Objective: To understand the effect of sirolimus on the erythropoiesis of K562 cell line and bone marrow cells from pure red cell aplasia (PRCA) patients and normal controls. Methods: Different concentrations (10, 100, 1 000 nmol/L) of sirolimus were added to the K562 cell line or bone marrow cells from PRCA patients or normal controls and cultured 14 days for BFU-E formation. Meanwhile, sirolimus was also added to the serum treated PRCA bone marrow cells to cultivate for the same priod of time. Results: Neither K562 cells, bone marrow cells from PRCA patients or normal controls showed any difference when sirolimus was added to the culture system for BFU-E. However, BFU-E formation decreased after serum was added in PRCA patients (76.40±22.48 vs 136.33±12.58, t=-4.329, P=0.001) and this suppression of BFU-E was partly corrected by 1 000 nmol/L sirolimus treatment (97.14±15.83 vs 76.40±22.48, P=0.038). Conclusions: Sirolimus may modulate the suppression of erythropoiesis by serum instead of directly stimulate the growth of red blood cells in PRCA patients.
Erythroid Precursor Cells ; Erythropoiesis ; Humans ; K562 Cells ; Red-Cell Aplasia, Pure ; Sirolimus

No abstract available.
Bone Marrow Transplantation* ; Bone Marrow* ; Erythroid Precursor Cells* ; Pathology*

PURPOSE: Because of the unavailability of marrow transplantation, umbilical cord blood (CB) is increasingly being used. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from cord blood source and mobilized peripheral blood (PB) in a serum-free media. METHODS: The CD34+ cells, selected from CB and mobilized PB, were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at culture days 0, day 4, day 7, and day 14 with various growth factors. RESULTS: The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the PB at day 7 (2 fold increase than PB). The CB-selected CD34+ cells produced more BFU-E colonies than did the PB on culture at days 7 and at day 14. Also, the CB-selected CD34+ cells produced more CFU-Mk colonies than did the PB on culture at day 4 and at day 7. CONCLUSION: The ex vivo expansion of the CB cells may be promising in producing total cellular expansion, CFU-Mk and BFU-E compared with PB for 7 to 14 days. The growth factors combination including megakaryocyte growth and development, flt3-ligand and interleukin-3 showed more expansion in the view of total cells and clonal maintenance compared with less combination.
Bone Marrow ; Culture Media, Serum-Free ; Erythrocytes ; Erythroid Precursor Cells ; Fetal Blood* ; Granulocytes ; Growth and Development ; Intercellular Signaling Peptides and Proteins ; Interleukin-3 ; Megakaryocytes ; Monocytes ; Stem Cells

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

A patient presenting paroxysmal nocturnal hemoglobinuria (PNH) cloned cells in the course of myelodysplastic syndrome (MDS) with reticulocytosis is described. The bone marrow biopsy demonstrated erythroid hyperplasia and moderate dysplasia. Mild hemoglobinuria was detected but the Ham test was negative. The reticulocyte survival test revealed sustained survival curve indicating delayed reticulocyte maturation regarded as the characteristic of MDS cloned erythroid cells. The glycosylphosphatidylinositol-linked protein deficient neutrophils and erythrocytes population regarded as PNH clones were identified by flow cytometric analysis using monoclonal antibody. From these results, we concluded that MDS and PNH cloned cells were coexisited in this patient. In this patient, long-term follow-up observation could clarify whether MDS and PNH were arising from the same clone or from two distinct clones.
Biopsy ; Bone Marrow ; Clone Cells ; Erythrocytes ; Erythroid Cells ; Follow-Up Studies ; Glycosylphosphatidylinositols* ; Hemoglobinuria ; Hemoglobinuria, Paroxysmal* ; Humans ; Hyperplasia ; Myelodysplastic Syndromes* ; Neutrophils ; Reticulocytes* ; Reticulocytosis

PURPOSE: Many studies for hematopoietic stem cell have investigated CD133, instead of CD34, as a new surrogate stem cell marker. Counterflow centrifugal elutriation (CCE) is a physical separation of a homogeneous cell population through cell sedimentation characteristics. We evaluated the stem cell distribution and hematopoietic function from cord blood (CB) and bone marrow (BM) through CCE. METHODS: We obtained total nucleated cells from CB and BM, and separated the cell fractions according to media infusion flow rates (17 mL/min (FR 17), 24 mL/min (FR 24), 29 mL/min (FR 29), and rotor off (R/O) ) by CCE. We analyzed the proportion of CD34+ and CD133+ cells in each fraction, and performed methylcellulose-based colony assay. RESULTS: In CB, the cell recovery rates after CCE were 5.9+/-4.3% in FR 17, 4.2+/-2.1% in FR 24, 19.4+/-11.9% in FR 29, and 61.9+/-11.7% in R/O. In BM, they were 14.9+/-8.2% in FR 17, 17.4+/-13.4% in FR 24, 23.6+/-6.11% in FR 29, and 27.1+/-8.9% in R/O. The distributions of CD133+ and CD34+ cells in CB were more abundant in R/O (2.91%, 1.85%) than in other fractions. In BM, CD133+ and CD34+ cell rates in R/O (5.40%, 2.75%) were similar with those in unmanipulated BM (5.48%, 2.78%). In both CB and BM, there was more CFU-GM and BFU-E in R/O than in other fractions. CONCLUSION: We suggested that the distribution of CD34+ and CD133+ cells might be different between CB and BM. However, the R/O containing relatively large cells could have an effective clonogenicity compared with the unmanipulated sample in both CB and BM.
Bone Marrow* ; Erythroid Precursor Cells ; Fetal Blood* ; Granulocyte-Macrophage Progenitor Cells ; Hematopoietic Stem Cells ; Stem Cells

OBJECTIVE: To determine the expression of DLK1 gene in acute leukemias (AL) and its function in erythroid differentiation of K562 cells. METHODS: We detected the expression of DLK1 gene in 65 different acute leukemia categories (a test group) and 34 normal bone marrow controls (a control group) with RT-PCR. DLK1 protein in 20 out of the 65 AL patients and 13 of the 34 controls was assayed by Western blot. The K562 cell line was induced to erythroid differentiation by hemin. We observed the relationship between its expression and erythroid differentiation. RESULTS: Both leukemia cells and normal marrow cells expressed DLK1. The expression of DLK1 mRNA in patients in the test group was higher than that in the control group (P=0.018), while there was no significance between acute lymphoblastic leukemia and acute myelogenous leukemia (P>0.05).The expression of DLK1 mRNA in the test group at onset had no relation with the WBC and platelet count in the total peripheral blood, and the same was true for blast cell rates in bone marrow cells.The level of DLK1 protein in the test group was higher than that in the control group, which was consistent with the mRNA expression (P=0.042). The expression of DLK1 mRNA decreased gradually with K562 cells towards hemin-induced erythroid differentiation. CONCLUSION DLK1 gene may be involved in leukemia,but the mRNA level of DLK1 has no relation with some clinical characteristics of AL patients at onset. DLK1 may inhibit the erythroid differentiation of K562 cells.
Acute Disease ; Adolescent ; Adult ; Aged ; Calcium-Binding Proteins ; Case-Control Studies ; Cell Differentiation ; drug effects ; genetics ; Cell Transformation, Neoplastic ; genetics ; Child ; Child, Preschool ; Erythroid Cells ; pathology ; Erythroid Precursor Cells ; pathology ; Female ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; K562 Cells ; Leukemia ; genetics ; metabolism ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Young Adult

OBJECTIVE: To compare the efficacy of directional erythroid differentiation in different serum free culture systems and to screen the optimal culture systems for inducing the differentiation of umbilical cord blood hematopoietic stem and progenior cells (HSPC) to erythroid cells. METHODS: The CD34 cells from umbilical blood munonuclear cells were sorted by using the magnetic beads, and were inoculated into 3 different of culture systems (system 1, 2 and 3 respectively), to induce erythrold differentiation by 3 stage culture. The living cells were counted in different differentiation stages and were observed by Wright-Giemsa staining; the expression of CD71 and CD235a on cell surface was detected by flow cytometry, the erythroid differentiation pteency was detected via colony-forming test. RESULTS: The ability of system 2 to promote the HSPC proliferation was the strongest, the efficacy of system 3 to promote the erythroid differentiation of HSPC was the most optimal; the proliferation ability of cells cultured in system 2 for 2-15 days all was higher than that of cells cutured in system 1 and 3 (P＜0.05). The flow cytometry detection showed that the expression of CD71 and CD235a on surface of cells cultured in system 3 was the highest, the CD235a percentage on day 15 of differentiation in system 3 was (92.33±3.89)%, that in system 2 was (84.67±3.12)%, while that in system 1 was (72.17±6.83)% (P＜0.05). Cell morplologic detection showed that throid differentiation was accelerated on day 12, the percentage of orthochromatic erythrocytes in system 3 was (67.67±2.08)% which was 10.69 and 25.34 times higher than that in system 2 and 1 respectively (P＜0.05). The colony-forming test showed the ratio of BFU-E in system 3 increased gradually on day 3-9 (r=0.99, P＜0.05), which was significanlly higher than that in system 2 and 1 on day 9 (90.35±5.52% vs 77.06±2.26% and 74.50±3.95%). CONCLUSION Culture system 3 is the most effective serum-free erythroid differentiation system, and the culture system 2 is the most powerful HSPC proliferation system. This study results provide a technical basis for further efficiently increasing and inducing the erythroid proliferation and differentiation of HSPC, and also provide culture system in vitro for the clinical application and basic research.
Antigens, CD34 ; Cell Differentiation ; Cells, Cultured ; Culture Media, Serum-Free ; Erythroid Precursor Cells ; Fetal Blood ; Humans

The effects of the Smad3- knockout on the hematopoiesis of mouse were investigated in this work. Five pairs of wild type and Smad3- null mice were studied. White blood cell(WBC), red blood cell(RBC) and platelet (PLT) counting of peripheral blood cells were performed with blood obtained from tails. And white blood cells were classified by their morphology. Bone marrow nucleated cells (BMNCs) were counted and classified. The CFU-GM, BFU-E, CFU-GEMM yields were measured in each pair of mice. CFU-S yield of each mouse was measured by injecting bone marrow cells into lethally irradiated 8-10 weeks old wild type female mice. And the pathomorphism of their bone marrows, spleens and livers were observed. As a result, WBC and PLT of Smad3- null mice were significantly higher than those in wild type mice. Smad3- null mice had much more proportion of granulocytes in classification. There wasn't any difference in RBC counting and BFU-E measurement. The yield of CFU-GM increased, while the yields of CFU-GEMM and CFU-S markedly reduced. Bone marrows are actively proliferative, with granulocytosis. The granulocyte/erythrocyte ratio increased. There were no obviously alterative in spleen and liver. Thus Smad3- knockout results in a decreased number of stem and progenitor cells. Moreover hematopoietic differentiation is abnormal with a tendency to forming more granulocytes and platelets. The effect of Smad3 on hematopoiesis is correlative to that of TGF-beta.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Erythrocytes ; cytology ; metabolism ; Erythroid Precursor Cells ; cytology ; metabolism ; Female ; Granulocyte-Macrophage Progenitor Cells ; cytology ; metabolism ; Granulocytes ; cytology ; metabolism ; Hematopoiesis ; genetics ; Mice ; Mice, Knockout ; Myeloid Progenitor Cells ; cytology ; metabolism ; Smad3 Protein ; genetics

Among the causes of pure red cell aplasia, human parvovirus B19 has been shown to be cytotoxic to erythroid progenitor cells in the bone marrow associated with chronic hemolytic anemia with rapidly dividing erythroids and persistently to be suppression of erythropoiesis in immunocompromised individuals related with failure to produce neutralizing antibody to the virus. In a patient with hereditary spherocytosis presenting acute onset of reticulocytopenia during hospitalization, who had shown severe anemia and prodromal symptoms including fever, fatigue and dizziness, infection of parvovirus Bl9 was proven by the presence of IgM and IgG antibodies to parvovirus Bl9, the detection of viral DNA using PCR technique in her serum and the decreased erythroid cells, especially late normoblasts in bone marrow, Also in the other who was diagnosed as hereditary elliptocytosis and complained of fever, headache, abdominal pain and diarrhea, an episode of reticulocytopenia and the nearly absence of late normoblasts in the bone marrow were observed. IgM antibodies to parvovirus Bl9 and the viral DNA were detected in her serum, too.
Abdominal Pain ; Anemia ; Anemia, Hemolytic ; Antibodies ; Antibodies, Neutralizing ; Bone Marrow ; Diarrhea ; Dizziness ; DNA, Viral ; Elliptocytosis, Hereditary* ; Erythroblasts ; Erythroid Cells ; Erythroid Precursor Cells ; Erythropoiesis ; Fatigue ; Fever ; Headache ; Hospitalization ; Humans* ; Immunoglobulin G ; Immunoglobulin M ; Parvovirus B19, Human ; Parvovirus* ; Polymerase Chain Reaction ; Prodromal Symptoms ; Red-Cell Aplasia, Pure