Hypoxia in bone marrow is suitable for the perfect preservation of biological functions of bone marrow hematopoietic stem cells (BM HSC). It is deserved to study whether the biological functions of BM HSC are influenced when being exposed to environment of oxygen at various concentration during amplification of BM HSCs in normal oxygen condition in vitro and process of peripheral blood hematopoietic stem cell transplantation (PBSCT). This study was purposed to investigate the effects of various oxygen concentrations on biological functions of human BM HSCs. The BM HSCs were amplified in vitro, the amplification level of CD34(+) HSCs and CD34(+)AC133(+) HSCs were detected by flow cytometry, the apoptosis and cell cycle distribution of CD34(+) HSCs amplified in various oxygen concentrations were assayed by flow cytometry with Annexin V/PI double staining as well as PI and Ki-67 antibody, respectively, the differentiation of amplified CD34(+) HSCs in vitro was determined by direction differentiation assay, the migration ability of amplified CD34(+)AC133(+) HSCs was measured by migration test. The results indicated that the oxygen environment below normal oxygen, especially hypoxia, could amplify more primitive CD34(+)AC133(+) HSCs and CD34(+) HSCs with activity, arrest more HSCs in G₀/G₁ phase, promote the generation of BFU-E, CFU-GM, CFU-GEMM, and better preserve the migration ability of HSCs. While the above functional indicators of BM HSCs were poor when HSCs exposed to normoxia, oxygen-unstable and oxygen-severe changeable environments. It is concluded that the biological functions of BM HSCs in PBSCT are related with oxygen concentration and its stability, the culture of BM HSCs in lower oxygen environment may be more beneficial for PBSCT.
Bone Marrow Cells ; cytology ; drug effects ; Bone Marrow Transplantation ; Cell Hypoxia ; Cells, Cultured ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Oxygen ; administration & dosage ; pharmacology

OBJECTIVETo explore the effects of a novel human chemokine-like factor 1 (CKLF1) on stem cell/progenitor cells.

METHODSHuman bone marrow mononuclear cells were separated by Ficoll (1.077 g/ml), and cultured in suspension and semisolid colony culture. The effects of CKLF1 on CFU-GM and CFU-Mix were observed.

RESULTSThe number of CFU-GM was significantly increased in 10 groups by addition of CKLF1 plasmid supernatant. The mean value was 234.81 +/- 98.71/1 x 10(5) cells, 2.42 fold of control group (P < 0.05). The mean value of CFU-Mix in groups of negative control, CKLF1, PHA, GM-CSF and G-CSF were 82.00 +/- 20.25, 105.76 +/- 36.70, 146.63 +/- 27.09, 143.33 +/- 60.23 per 1 x 10(5) cells, respectively, no statistical differences were seen between control and CKLF1 groups. The CD(34)(+) cells were detected by FACS. The average percentage in the groups of normal control, CKLF1, PHA and GM-CSF were (0.75 +/- 0.62)%, (1.32 +/- 0.87)%, (0.15 +/- 0.02)%, and (0.29 +/- 0.23)%, respectively. Compared with control, no significant differences were seen between each group (P > 0.05).

CONCLUSIONNovel chemokine-like factor 1 can increase the proliferation of CFU-GM, which indicated that CKLF1 could increase the proliferation of human bone marrow hematopoietic progenitor cells and colony formation.

Adult ; Cells, Cultured ; Chemokines ; pharmacology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; MARVEL Domain-Containing Proteins

OBJECTIVETo investigate the roles of telomere and telomerase in the effect of ginsenoside Rg1 to delay hematopoietic stem cell senescence.

METHODSca-1(+) HSC was isolated by magnetic cell sorting(MACS) and divided into five groups: the control group, the aged model group, the Rg1 group, the Rg1 treated aged group and the Rg1 delayed aged group. The changes of cells were observed by senescence-associated beta-Galactosidase (SA-beta-Gal) staining. Cell cycle assay and culture of mixed hematopoietic progenitor cell were used to investigate the effect of ginsenoside Rg1 to delay Sca-1(+) HSC senescence. Telomere length and telomerase activity were detected by southern blotting and TRAP-PCR-SYBR Green staining.

RESULTCompared with aged model group, the percentage of positive cells expressed SA-beta-Gal and the number of cells entered G1 phase were decreased and the number of colony of mixed hematopoietic progenitor was increased. It showed markedly decreased in the shortening of telomere length and reinforcing in the telomerase activity to Rg1 treated aged group and Rg1 delayed aged group. The change of Rg1 delayed aged group was significantly higher than Rg1 treated aged group.

CONCLUSIONActivation of telomerase and prolonging of telomere length might be involved in the process of ginsenoside Rg1 to delay and treat the senescence of Sca-1(+) HSC.

Cellular Senescence ; drug effects ; Ginsenosides ; pharmacology ; Hematopoietic Stem Cells ; drug effects ; physiology ; Telomerase ; metabolism ; Telomere ; drug effects

AIMTo explore proper cryopreservative systems for hematopoietic stem cells.

METHODSPeripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation.

RESULTSThe recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group.

CONCLUSION5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.

Blood Preservation ; methods ; Cell Survival ; drug effects ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans

OBJECTIVEEmbryonic stem cells (ESCs) are derived from totipotent cells of early embryo and they are potential to differentiate to any kind of cells of tissues in the body. Some reports showed that ESCs had broad capabilities of differentiating to variety of hematopotietic cells, such as erythroid, granulocyte/macrophage, megakaryocyte, mast and lymphocyte precursors. However, it is very difficult to control the phase of differentiation for ESCs in vitro. There is few report about hematopotietic stem cells (HSCs) from ESCs. Therefore, this research was designed to establish a culture system for generation of CD(34)(+)/Sca-1(+) HSC from ESC in vitro.

METHODSSingle mouse E 14.1 cells were suspended in methylcellulose medium, containing 40 ng/ml stem cell factor (SCF) and 20 ng/ml vascular endothelial growth factor (VEGF) and incubated at 37 degrees C with 5% CO2. In order to ensure the viability of the primary differentiation cultures over an extended period of time, the cultures were fed on day 7 with a dilute methylcellulose medium containing VEGF, SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (EPO), which promoted their primary differentiation into embryoid bodies (EBs) with more CD(34)(+)/Sca-1(+) cells. Then, EBs with peak level of CD(34)(+)/Sca-1(+) cells were dispersed into single cells and replanted either in methylcellulose medium or in bone marrow stromal cells differentiation system containing 15% fetal bovine serum (FBS), 160 ng/ml SCF, 20 ng/ml VEGF, 30 ng/ml IL-3, 30 ng/ml IL-6, 3 U/ml EPO and 20% BIT for HSC into second-step differentiation. The HSCs were characterized by flow cytometric analysis, colonogenic cell assay and Wright-Giemsa stains.

RESULTSVEGF had the strongest stimulatory effect on the enhancement of the CD(34)(+)/Sca-1(+) cells population when combined with SCF, IL-3, IL-6 and EPO. It could markedly accelerate mouse E14.1 cells to differentiate into EB with more CD(34)(+)/Sca-1(+) cells. Cell cytometric analysis showed CD(34)(+)/Sca-1(+) cells were up to (1.91 +/- 0.40)% by day 5 and (8.11 +/- 1.17)% by day 8, and the peak level of CD(34)(+)/Sca-1(+) cells was (13.72 +/- 1.92)% by day 12. However, CD(34)(+)/Sca-1(+) cells could not increase in number with the prolongation of differentiation. So renewal single cells suspension from EB by day 12 was dispersed into the second step differentiation. The results showed that HSC was slowly generated with a few hematopoietic colony formations in methylcellulose medium differentiation system. CD(34)(+)/Sca-1(+) cells got (2.74 +/- 0.80)% by day 5 and (11.37 +/- 1.84)% by day 10, and apex percentage of CD(34)(+)/Sca-1(+) cells was about (20.52 +/- 2.78)% by day 14. However, EBs generated quickly for HSC with increased hematopoietic cell population by co-culture on bone marrow stromal cells feeder. Flow cytometric analysis showed that the percentages of CD(34)(+)/Sca-1(+) cells was (7.33 +/- 1.61)% by day 5, (13.28 +/- 2.59)% by day 8, and (20.81 +/- 3.19)% by day 10. EB cells were induced after 12 days to reach the peak level of (34.60 +/- 3.71)%. Hematopoietic colony formation unit (CFU) analysis showed that CFU was sufficient from cells on bone marrow stromal cells differentiation system in the second step compared to that in methylcellulose medium differentiation system, and Wright-Giemsa stain could confirm its characteristics of hematopoietic progenitors.

CONCLUSIONUsing two-step differentiation, the investigators got a good way to control the phase of differentiation from ESC to HSC. The bone marrow stromal cell differentiation system combining with VEGF, SCF, IL-3, IL-6 and EPO was an optimal system for the generation of HSC with CD(34)(+)/Sca-1(+) surface marker from ESC differentiated in vitro. This study demonstrated that these cells could form more hemopoietic colonies.

Animals ; Antigens, CD34 ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Survival ; Cells, Cultured ; Embryonic Stem Cells ; drug effects ; immunology ; Hematopoietic Stem Cells ; physiology ; Mice

The objective of this study was to explore the effects of BMP-4 and VEGF on the development of primary hematopoietic stem cells during the differentiation of embryonic stem cells (ESCs) into embryoid body (EB). Murine E14 ESCs were seeded into semisolid methylcellulose-based medium for EB formation. According to added or not cytokines, experiments were divided into: (1) group of spontaneous differentiation without cytokine as control; (2) group of BMP-4 in different concentrations (0, 5, 15, 25 and 50 ng/ml); (3) group of BMP-4 combined with VEGF; (4) group of VEGF alone. EBs were collected on days 3, 6, 9, 12, 15, and the proportion of Flk-1(+) cells were assayed by flow cytometry. The results showed that in the different BMP-4 concentration groups, the proportions of Flk-1(+) cells were significantly different, and it reached the peak values in 25 ng/ml BMP-4 group as 6.51 +/- 1.02% at day 3 and 7.70 +/- 1.12% at day 6 respectively, which were statistically higher than those in control group without-BMP-4 and in 5 ng/ml BMP-4 group (p < 0.05). When BMP-4 was used in combination with VEGF, Flk-1(+) cells went to peak proportion value at day 9 as 27.53 +/- 8.14%, which was statistically higher than that in spontaneous differentiation group as 8.77 +/- 2.35% (p < 0.05) and VEGF treatment group as 11.21 +/- 2.23% (p < 0.05). It is concluded that BMP-4 in combination with VEGF can promote Flk-1(+) cells genesis during EB formation in vitro, which provides experimental evidence for researches on directed differentiation of ESCs into hematopoietic stem cells simulating the microenvironment in vivo.
Animals ; Bone Morphogenetic Protein 4 ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; drug effects ; Hematopoietic Stem Cells ; cytology ; Mice ; Vascular Endothelial Growth Factors ; pharmacology

This study was aimed to investigate the effect of 1,4-benzoquinone (1,4-BQ) on growth of myeloid progenitor cells with IFN-gamma different genotypes and to compare its differences. Polymerase chain reaction (PCR) was used to amplify the polymorphism gene segment of IFN-gamma +874 A/T in 36 cord blood (CB) specimens. The specimens were divided into three groups (AA, AT and TT group). MNCs were planted on complete methylcellulose medium containing different concentrations of 1,4-BQ. The colony-forming units (CFU) were assayed, the differences of colony growth in specimens with different genotypes (AA, AT and TT) under 1,4-BQ exposure were analyzed. The results showed that frequencies of AA, AT and TT genotypes were 5.56%, 88.89% and 5.56% in the 36 CB samples respectively. Comparing colony numbers of IFN-gamma +874 AA, AT and TT genotype indicated that there was significant difference (p(AA) = 0.033, p(AT) = 0.009, p(TT) = 0.001, < 0.05). Significant cytotoxicity was observed after exposure to concentrations of 1,4-BQ > or = 5 micromol/L. Cytotoxic response of 1,4-BQ was dose-dependent. Under the same concentration of 1,4-BQ, there were no significant differences in capacity of cell colony growth between 3 groups (AA, AT and TT). Colony numbers of specimen No 3 in AT group and specimen No 2 in TT group were less than those of other specimens significantly. It is concluded that the hematopoietic myeloid progenitor cells cultured in the presence of 1,4-BQ show a dose-dependent cytotoxic response, but there are no significant differences in colony growth of IFN-gamma different genotypes (AA, AT and TT) under the same concentration of 1,4-BQ.
Benzoquinones ; pharmacology ; Bone Marrow Cells ; drug effects ; Fetal Blood ; cytology ; Genotype ; Hematopoietic Stem Cells ; drug effects ; Humans ; Interferon-gamma ; genetics ; Stem Cells

To investigate the effects of Ligustrazine on histogenesis of bone marrow in the early phase of hematopoietic reconstruction in bone marrow transplantation (BMT) mice. The syngeneic BMT mice model was established. The syngeneic BMT mice were orally given 2 mg Ligustrazine twice a day. 1, 3, 5, 7, 10, 15 and 21 day(s) after BMT, peripheral blood granulocytes and bone marrow nucleated cells (BMNC) were counted and the diameter of central vein and the area of micro-vessel in femur were measured. The effect of Ligustrazine on hematopoietic stem cells was observed by colony forming unit of spleen (CFU-S). The effect of Ligustrazine on hemopoietic progenitors was studied by observing the number of progenitors of Granulocytes/Macrophage on day 10 and day 20 after BMT. In Ligustrazine-treated group, the diameter of center veins and the area of micro-vessel of femur were all significantly less than the control group 7, 10, 15, 21 days after BMT (P < 0.01). In addition, Ligustrazine significantly increased the number of CFU-S on day 10 and the number of CFU-GM on day 10, 20 after BMT. These results indicate that Ligustrazine can accelerate the histogenesis of hemopoietic bone marrow, which may be one mechanism by which Ligustrazine promotes hematopoietic reconstitution after BMT.
*Bone Marrow Transplantation ; Hematopoiesis/*drug effects ; Hematopoietic Stem Cells/*drug effects ; Mice, Inbred BALB C ; Pyrazines/*pharmacology ; Time Factors

OBJECTIVETo investigate the effect of PD0332991 (C24H29N7O2) on cell cycle, apoptosis, differentiation and self-renewal of hematopoietic stem cells (HSC) in mice.

METHODSThe self renewal ability of HSCs was measured by cobblestone forming cell assay (CAFC). The colony-forming cell (CFC) assay was used to quantify the changes of numbers and functions of HPC after the treatment of the compound. The expressions of self-renewal regulation genes, cell cycle-related genes, apoptosis-related genes were measured by real-time PCR. The cell cycle status and apoptosis of HSC and HPC were analyzed by flow cytometry.

RESULTSThere were obvious changes in cell cycle regulation between control and PD0332991 groups. HSCs in G1 phase increased significantly from 76.3% to 89.5% after treatment of PD0332991 (P<0.05) while cells in S, G2 and M phase reduced from 20.5% to 7.3% (P<0.05). HPCs in G1 phase also increased from 74% to 87.4% after treatment of PD0332991 (P<0.05) while cells in S, G2 and M phase reduced from 25.54% to 11.6% (P<0.05). The apoptotic fractions between control and PD0332991 groups had no statistical difference (P>0.05). After cultured with PD0332991, the expression levels of cell cycle genes CDK1, CyclinA2, CyclinF, p18, p19 and p27 decreased by 58.77%, 66.35%, 56.33%, 62.18%, 32.28% and 36.53% respectively, while expression of CDK7 increased by 27.27% (P<0.05). No visible expression difference was observed in apoptosis and self-renew related genes. After treatment of PD0332991, the self-renewal ability of HSC decreased significantly. There were almost no CFCs in PD0332991 group in CAFC assay. Similarly, the frequency of CFCs was dramatically lower in PD0332991 group.

CONCLUSIONThese results suggested that PD0332991 affected HSC/HPC from mice mainly through inhibiting the cell cycle rather than apoptosis. It also suggested that CDK4/6 might play a key role in the regulation of HSC/HPC.

Animals ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Mice ; Mice, Inbred C57BL ; Piperazines ; pharmacology ; Pyridines ; pharmacology

Animals ; Drugs, Chinese Herbal ; pharmacology ; Erythroid Precursor Cells ; drug effects ; Female ; Hematopoietic Stem Cells ; drug effects ; Mice ; Radiation Injuries, Experimental ; drug therapy ; pathology ; Salvia miltiorrhiza