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

OBJECTIVE: To investigate the effect of Rheb1 in the development of mouse megakaryocyte-erythroid progenitor cells and its related mechanism. METHODS: Rheb1 was specifically knocked-out in the hematopoietic system of Vav1-Cre;Rheb1^fl/fl mice(Rheb1^Δ/Δ mice). Flow cytometry was used to detect the percentage of red blood cells in peripheral blood and erythroid cells in bone marrow in Vav1-Cre;Rheb1^fl/fl mice and control mice. The CFC assay was used to detect the differentiation ability of Rheb1 KO megakaryocyte-erythroid progenitor cells and control cells. Real-time fluorescence quantification PCR was used to detect the relative expression of PU.1,GATA-1,GATA-2,CEBPα and CEBPβ of Rheb1 KO megakaryocyte-erythroid progenitor cells and control cells. Rapamycin was added to the culture medium, and it was used to detect the changes in cloning ability of megakaryocyte-erythroid progenitor cells from wild-type mice in vitro. RESULTS: After Rheb1 was knocked out, the development and stress response ability of megakaryocyte-erythroid progenitor cells in mice were weaken and the differentiation ability of megakaryocyte-erythroid progenitor cells in vitro was weaken. Moreover, the expression of GATA-1 of megakaryocyte-erythroid progenitor cells was decreased. Further, rapamycin could inhibit the differentiative capacity of megakaryocyte-erythroid progenitor cells in vitro. CONCLUSION Rheb1 can regulate the development of megakaryocyte-erythroid progenitor cells probably through the mTOR signaling pathway in mice.
Animals ; Cell Differentiation ; Erythrocytes ; Flow Cytometry ; Megakaryocyte-Erythroid Progenitor Cells ; Megakaryocytes ; Mice ; Signal Transduction

Self-renewal and differentiation are hallmarks of stem cells and controlled by various intrinsic and extrinsic factors. Increasing evidence indicates that estrogen (E2), the primary female sex hormone, is involved in regulating the proliferation and lineage commitment of adult and pluripotent stem cells as well as modulating the stem cell niche. Thus, a detailed understanding of the role of E2 in behavior of stem cells may help to improve their therapeutic potential. Recently, it has been reported that E2 promotes cell cycle activity of hematopoietic stem and progenitor cells and induces them to megakaryocyte-erythroid progenitors during pregnancy. This study paves the way towards a previously unexplored endocrine mechanism that controls stem cell behavior. In this review, we will focus on the scientific findings regarding the regulatory effects of E2 on the hematopoietic system including its microenvironment.
Adult ; Cell Cycle ; Estrogens* ; Female ; Hematopoiesis ; Hematopoietic Stem Cells* ; Hematopoietic System ; Humans ; Megakaryocyte-Erythroid Progenitor Cells ; Pluripotent Stem Cells ; Pregnancy ; Stem Cell Niche ; 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

Pure red cell aplasia (PRCA) is a rare disorder, which is characterized by severe anemia associated with reticulocytopenia and absence of erythroid precursor cells in the bone marrow. Recently we experienced a patient with B-cell chronic lymphocytic leukemia associated with PRCA, which was successfully treated with prednisone. A 50-year-old man was admitted because of lymphocytosis. The leukocyte count was 26,800/nL with 82% abnormal mature lymphocytes. Neither anemia nor thrombocytopenia was present. Many abnormal lymphocytes and erythroblasts were seen in the bone marrow. The surface markers of these cells were positive for CD5, CD19, CD20, CD22, and surface immunoglobulin. The patient was diagnosed of B-CLL in Binet-Rai stage A (II), and was treated with chlorambucil. Six months later, severe anemia (hemoglobin 5.9g/dL) with reticulocytopenia developed. In the bone marrow at that time, neutrophils and megakaryocytes besides leukemic cells were preserved, but marked decrease of erythroblasts, a pattern of PRCA was observed. After one-month's observation, we prescribed prednisone. After four weeks of such therapy the patient's hemoglobin reached 10.9g/dL, and he needed no further transfusion.
Anemia ; B-Lymphocytes* ; Bone Marrow ; Chlorambucil ; Erythroblasts ; Erythroid Precursor Cells ; Humans ; Immunoglobulins ; Leukemia, Lymphocytic, Chronic, B-Cell* ; Leukocyte Count ; Lymphocytes ; Lymphocytosis ; Megakaryocytes ; Middle Aged ; Neutrophils ; Prednisone ; Red-Cell Aplasia, Pure* ; Thrombocytopenia

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

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

Liver tissuses obtained from 5 human fetuses between 11 weeks and 23 weeks of gestation during the high activity of hepatic hemopoiesis were observed with transmission electron microscope using continuous series of thin sections. The objective of present study was to evaluate ultrastructures of megakaryopoietic cells, the migration of extravascular megakaryocyte into the sinusoidal lumen and the relevence between a migrated megakaryocyte and a Kupffer cell. Immature megakaryocytes were usually observed between growing hepatic laminae and within hepatic sinusoids. A megakaryoblast contained numerous polyribosomes, rather large mitochondria, short tubular elements of rough endoplasmic reticulum and small granules. Moreover, demarcation tubules and a few small specific granules were observed in immature megakaryocytes. The nucleus was mononuclear but frequently indented. With maturation, the nuclei were multilobulated. In the cytoplasm, in contrast to the decrease in polyribosomes and rough endoplasmic reticulum, the numerous specific granules and well -developed demarcation membrane system were predominant. Thereafter cytoplasmic zonation was observed clearly in maturing and mature megakaryocytes. Some megakaryocytes passed through the sinusoidal lining epithelium and into the hepatic sinusoids. The cell to cell interaction was often found as adhesion between migrated megakaryocyte and Kupffer cell, and erythroblasts within megakaryocyte (emperipolesis). These results suggest that intravascular megakaryopoiesis in addition to extravascular megakaryopoiesis occurs to produce platelet during the human fetal liver.
Blood Platelets ; Cell Communication ; Cytoplasm ; Emperipolesis ; Endoplasmic Reticulum, Rough ; Epithelium ; Erythroblasts ; Fetus* ; Humans* ; Liver ; Megakaryocyte Progenitor Cells ; Megakaryocytes* ; Membranes ; Mitochondria ; Polyribosomes ; Pregnancy ; Thrombopoiesis

Embryonic hematopoiesis in mammals is characterized by successive temporal and spatial changes. Previous investigations indicate that in vitro differentiation of embryonic stem cells (ES cells) derived from 129 mice can mimic embryonic hematopoiesis to some extent. To investigate the in vitro hematopoietic differentiation capacity of ES cells derived from C57BL/6 mice, the authors initially established the murine ES cell line with standard identification methods employed. Next, two-step culture system was utilized for embryoid bodies formation and the appearance of different hematopoietic precursors was confirmed by CFC assay, cellular chemical staining as well as RT-PCR. The results demonstrated that the ES cell line MES-1 fulfilled the criteria of ES cell line and its progeny after in vitro differentiation included primitive and definitive erythrocyte precursors, mixed colony-forming cells and granulocyte/macrophage colony-forming cells. RT-PCR analysis revealed the molecular consistence of transcription factors and hematopoietic markers with cellular event. In conclusion, MES-1 established from C57BL/6 mice was able to differentiate in vitro to a variety of hematopoietic precursors, thus could partly recapitulate embryonic hematopoiesis.
Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; genetics ; Cell Line ; Colony-Forming Units Assay ; DNA-Binding Proteins ; genetics ; Embryo, Mammalian ; cytology ; Erythroblasts ; cytology ; metabolism ; Erythroid Precursor Cells ; cytology ; metabolism ; Erythroid-Specific DNA-Binding Factors ; Gene Expression ; Hematopoietic Stem Cells ; cytology ; metabolism ; Mice ; Mice, Inbred C57BL ; Mice, Inbred Strains ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; cytology ; metabolism ; Time Factors ; Transcription Factors ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; genetics

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