effects of stem cell factor on the adhesion of hematopoietic cells to fibronectin were observed by using a hematopoietic growth factor dependent cell line-Mo7e. Results showed that Mo7e cells express the very late antigen VLA-4 (beta1 alpha4) and VLA-5 (beta1 alpha5) integrins. The expression of the SCF receptor (c-kit) was also detected in the Mo7e cells. SCF enhances the adhesion of Mo7e cells to fibronectin in a concentration dependent manner. SCF enhanced adhesion of Mo7e cells to fibronectin was blocked by anti-beta1, alpha4 and alpha5 antibodies. Addition of PI-3 kinase inhibitors also blocked the adhesion of Mo7e cells to fibronectin induced by SCF. It was concluded that SCF enhances the adhesion of Mo7e cells to fibronectin, and this process is mediated by integrins and PI-3 kinase pathway.

To identify differentially expressed genes between fetal mesenchymal stem cell (MSC) and adult MSC, especially specified genes expressed in fetal MSC, a cDNA subtractive library of fetal MSC was constructed using suppression subtractive hybridization (SSH) technique. At first, total RNA was isolated from fetal and adult MSC. Using SMART PCR synthesis method, single-strand and double-strand cDNAs were synthesized. After Rsa I digestion, fetal MSC cDNAs were divided into two groups and ligated to adaptor 1 and adaptor 2 respectively. Results showed that the amplified library contains 890 clones. Analysis of 890 clones with PCR demonstrated that 768 clones were positive. The positive rate is 86.3%. The size of inserted fragments in these positive clones was between 0.2 - 1 kb, with an average of 400 - 600 bp.

The aim of this study was to investigate the cryobiological characteristics of placental cord blood (PCB) cryopereserved by using BioArchive auto-preserved liquid nitrogen system (BioArchive system). After Hespan depletion of red blood cells, 5 ml mixture of DMSO and 10% Dextran 40 were added into 20 ml of enriched leukocyte. 53 PCB units were cryopreserved as following protocol: pre-freeze rate 10 degrees C/min, start freeze temperature -3 degrees C, end freeze temperature -10 degrees C to -15 degrees C, post freeze rate 2 degrees C/min, and end temperature -50 degrees C. After rapid thawing at 38 degrees C, the PCB were washed with 5% human serum albumin -10% Dextran 40 and centrifuged at 400 x g, 10 degrees C for 20 minutes. The results showed that the viability of nucleated cells post-thaw was (73.3 +/- 12.5)%, the CD34(+) cell content was (0.3 +/- 0.21)% for pre-freeze PCB and (0.45 +/- 0.36)% for post-t haw PCB. No significant difference for CFU-GM/-G/-GEMM counts was found between pre-freeze and post-thaw PCB. Thawed PCB contained in two compartments (20 ml and 5 ml) of a freezing bag showed similar viability and clonogenic capacity. Differential count of white blood cell was significantly changed. For post-thaw PCB, it was dramatically decreased for the percentage of granulocytes, and highly increased for the percentage of lymphocytes and monocytes. It was concluded that the condition for cryopreservation and thawing of PCB may be harmful to mature cells, and cells with large size, such as granulocyte, but suitable to lymphocyte and monocyte, especially for the cells with small size, such as CD34(+) cells.

To explore the dynamic change of CD34(+) cell expressing adhesion molecules in bone marrow and peripheral blood during mobilization with combination of chemotherapy and G-CSF and its clinical significance, mononuclear cells of bone marrow and peripheral blood from malignant hematopathy cases before and after mobilization with G-CSF were labeled by CD45-CY-Chrome, PE conjugated anti-CD34, and FITC conjugated anti-CD44, anti-CD49d, anti-CD62L and anti-CXCR4. For three-color fluorescence analysis by flow cytometry was performed on a FACScalibur. Also the relationship between the number of subpopulations in different expressions of adhesion molecules infused and the time of recovery in different blood cells after transplantation was evaluated. Results showed that a significantly lower expression of CD44(+) and CD49d(+) on CD34(+) cells in bone marrow after mobilization compared to that before mobilization, whereas great higher expression of CD44(+), CD49d(+), anti-CD62L(+) and lower of anti-CXCR4(+) in peripheral blood were observed after mobilization. No significant relations were found between expression of different adhesion molecules on CD34(+) cells infused and the time of reconstitution in blood cells after transplantation. It was concluded that this mobilizing regimen could downregulate the expressions of CD44, CD49d, CD62L, and anti-CXCR4 on CD34(+) cells in bone marrow, it may related to mobilization of CD34(+) cells from marrow to blood, and homing of blood CD34(+) cells into marrow.

Using transplantable erythroblastic leukemia cells of EL9611(H-2d), the cells were inoculated to CB6F(1)(H-2d/b) generation of BALB/c x C57BL/6 mouse, the biological characterization of erythroblastic leukemia in haploidentical mouse was studied, that provides an experimental model for the study of graft-versus leukemia (GVL) with bone marrow or stem cell transplantation. When 10(3) - 10(8) of the spleen cells of EL9611(H-2d) had been intravenously inoculated to CB6F(1) mouse, the erythroblastic leukemia cells were transplanted successively and the F(1) generation of erythroblastic leukemia model in mice was established with 100% incidence of erythroblastic leukemia. There was a linear relationship between the survival time and the number of leukemic cell. The survival time of the mice was (9.6 +/- 0.8) days when 10(6) cells were inoculated. If the CB6F(1) mouse was transplanted successively for four generations, the incidence was 100%. The main targets for the leukemic EL9611(H-2d) cells were liver, spleen and marrow. The reaction of the erythroblastic leukemia cells for hemoglobin staining was positive, while the peroxidase reaction was negative. These cells were sensitive to some chemotherapeutic drugs, such as cytosine arabinoside and cyclophosphamide. This study presents the convenience for the studies on the GVL with haplo-allogeneic transplantation, in the F(1) generation of erythroblastic leukemia model of the commonly-used CD57BL/6 x BALB/c mouse.

Hematopoietic stromal cells, being the essential ingredient of the hematopoietic microenvironment, play very important roles in the control and regulation of self-renewal, proliferation and differentiation of hematopoietic stem cells (HSC) via complex interactions of cell-cell, cell-humoral and cell-extracellular matrix. Evidence from in vivo experiment has proved that HSC derived from normal mice could reconstitute hematopoiesis of mice with HSC defects but failed to reconstitute hematopoiesis of those mice with microenvironment defects, showing the importance of hematopoietic microenvironment in the maintenance of hematopoiesis in vivo. A well-known long-term culture (LTC) system established by Dexter demonstrated in another way that stromal cell layer in the system could support ex vivo hematopoiesis for several months, even more than one year under the optimal conditions. It, however, has not been demonstrated that what is the key elements and in which way the ex vivo hematopoiesis could be maintained for so long time. As the inventions for the large-scale screening methodologies the suppression subtractive hybridization (SSH) was chosen for the screening differentially expressed genes expressed by LTC cultured stromal cells but not by the uncultured bone marrow cells (BMC). mRNA extracted from both cultured adherent cells (tester) and BMC (driver) were hybridized according to the protocol provided by CLONTECH. Total of 130 clones differentially expressed by cultured cells were randomly picked up and 106 ESTs were obtained after sequencing. They represent 26 identical or similar genes and 7 novel genes after the bioinformatics analysis. 5 of the novel genes with the entire open reading frame, without functional clues, have been cloned into the mammalian expression vectors and the functions of them in the control of proliferation and differentiation of HSC will be further exploring. The most interesting discovery is that 3 novel genes have signal peptides, implying the potential discovery of novel growth factors as 80% known growth factors have signal peptides. Our experimental results suggest that: (a) based on the results of subtractive efficiency, the SSH could be a reliable method to screen differentially expressed genes; (b) gene expression may be regulated by multiple factors, even conditioning-dependent, in this experiment the genes expressed by bone marrow stromal cells are LTC-cultivation inducible; (c) it is possible to find interesting genes or special gene after relatively large-scale screen.

To explore a simple and effective method to determinate the volume of CD34(+) cells in the peripheral blood of donors received drug mobilization for stem cell transplantation by using flow cytometry, the mobilized peripheral blood from donors and 100 micro l fresh whole blood were labeled with monoclonal antibodies Anti-CD34-PE and Anti-CD45-FITC, after lying the red blood cells, and assessed with flow cytometer FL2 (log) vs SSC (log) and FL1 (log) vs SSC (log) were mainly used for analysis windows. The results showed that a level of CD34(+) cells in whole nucleated cells as low as 0.05% - 0.1% can be detected effectively using this method when 10(5) nucleated cells were counted. At day 5 or day 6, the level of CD34(+) cells in most samples of patients reached a peak volume, some of samples and the levels were more than one percent in. It was concluded that CD34(+) cells can be effectively determined by using this method. According to the relative rate of CD34(+) cells, the time to harvest the stem cells in blood can be determined.

To investigate the mechanisms underlying the issue that bone marrow mesenchymal stem cells (MSC) could trigger low responsiveness of allogeneic T lymphocytes against alloantigens, phenotypes of T cells were analysed with flow cytometry before and after coculture of allogeneic T lymphocytes with MSC. Further, expression of cytokines including IL-4, IFN-gamma and TGF-beta1 was evaluated by RT-PCR and ELISA as well. The results showed that CD8(+) subpopulation was increased and CD25(+) subset was decreased in proportion after coculture of allogeneic T lymphocytes with MSC for 2 weeks. RT-PCR assay showed that MSC expressed TGF-beta1 but not IL-4 and IFN-gamma, and the result was further proved by ELISA assay showing that the secretion of TGF-beta1 reached to 1 ng/ml in 72 hours. It was concluded that allogeneic MSC suppressed T cells activation by alteration of T cell subpopulations. The suppressive effect was at least in part due to the secretion of TGF-beta1. The results indicate that MSC pretreatment might be useful in the prevention of GVHD in HLA-mismatched bone marrow transplantation and further donors for hematopoietic stem cells could be selected from greater potentials.

To investigate the expression of telomerase in cord blood hematopoietic stem/progenitor cells during their committed differentiation in vitro and provide an index of monitoring the proliferating potential of the hematopoietic stem/progenitor cells and security for clinical application. Human CD34 positive cells were isolated from umbilical cord blood by using magnetic cell sorting system (MACS), and were induced to differentiation with hematopoietic growth factors (SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO) in a liquid culture system. The telomerase activity and the cytalytic subunit of telomerase (hTERT) of the cells were analysed during different periods of culture by using TRAP-PCR, TRAP-ELISA, Western blot and RT-PCR techniques, respectively. The results showed that a peak of cell growth was achieved on day 14 - 21 during induction of differentiation in vitro. Total cell number could increase 1006.4 +/- 103.2 times and could not increase there after. Telomerase activity and hTERT expression were low in freshly isolated cord blood CD34(+) cells and increased after about 7 days of culture in addition of cytokine combinations of SCF + IL3 + IL6 + GCSF and SCF + IL3 + IL6 + EPO, respectively. The telomerase activity and hTERT decreased after 14 days of culture and were not detected after 28 days of culture. It was concluded that the hematopoietic stem/progenitor cells can be expanded in large number in vitro and do not have the character of immortality and the telomerase activity could be a useful index in hematopoiesis regulation.

To investigate the expression and function of the receptors of early-acting cytokines on cord blood CD34(+) hematopoietic stem/progenitor cells, the studies of Flt3 and c-kit were undertaken at the gene and protein levels. Fresh and cultured cord blood CD34(+) stem/progenitor cells were analyzed by flow cytometric two-color direct immunofluorescence methods and RT-PCR, while function of receptors was studied in vitro. It was found that (68.8 +/- 15.4)% of CD34(+) cells expressed Flt3, (50.6 +/- 12.7)% of CD34(+) cells expressed c-kit, the proportion of CD34(+) cells expressing Flt3 and c-kit decreased as in vitro culture time extended. It was concluded that cord blood CD34(+) stem/progenitor cells are capable of expressing Flt3 and c-kit for as long as 2 - 3 weeks in liquid medium, during the first week of culture, SCF and FL enhanced the generation of cells and progenitors notably.

Mesenchymal stem cells possess the ability to differentiate into osteoblasts, chondroblasts, lipoblasts, myoblasts and so on, which can be used in the formation of hematopoietic microenvironment, tissue repairing and gene therapy. Growth factors such as TGF-beta, IGF-I, BMP and FGF can influence on the differentiation of MSC and they cooperate with each other. MSCs support hematopoiesis by secreting cytokines including G-CSF, SCF, LIF, M-CSF, IL-6, IL-11 and are related to some diseases. MSC would demonstrate important effect on gene engineering.

To investigate the relationship between interleukin-18 (IL-18) and human acute graft-versus-host disease (aGVHD), 26 patients undergoing allogeneic peripheral blood stem cell transplantation (allo-PBSCT) were included in this study. IL-18 secreted by peripheral blood mononuclear cells (MNCs) was measured by enzyme-linked immunosorbent assay (ELISA) before transplantation and during aGVHD. The results showed that grade I GVHD and grades III-IV GVHD developed in 10 and 5 cases, respectively. The levels in the supernatants of MNCs from patients with aGVHD were significantly higher than those in the cases without aGVHD. The levels of IL-18 were correlated with the severity of aGVHD. It is concluded that IL-18 plays an important role in the development of aGVHD.