Humans with chronic psychological stress are prone to develop multiple disorders of body function including impairment of immune system. Chronic psychological stress has been reported to have negative effects on body immune system. However, the underlying mechanisms have not been clearly demonstrated. All immune cells are derived from hematopoietic stem cells (HSC) in the bone marrow, including myeloid cells which comprise the innate immunity as a pivotal component. In this study, to explore the effects of chronic psychological stress on HSC and myeloid cells, different repeated restraint sessions were applied, including long-term mild restraint in which mice were individually subjected to a 2 h restraint session twice daily (morning and afternoon/between 9:00 and 17:00) for 4 weeks, and short-term vigorous restraint in which mice were individually subjected to a 16 h restraint session (from 17:00 to 9:00 next day) for 5 days. At the end of restraint, mice were sacrificed and the total cell numbers in the bone marrow and peripheral blood were measured by cell counting. The proportions and absolute numbers of HSC (LinCD117Sca1CD150CD48) and myeloid cells (CD11bLy6C) were detected by fluorescence activated cell sorting (FACS) analysis. Proliferation of HSC was measured by BrdU incorporation assay. The results indicated that the absolute number of HSC was increased upon long-term mild restraint, but was decreased upon short-term vigorous restraint with impaired proliferation. Both long-term mild restraint and short-term vigorous restraint led to the accumulation of CD11bLy6C cells in the bone marrow as well as in the peripheral blood, as indicated by the absolute cell numbers. Taken together, long-term chronic stress led to increased ratio and absolute number of HSC in mice, while short-term stress had opposite effects, which suggests that stress-induced accumulation of CD11bLy6C myeloid cells might not result from increased number of HSC.
Animals ; Antigens, Ly ; metabolism ; Bone Marrow Cells ; cytology ; CD11b Antigen ; metabolism ; Cell Proliferation ; Hematopoietic Stem Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Restraint, Physical ; Stress, Psychological

Human cytomegalovirus (HCMV) infection, a common complication, remains a major risk factor related with patient death after hematopoietic stem cell transplantation (HSCT). Cytotoxic T lymphocytes (CTL) which is crucial to control HCMV infection, can prevent or treat HCMV infection safely and effectively after adoptive infusion. Many studies have been focussed on exploring different methods for preparation of CTL. The method of using antigen presenting cells to stimulate peripheral blood mononuclear cells is simple to operate, easy to conduct large-scale clinical trials. Isolation of CTL from donor-derived PBMC by peptide-tetramer or INF-γ antibody requires a large volume of peripheral blood and high cost for preparation. Third-party CTL can provide an "off-the-shelf" product, but the problem of HLA-mismatch still would be solved. In addition, the clinical efficacy and safety of different methods also vary. This article reviews and compares the current methods to generate CTL and efficacy of the cells after infusions.
Adoptive Transfer ; Antigen-Presenting Cells ; cytology ; Cytomegalovirus ; Cytomegalovirus Infections ; therapy ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Leukocytes, Mononuclear ; cytology ; T-Lymphocytes, Cytotoxic ; cytology

OBJECTIVETo evaluate the therapeutic effects of combined administration of recombinant human granulocyte colony-stimulating factor (rhG-CSF), recombinant human thrombopoietin (rhTPO) and recombinant human interleukin-2 (rhIL-2) on radiation-induced severe haemopoietic acute radiation sickness (ARS) in rhesus monkeys, so as to provide experimental evidences for the effective clinical treatment.

METHODSSeventeen rhesus monkeys were exposed to 7.0 Gy (60)Co γ-ray total body irradiation (TBI) to establish severe haemopoietic ARS model, and were randomly divided into supportive care group, rhG-CSF+rhTPO treatment group and rhG-CSF+rhTPO+rhIL-2 treatment group. Survival time, general signs such as bleeding and infections, and peripheral blood cell counts in each group were monitored. Bone marrow cells were cultivated to examine the colony formation ability. The histomorphology changes of bone marrow were observed at 45 d post irradiation.

RESULTSAfter 7.0 Gy (60)Co γ-ray TBI, monkeys of supportive care group underwent tarry stool and emesis, then died in 12~18 d. The overall survival rate in this group was 16.7%. Gastrointestinal reactions of monkeys in two combined-cytokines treatment groups were inapparent. Combined-cytokines treatment induced 100% survival. Complete blood cells declined sharply after irradiation in each group, but two combined-cytokines treatment schemes could elevate the nadir of all blood cells, shorten the duration of pancytopenia and accelerate the recovery of hemogram. Compared with rhG-CSF+ rhTPO treatment, rhG-CSF+ rhTPO+ rhIL-2 treatment could increase the counts of lymphocytes and monocytes. The colony-formation rate of haemopoietic stem/progenitor cells in bone marrow dropped markedly at 2 d after irradiation. Combined-cytokines treatment promoted the ability of colony formation on day 29. Hematopoietic cells mostly disappeared in bone marrow of animals in supportive care group, but hematopoietic functions were recovered after cytokines were administrated.

CONCLUSIONrhG-CSF+ rhTPO and rhG-CSF+ rhTPO+ rhIL-2 treatment can significantly promote hematopoiesis recovery, improve the quantity of life, simplify the supportive therapy, and enhance the survival rate of rhesus monkeys with severe haemopoietic ARS induced by 7.0 Gy (60)Co γ-ray exposure. Especially the application of rhIL-2 can accelerate the recovery of lymphocytes and monocytes and restore the immunological function. Thus, combination of rhG-CSF, rhTPO and rhIL-2 on the basis of supportive care is an efficient strategy to treat severe haemopoietic ARS.

Animals ; Bone Marrow ; pathology ; Bone Marrow Cells ; pathology ; Gamma Rays ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Hematopoiesis ; drug effects ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-2 ; pharmacology ; Macaca mulatta ; Radiation Injuries ; drug therapy ; Random Allocation ; Recombinant Proteins ; therapeutic use ; Thrombopoietin ; pharmacology ; Whole-Body Irradiation

OBJECTIVETo analyze the influential factors related to mobilization and collection of stem cells so as to improve the collection efficiency of autologous peripheral stem cell transplantation in lymphoma patients.

METHODSThe peripheral blood stem cell collection data of 151 cases of lymphoma in our hospital was analyzed retrospectively. The relationship between the harvested CD34(+) stem cells and some factors, such as age, sex, height, weight, histological type, staging, mobilization programs, collecting days, blood transfusion, time and duration of chemotherapy, was analyzed.

RESULTSThe single factor analysis showed that sex, height, weight, histological type, staging, mobilization program, collecting days, blood transfusion were not significantly associated with CD34(+) stem cells collection, respectively. Age (r = -0.248, P = 0.002), duration of sick and cycles of chemotherapy were significantly associated with CD34(+) cell collection. At the age older than 50 years, the collected CD34(+) cell number decreased significantly; and at the age older than 60 years, the CD34(+) cell number was greatly reduced; CD34(+) cells non-significantly correlated with peripheral blood WBC (r = 0.053, P = 0.527), but significantly with the percentage of mononuclear cells (r = 0.260, P = 0.002) and the absolute value of mononuclear cells (r = 0.338, P = 0.00003) .

CONCLUSIONThe patients less than 60 years old, fewer chemotherapy cycles, shorter duration time or PB mononuclear cells between (2-6) × 10(9)/L may contribute to the better mobilization and collection of peripheral blood stem cells.

Age Factors ; Antigens, CD34 ; metabolism ; Blood Transfusion ; Cell Count ; Hematopoietic Stem Cell Mobilization ; Hematopoietic Stem Cells ; cytology ; Humans ; Lymphoma ; therapy ; Peripheral Blood Stem Cell Transplantation ; Retrospective Studies ; Transplantation, Autologous

OBJECTIVETo explore the effect of ligustrazine on the migration of bone marrow mesenchymal stem cells (BMSCs) and protein expressions of matrix metalloproteinase-2 and-9 (MMP-2 and MMP-9) in vitro.

METHODSBMSCs were in vitro isolated and cultured using whole bone marrow adherent method, and phenotypes [surface positive antigens (CD29 and CD90) and negative antigens (CD34 and CD45)] identified using flow cytometry. BMSCs were divided into the blank control group, 25, 50, 100 µmol/L ligustrazine group, and the GM6001 group (100 µmol/L ligustrazine +MMPs inhibitor GM6001 ). The migration of BMSCs was tested by Transwell chamber test and wound healing assay after treated with ligustrazine for 24 h. The protein expressions of MMP-2 and MMP-9 were detected by Western blot.

RESULTSThe third passage BMSCs grew well in uniform morphology. The expression rate of CD29, CD90, CD34, and CD45 was 96.9%, 97.3%, 0.2%, and 3.0%, respectively. Compared with the blank control group, the number of migrated cells and relative distance of cell invasion increased, and the protein expressions of MMP-2 and MMP-9 were elevated in each ligustrazine group (P < 0.05, P < 0.01). Compared with 100 µmol/L ligustrazine group, the number of migrated cells and relative distance of cell invasion decreased in 25 and 50 µmol/L ligustrazine groups and the GM6001 group (P < 0.01). Protein expression of MMP-2 decreased in 25 and 50 µmol/L ligustrazine groups (P < 0.01).

CONCLUSIONLigustrazine could promote the migration of BMSCs in vitro, and its mechanism might be related to up-regulating expression levels of MMP-2 and MMP-9 protein.

Cell Movement ; Cells, Cultured ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Pyrazines ; pharmacology ; Up-Regulation

OBJECTIVETo compare the biological characteristics and immunosuppressive activity between human amniotic mesenchymal stem cells (hAMSC) and human bone marrow mesenchymal stem cells (hBMMSC).

METHODSMSC from human amnion and bone marrow were isolated using enzymatic digestion and Ficoll-Hypaque density gradients, respectively. Their biological characteristics were compared by morphology, cell growth curves, cell cycle profile analysis, immunophenotype and immunofluorescence assay. Their immunosuppressive activities were studied on total activated T-cells with phytohemagglutinin (PHA-PBMSC). An in vitro co-culture was performed to compared the lymphocyte proliferation and the supernatant level of IFN-γ were measured by CCK-8 method and ELISA, respectively.

RESULTSBoth hAMSC and hBMMSC demonstrated fibroblast-like morphology. The hAMSC were able to be amplified for at least 15 passages, while the hBMMSC only for 6-7 passages. There was no significant difference in the proportion of G2/M phase cells of the 2 cells types (P>0.05). By FACS analysis for immunophenotype, both MSC were shown to be positive for CD105, CD90, CD73 and negative for CD34, CD45, CD11b, CD19, HLA-DR, but hAMSC were positive for Oct-3/4, which was in contrast to hBMMSC. Both of them expressed vimentin. Both the cells exhibited a inhibitory role on the lymphocyte proliferation induced by PHA in co-culture conditions, that was increased with the increase MSC proportion and both the suppressing effecs were enhanced. The supernatant IFN-γ levels of hAMSC co-cultured with lymphocyte at a ratio of 1:1 after 72 hours were measured by ELISA, and the level of IFN-γ was significantly lower than that in the same co-culture system of hBMMSC. In contrast to the IFN-γ in the PHA-stimulated group, the IFN-γ level in both co-culture groups was significantly lower.

CONCLUSIONMSC from amnion displayed a higher proliferative capacity and stem cell properties, compared with hBMMSC. Both MSC can inhibit lymphocyte proliferation and suppress IFN-γ secretion induced by PHA in vitro.

Amnion ; cytology ; Bone Marrow Cells ; cytology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Hematopoietic Stem Cells ; cytology ; Humans ; Immunophenotyping ; Immunosuppression ; Lymphocyte Activation ; Mesenchymal Stromal Cells ; cytology ; T-Lymphocytes ; cytology

OBJECTIVETo explore an efficient, stable system and method to verify the regulation effect of small molecule compounds on human hematopoietic stem cells (hHSC).

METHODSBy using combination of flow cytometry with study results of surface markers on hHSC, and optimation of sorting process for further studying the effect of small molecular compounds on stem property of hHSC, the single hHSC was treated with published small molecular compounds such as SR1 and UM171 which possess the expansion effect. After treating with hHSC for 14 d, the flow cytometric analysis of cell phenotypes and cell morphologic observation were performed, at the same time the hematopoietic function of cultured hHSC was verified by colony-forming cell (CFC) test and cobblestone area forming cell (CAFC) test.

RESULTSThe effects of SR1 and UM171 and their compositions in multi-cell culture were consistent with the published data, therefore the useful concentration of compounds were obtained. The results of multiparameter sorting of single cell (CD34+ CD38- CD45RA- CD90+ CD49f+) and ex vivo culture were consistent with the results of bulk cell culture. The results of cell phenotype analysis was in accordance with flow cytometric results. In addition, CFC test and CAFC test revealed that the colony-forming ability in treated group was significantly higher than that in control group (P<0.05).

CONCLUSIONThe rapid, efficient stably amplified and short-time culture system for single hHSC and method for varifying the effect of small molecular compounds are established, which provides platform for screening small molecular compounds and lays the foundation for further study of hHSC expansion.

Cell Culture Techniques ; Cell Separation ; Flow Cytometry ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Indoles ; pharmacology ; Pyrimidines ; pharmacology

OBJECTIVETo investigate the effects of oxygen concentration and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and to analyzed the relationship among the oxygen concentration, ROS and the biological characteristics of mouse HSC through simulation of oxygen environment experienced by PB HSC during transplantation.

METHODSThe detection of reactive oxygen species (ROS), in vitro amplification, directional differentiation (BFU-E, CFU-GM, CFU-Mix), homing of adhesion molecules (CXCR4, CD44, VLA4, VLA5, P-selectin), migration rate, CFU-S of NOD/SCID mice irradiated with sublethal dose were performed to study the effect of oxgen concentration and reactive oxygen species on the biological characteristics of mouse BM-HSC and the relationship among them.

RESULTSThe oxygen concentrations lower than normal oxygen concentration (especially hypoxic oxygen environment) could reduce ROS level and amplify more Lin(-) c-kit(+) Sca-1(+) BM HSC, which was more helpful to the growth of various colonies (BFU-E, CFU-GM, CFU-Mix) and to maintain the migratory ability of HSC, thus promoting CFU-S growth significantly after the transplantation of HSC in NOD/SCID mice irradiated by a sublethal dose. BM HSC exposed to oxygen environments of normal, inconstant oxygen level and strenuously thanging of oxygen concentration could result in higher level of ROS, at the same time, the above-mentioned features and functional indicators were relatively lower.

CONCLUSIONThe ROS levels of BM HSC in PB HSCT are closely related to the concentrations and stability of oxygen surrounding the cells. High oxygen concentration results in an high level of ROS, which is not helpful to maintain the biological characteristics of BM HSC. Before transplantation and in vitro amplification, the application of antioxidancs and constant oxygen level environments may be beneficial for transplantation of BMMSC.

Animals ; Cell Differentiation ; Culture Media ; chemistry ; Erythroid Precursor Cells ; cytology ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Oxygen ; chemistry ; Reactive Oxygen Species ; metabolism

OBJECTIVE: To explore the effect of bone marrow mesenchymal stem cells (BM-MSCs) on angiogenesis in rats after brain injury. 
 METHODS: Brain injury model of rats was established with freely fall method. A total of 50 Sprague-Dawley (SD) rats were randomly divided into a transplanted group and a control group (n=25 in each group). BM-MSCs were injected in lateral ventricle in the transplanted group, and normal saline was injected in the control group. Modified method for neurological deficit scores (mNSS) was performed at the 1, 3, 7, 21, 14 d after the operation. Flow cytometry were performed to detect CD34 and CD133 double-labeled peripheral blood cells in preoperative or 3, 6, 12, 24 h, and 3, 7 d after the operation. Expression of neuron-specific enolase (NSE) and CD31 in the brain tissues near injury area was detected by immunohistochemical SP method. 
 RESULTS: There was significant difference in the MNSS scores between the 2 groups (F=5.997, P<0.05), and the difference at the different time points in each group was significant (F=37.106, P<0.01). The mNSS scores in the control group were higher than those in the transplanted group at the 7, 14, 21 d after the operation (P<0.05). The CD34 and CDl33 double positive cells (DPCs) were present in rats' peripheral blood. DPCs's numbers in peripheral blood in the control group were declined at 3 h after the sugery, they were increased and reached the highest point at 6 h after the surgery, and decreased gradually and reached normal levels at 24 h after the surgery. The same tendency was achieved in the transplanted group, and the DPCs's numbers were increased until 24 h after the surgery, which were significantly higher in the transplanted group than those in the control group at 24 h after the surgery (P<0.05). The NSE expression in the transplanted group was significantly greater than that in the control group in 7 and 14 d after the surgery (P<0.05). The expression of CD31 in the transplanted group was significantly higher than that in the control group in 3 and 7 d after the surgery (P<0.05).
 CONCLUSION BM-MSCs transplantation can increase the number of peripheral blood endothelial progenitor cells after traumatic brain injury in rats and sustain for 24 h, which in turn up-regulate the angiogenesis and neuronal marker, and improve the neurological function.
AC133 Antigen ; Animals ; Antigens, CD ; metabolism ; Antigens, CD34 ; metabolism ; Brain Injuries ; therapy ; Glycoproteins ; metabolism ; Hematopoietic Stem Cells ; cytology ; Mesenchymal Stem Cell Transplantation ; Neovascularization, Physiologic ; Peptides ; metabolism ; Phosphopyruvate Hydratase ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Muscle-derived stem cells (MDSC) are defined as myogenic stem cells endowed with their ability to self-renew and differentiate into multiple cell types of their derivative tissue, and are proved to be over 10 times more efficient in hematopoiesis than hematopoietic stem cells (HSC). Although the mechanism which MDSC differentiate into blood cells is still unclear, MDSC were considered to replace HSC to treat the patients suffering from bone marrow diseases such as aplastic anemia and tumor. MDSC are different from HSC in a variety aspects like biological characteristics, protein expression and cell proliferation. On the other hand, MDSC contain multiple distinct stem cell populations. Among these, there is only a small part with the ability to repopulate hematopoietic cells, and it is still uncertain whether their origin is same as HSC. This review summarizes the difference between MDSC and HSC, the ability of MDSC to repopulate hematopoietic cells, and the prospect of MDSCs' transplantation.
Anemia, Aplastic ; Cell Differentiation ; Cell Proliferation ; Hematopoiesis ; Hematopoietic Stem Cells ; cytology ; Humans ; Muscle, Skeletal ; cytology