Mitochondrial metabolism is crucial for providing energy and heme precursors during erythroid development. Oxoglutarate dehydrogenase complex (OGDC) is a key enzyme in the mitochondrial tricarboxylic acid (TCA) cycle, and its level gradually increases during erythroid development, indicating its significant role in erythroid development. The aim of the present study was to explore the role and mechanism of OGDC in erythroid development. In this study, we treated erythroid progenitor cells with CPI-613, a novel lipoic acid analog that competitively inhibits OGDC. The results showed that CPI-613 inhibited erythropoietin (EPO)-induced differentiation and enucleation of human CD34⁺ hematopoietic stem cells into erythroid cells, suppressed cell proliferation, and induced apoptosis. The results of in vivo experiments showed that CPI-613 also hindered the recovery of mice from acute hemolytic anemia. Further mechanism research results showed that CPI-613 increased reactive oxygen species (ROS) in erythroid progenitor cells, inhibited mitochondrial respiration, caused mitochondrial damage, and suppressed heme synthesis, thereby inhibiting erythroid differentiation. Clinical research results showed that oxoglutarate dehydrogenase (OGDH) protein expression levels were up-regulated in bone marrow cells of polycythemia vera (PV) patients. Treatment with CPI-613 significantly inhibited the excessive proliferation and differentiation of erythroid progenitor cells of the PV patients. These findings demonstrates the critical role of OGDC in normal erythroid development, suggesting that inhibiting its activity could be a novel therapeutic strategy for treating PV.
Animals ; Humans ; Mitochondria/metabolism* ; Mice ; Ketoglutarate Dehydrogenase Complex/physiology* ; Cell Differentiation/drug effects* ; Cells, Cultured ; Erythropoiesis/drug effects* ; Reactive Oxygen Species/metabolism* ; Cell Proliferation/drug effects* ; Erythroid Precursor Cells/cytology* ; Apoptosis/drug effects* ; Thioctic Acid/pharmacology* ; Caprylates ; Sulfides

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

OBJECTIVETo explore the value of morphological examination, cytochemical staining combined with bone marrow biopsy in the differential diagnosis between myelodysplastic syndrome (MDS) with low blasts and hemolytic anemia (HA).

METHODSThe clinical data of 85 cases of myelodysplastic syndrome with low blasts (< 5%) and 61 patients with hemolytic anemia in Chinese PLA's Gerneral hospital from September 2009 to March 2015 were retrospectively analysed. The clinical characteristics, cytogenetic and molecular features, bone marrow cell count and morphology features, cytochemical staining results and bone marrow biopsy features of above-methioned patients were compared.

RESULTSThere was no significant difference (P > 0.05) in clinical data between MDS group and HA group. Megakaryocytic dysplasia-positive rate, and ring sideroblasts positive rate, and PAS positive rate were significantly higher in MDS group than those that in HA group (P < 0.05). Abnormal localization of immature precursors (ALIP) and megakaryocytic dysplasia positive rate in bone marrow biopsy were significantly higher in MDS group than those that in HA group (P < 0.05), 90.6% of MDS with low blasts patients were identifiable by combined detections.

CONCLUSIONCombining detection of morphology, cytochemistry staining and bone marrow biopsy has been confirmed to be more useful for differential diagnosis between MDS with low blasts and HA.

Anemia, Hemolytic ; complications ; diagnosis ; Biopsy ; Bone Marrow Cells ; cytology ; Diagnosis, Differential ; Erythroid Precursor Cells ; cytology ; Humans ; Megakaryocytes ; cytology ; Myelodysplastic Syndromes ; complications ; diagnosis ; Retrospective Studies ; Staining and Labeling

OBJECTIVETo discover the techniques for ex vivo generation and cryopreservation of erythroid progenitor cells (EPCs)derived from umbilical cord blood (UCB)mononuclear cells (MNCs).

METHODSUCB was chosen as the source of EPCs. Erythrocytes were precipitated by hydroxyethyl starch (HES). MNCs were separated by Ficoll density gradient centrifugation. Erythroid progenitor cell were generated from MNC ex vivo in suspension culture supplemented with stem cell growth factor, insulin growth factor, erythropoietin, Fms- liketyrosinekinase ligand, transferrin and dexamethasone. Cell maturation was evaluated by morphologic analysis and CD71/CD235a expression profiling. In vitro induced cells were cryopreserved using different cryopreservation media. The cell survival rate, phenotype and proliferation curves were detected after cell thawing.

RESULTSWith the extension of culture time, the total number of cells increased significantly accompanied with the elevation of CD71 and CD235 positive populations. After 14- day inducing, the cells reached to approximately 110 times of the starting number with the cell viability as (88.92±0.95)%. The percentages of cell surface markers were (86.77±9.11)% for CD71 and (64.47±16.67)% for CD71/CD235, respectively. With the extension of inducing time, wright- Giemsa staining showed that the middle erythroblasts appeared mostly at day 10, and the late erythroblasts were seen at day 14. The red pellets were present at day 14, which indicated the more production of hemoglobin. Colony forming assay showed that erythroid colonies at induction day 7 were higher than that for non-induced cells (326.00±97.96vs 61.60±20.03 per 2 000 cells). With the extension of culture time, the number of erythroid colonies decreased. Induced EPCs were preserved with different cryopreservation solutions, in which 10% DMSO were better than 5% DMSO. Additionally, 10% DMSO + 2% HSA showed no different with 10% DMSO + 5% HSA. Combined 50% plasma with 2% HSA was more effective.

CONCLUSIONSThis non- serum culture media could effectively induced and expanded EPCs, and 10% DMSO + 2% HSA + 50% plasma appeared to be a desirable cryopreservation solution for EPCs from UCB.

Cell Culture Techniques ; Cell Differentiation ; Cell Survival ; Cells, Cultured ; Cryopreservation ; methods ; Erythroblasts ; cytology ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Umbilical Cord

OBJECTIVETo explore the influence of 1,4-benzoquinone (1,4-BQ) on proliferation of human bone marrow haematopoietic stem cells (hBM-HSCs) and human bone marrow mesenchymal stem cells (hBM-MSCs).

METHODSThe bone marrow samples were collected from a healthy donor. Methylcellulose semi-solid culture medium was used to culture the mononuclear cells of bone marrow in different culture systems. Colony-forming unit (CFU) assay was utilized to evaluate the proliferation of hBM-HSCs exposed to 1,4-BQ at the doses of 10, 25, 50 and 100 µmol/L and to observe the influence of 1,4-BQ on the Colony-forming unit-erythroid (CFU-E)/Burst-forming unit-erythroid (BFU-E), Colony-forming unit-granulocyte, macrophage (CFU-GM), Colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) in hBM-MSCs. MTT assay was used to detect the proliferation of hBM-MSCs exposed to 1,4-BQ at the doses of 1, 5, 10, 25, 50, 100, 200, 500 and 1000 µmol/L for 24 h, respectively, after hBM-MSCs were isolated, cultured and expanded.

RESULTSThe results of CFU assay indicated that numbers of CFU-E/BFU-E, CFU-GM and CFU-GEMM in 25, 50 and 100 µmol/L groups significantly decreased, as compared with control group (P < 0.05). However, no significant difference was found between the 10 µmol/L group and the control group. The results of MTT assay showed that the cellular viability of hBM-MSCs exposed to 1,4-BQ at the doses of 50 ∼ 200 µmol/L for 24 h significantly decreased in a dose-depended manner. When the exposure dose was higher than 200 µmol/L, the cellular viability of hBM-MSCs was lower than 5% which was significantly lower than that of control group (P < 0.05). When the exposure dose was lower than 25 µmol/L, there was no significant difference of cellular viability between exposure group and control group (P > 0.05).

CONCLUSIONThe results of the present study demonstrated that 1,4-BQ could inhibit the colony forming of hBM-HSCs and the relative viability of hBM-MSCs in vitro. The hematotoxicity induced by 1,4-BQ may be related to inhibiting the proliferation capacity of hBM-HSCs.

Benzoquinones ; toxicity ; Bone Marrow Cells ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Erythroid Precursor Cells ; Granulocyte-Macrophage Progenitor Cells ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology

OBJECTIVETo investigate the effects of combined use of low-dose hydroxyurea (HU) and sodium butyrate (NaB) on the expression of 7 globin genes (zeta, alpha, epsilon, Ggamma, Agamma, delta, and beta) in human erythroid progenitor cells.

METHODSHuman erythroid progenitor cells were cultured using a two-step liquid culture system and treated with HU and NaB either alone or in combination. The inhibitory effects of the agents on the cell growth were monitored with trypan blue exclusion assay, and the changes in the mRNA of the 7 globin genes were detected using RT-PCR.

RESULTSLow-dose HU combined with NaB resulted in significantly lower inhibition rate of the erythroid progenitor cells than routine dose HU and NaB used alone (28.56% and 38.80%, respectively, P<0.05). Compared with untreated cells (0.653-/+0.092 and 0.515-/+0.048), HU combined with NaB significantly increased the expression of Ggamma-and Agamma- mRNA (1.203-/+0.018 and 0.915-/+0.088, respectively, P<0.05), and HU and NaB used alone produced similar effects (1.305-/+0.016 and 0.956-/+0.029 for HU, and 1.193-/+0.070 and 0.883-/+0.012 for NaB, P>0.05). HU and NaB, either used alone or in combination or at different doses, caused no significant changes in the other globin genes (zeta, alpha, epsilon, delta and beta) (P>0.05).

CONCLUSIONLow-dose HU combined with NaB can up-regulate gamma globin gene expression, especially Ggamma-mRNA expression, to decrease the growth inhibition on human erythroid progenitor cells in vitro, but produces no significant effect on the expressions of zeta, alpha, epsilon, delta and beta genes.

Anemia, Sickle Cell ; genetics ; Butyrates ; administration & dosage ; pharmacology ; therapeutic use ; Cells, Cultured ; Drug Therapy, Combination ; Erythroid Precursor Cells ; cytology ; drug effects ; physiology ; Erythropoiesis ; drug effects ; Humans ; Hydroxyurea ; administration & dosage ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; gamma-Globins ; genetics ; metabolism

The purpose of this study was to explore the differences in megakaryocyte progenitor ex vivo expansion between CD34+ cells derived from human umbilical cord blood (CB) and bone marrow (BM). Mononuclear cells (MNCs) were obtained from CB or BM by Ficoll-Hypaque density gradient separation. CD34+ cells were purified by magnetic cell sorting (MACS). The selected CD34+ cells were seeded in serum-free conditions stimulated with thrombopoietin (TPO), TPO+interleukin 11 (IL-11), or TPO+IL11+heparin for 14 days. Amplification product (CD34+, CD41a+, and CD34+ CD41a+ cells) immunophenotypes, megakaryocyte apoptosis rates and the DNA content were measured by fluorescence-activated cell sorting (FACS). The colony-forming units of granulocytes and monocytes (CFU-GM), burst-forming units of erythrocytes (BFU-E), and colony-forming units of megakaryocytes (CFU-Mk) were also evaluated by the colony-forming units (CFU) assay. The results indicated that CD34+ cells derived from CB showed higher expansion ability of total cell counts, CD41a+ and CD34+ CD41a+ cells than those derived from BM for all days 14 of culture (p<0.05, respectively). There were no significant differences in CFU-GM, BFU-E, and total CFU-Mk counts between CB and BM-derived CD34+ cells on day 0 (p>0.05, respectively), but CB-derived CFU-Mk seemed mainly large colonies, and the number of large colonies was higher than that from BM (p<0.05) on day 0. There were no significant differences in expansion ability of CFU-GM between CB and BM-derived cells on days 7, 10, and 14 of culture (p > 0.05, respectively), but the expansion ability of BFU-E and CFU-Mk derived from CB cells was higher than that from BM (p<0.05, respectively). There were no significant differences in apoptosis rates of megakaryocyte from two source cells for days 14 of culture. Megakaryocytes derived from CB mostly showed the 2N DNA content (>90%) for days 14 of culture, while those cells derived from BM showed the increased DNA content, and 4N, 8N or more ploidy cells gradually increased with prolonging of culture time. It is concluded that CB-derived CD34+ cells have a greater proliferation potential than that derived from BM, which is therefore proven to be a better cell source for megakaryocyte progenitor expansion in vitro.
Antigens, CD34 ; Bone Marrow Cells ; cytology ; immunology ; Cell Culture Techniques ; methods ; Cell Differentiation ; Cell Division ; Cell Separation ; Cells, Cultured ; Erythroid Precursor Cells ; cytology ; Fetal Blood ; cytology ; immunology ; Humans ; Megakaryocyte Progenitor Cells ; cytology ; immunology

OBJECTIVETo explore the biological effect of Notch ligand Delta-1 (Notch L delta-1) on the sIL-6R during the differentiation of erythroid hematopoiesis.

METHODSMononuclear cells (MNCs) was isolated from the normal cord blood using Ficoll graduation solution. MNCs were enriched for CD34(+) CD38(-) cells by CD34 immunomagnetic beads and a FACS Vantage. CD34(+) CD38(-) cells was cultured for 7 days in the presence of SCF, Flt3L, TPO and IL-3 (4GFs). The cultured cells was detected for the expression of IL-6R and GPA. The subsequently enriched CD36(+) erythroid progenitors were sorted for cells with IL-6R(+) and IL-6R(-) using FACS Vantage. The CD36(+) GPA(-) IL-6R(-) cells were respectively cultured in the 4GFs, 4GFs + IL-6 or 4GFs + FP6 containing medium in the presence or absence of Notch L delta-1 for 14 days and CD36(+) GPA high red cells were counted.

RESULTSIL-6R cells accounted for 95% of CD36(+) GPA(+) cells. The CD36(+) GPA(-) cells was clearly divided into IL-6R(+) (46%) and IL-6R(-) (54%) subpopulations, the IL-6R(+) cell subpopulation formed only a few GM colonies (2.1 +/- 1.8) and a greater number of BFU-E colonies were generated from the IL-6R(-) subpopulation (58.2 +/- 18.1) (P < 0.05). The number of CD36(+) GPA high cell was (1.400 +/- 0.180) x 10(6) in the presence of FP6, lower than that [(2.460 +/- 0.190) x 10(6)] in the presence of FP6 + Notch L delta-1 (P < 0.05).

CONCLUSIONNotch L delta-1 enhances the sIL-6R-mediated effects of IL-6 on the generation of erythroid cells.

ADP-ribosyl Cyclase 1 ; Antigens, CD34 ; Cell Differentiation ; drug effects ; physiology ; Cells, Cultured ; Erythroid Precursor Cells ; cytology ; drug effects ; Humans ; Interleukin-6 ; metabolism ; physiology ; Intracellular Signaling Peptides and Proteins ; Membrane Proteins ; pharmacology ; Receptors, Interleukin-6 ; metabolism ; physiology

OBJECTIVETo explore the therapy to further elevate the efficacy of the treatment of chronic aplastic anemia (CAA).

METHODSForty-five patients with CCA were assigned into two groups, the 26 patients in the treated group were treated by Shengxuening (a Chinese herbal preparation) and cyclosporin A (CsA), and the 19 patients in the control group were treated with androgen alone, with the therapeutic course lasting for over 3 months. Changes of peripheral blood picture, and the colony productivity of burst forming unit-erythroid (BFU-E), colony forming unit-erythroid (CFU-E) and colony forming unit-granulocyte macrophage (CFU-GM) in bone marrow were observed before and after 3 months treatment. The amount of erythrocyte and platelet infusion, frequency of infection, condition of hemorrhage and relevant death were also observed. The follow-up study was conducted for over half a year.

RESULTSThe total effective rate in the treated group was 84.6%, which was significantly higher than that in the control group (52.6%, P < 0.05). Levels of hemoglobin, reticulocyte, neutrophil and platelet increased after treatment in the treated group, as compared with those before treatment, with significant difference (P < 0.05), and the colony productivity of BFU-E, CFU-E and CFU-GM in bone marrow also got significantly increased (P < 0.01), and showed significant difference from those in the control group (P < 0.05).

CONCLUSIONShengxuening-assisting CsA therapy is an effective measure for treatment of CAA.

Adult ; Aged ; Androgens ; therapeutic use ; Anemia, Aplastic ; drug therapy ; Chronic Disease ; Cyclosporine ; administration & dosage ; Drugs, Chinese Herbal ; administration & dosage ; Erythroid Precursor Cells ; Follow-Up Studies ; Hemoglobins ; analysis ; Humans ; Medicine, Chinese Traditional ; Middle Aged ; Neutrophils ; cytology ; Platelet Count ; Reticulocytes ; cytology ; Stanozolol ; therapeutic use ; Tablets

OBJECTIVEThe previous studies indicated that mesenchymal stem cells (MSCs) either from umbilical cord blood (UCB) or from bone marrow (BM) had the same biological characteristics and the function of secreting hematopoietic growth factors (HGFs). The present study aimed to understand the effects of human UCB MSCs on the expansion of CD(34)(+) cells from UCB.

METHODS1. Human UCB CD(34)(+) cells were incubated in the system containing UCB MSCs, HGFs and serum free medium. 2. The surface markers (CD(34)(+), CD(34)(+)CD(38)(-), CD(34)(+)CD(3)(+), CD(34)(+)CD(19)(+), CD(34)(+)CD(33)(+), CD(34)(+)CD(41a)(+)) on expanded UCB cells were examined by flow cytometry on the 6th and 12th days. 3. The expanded and unexpanded cells were cultured in semi-solid culturing system and checked for colony forming units of granulocyte and macrophage (CFU-GM), erythroid burst-forming unit (BFU-E), colony forming units of granulocyte- erythrocyte-megakaryocyte-macrophage (CFU-Mix) and colony forming units of high-proliferative potential (CFU-HPP).

RESULTS1. The expansion folds of CD(34)(+)CD(38)(-) cells from UCB MSCs + HGFs groups on the 6th and 12th days were 159.43 and 436.68, respectively. Interestingly, the percentage of CD(34)(+)CD(38)(-) cells declined in HGFs group after expanding for 12 days, but it rose to 9.98% in the UCB MSCs + HGFs group. 2. Colony forming capacity of expanded UCB cells showed that the folds of CFU-Mix and CFU-HPP of UCB MSCs + HGFs group increased from day 6 to day 12, but the folds decreased in the HGFs group. 3. From day 0 to day 12, CD(34)(+)CD(33)(+) cells and CD(34)(+)CD(41a)(+) cells were amplified gradually, but CD(34)(+)CD(19)(+) and CD(34)(+)CD(3)(+) cells decreased gradually, and in UCB MSCs + HGFs group this phenomenon was more significant than that in HGFs group.

CONCLUSION1. UCB MSCs containing system not only has the ability to expand the primitive HSCs but also has the ability to sustain the proliferation of HSCs. 2. UCB MSCs containing system amplified mainly myeloid and megakaryocytoid progenitor subsets. These may have clinical significance in reducing infection and hemorrhage.

Antigens, CD34 ; biosynthesis ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Cell Proliferation ; Culture Media, Conditioned ; Culture Media, Serum-Free ; Erythroid Precursor Cells ; Fetal Blood ; cytology ; Flow Cytometry ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Cell Growth Factors ; pharmacology ; Hematopoietic Stem Cells ; metabolism ; Humans ; Infant, Newborn ; Mesenchymal Stromal Cells ; immunology ; metabolism