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

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

OBJECTIVETo explore the differentiation-inducing potentiality of Pulsatilla saponin A on K562 cells.

METHODSPulsatilla saponin A of different concentrations was used to treat K562 cells; the benzidine staining and the hemoglobinometry were applied to measure the change of hemoglobin content; the flow cytometry (FCM) was used to detect the expression of CD71 and GPA on K562 cells.

RESULTSK562 cells treated with 4 µg/ml pulsatilla saponin A differentiated into the erythroid lineage. With the treatment of pulsatilla saponin A, the hemoglobin content in K562 cells increased significantly; CD71 and GPA expression on the K562 cell surface were up-regulated.

CONCLUSIONPulsatilla saponin A can induce K562 cells to differentiate into erythroid lineage.

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

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

Most protocols for in vitro producing red blood cells (RBC) use the CD34(+) cells or embryonic stem cells from cord blood, bone marrow or peripheral blood as the start materials. This study was purposed to produce the mature RBC in vitro by using peripheral blood mononuclear cells as start material. The peripheral blood mononuclear cells (PBMNC) were isolated from buffy coat after blood leukapheresis, the mature red blood cells (RBC) were prepared by a 4-step culture protocol. The results showed that after culture by inducing with the different sets of cytokines and supporting by mouse MS-5 cell line, the expansion of PBMNC reached about 1000 folds at the end of the culture. About 90% of cultured RBC were enucleated mature cells which had the comparable morphological characteristics with normal RBC. Colony-forming assays showed that this culture system could stimulate the proliferation of progenitors in PBMNC and differentiate into erythroid cells. The structure and function analysis indicated that the mean cell volume of in vitro cultured RBC was 118 ± 4 fl, which was slight larger than that of normal RBC (80-100 fl); the mean cell hemoglobin was 36 ± 1.2 pg, which was slight higher than that of normal RBC (27-31 pg); the maximal deformation index was 0.46, which approachs level of normal RBC; the glucose-6-phosphate dehydrogenase and pyrurvate kinase levels was consistant with young RBC. It is concluded that PBMNC are feasble, convenient and low-cost source for producing cultured RBC and this culture system is suitable to generate the RBC from PBMNC.
Animals ; Bone Marrow ; Cell Differentiation ; Cell Line ; Cytokines ; Erythrocytes ; cytology ; Erythroid Cells ; Leukocytes, Mononuclear ; cytology ; Mice

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

For studies on the developmental stages of migrating erythroid cells and the development of sinusoid, transmission and scanning electron microscopic observations were undertaken on rat fetal liver in 13, 15, 17, 19, 21 days of gestation. The results obtained were as follows. 1. The hepatic sinusoidal endothelium were mainly non-fenestrated cell and fenestrated cell with diaphragm before 17 days of gestation, but fenestrated cells without diaphragm began to appear after 17 days of gestation. Two types of fenestrae were observed, free and clustered fenestrae, but both types were not involved in migration of erythroid cells. 2. Endothelial cell was continuous with neighboring cells by intercellular junctions between lateral cytoplasmic processes with zonula adherens, and between perinuclear cytoplasms with macula adherens. 3. After 13 days of gestation, Kupffer cells showed as matured cell morphology of irregular shape with long cytoplasmic processes into hepatic cord and perisinusoidal space. 4. Migrating erythroid cells in rat fetal liver sinusoid were mainly consisted of immature erythroblasts from proerythroblast to acidophilic erythroblast. The migration occurred through the migrating pores formed on the various sites of the endothelial cytoplasm into the hepatic sinusoidal lumen with no relation to the maturation stages of erythroblast and endothelial cell. In summary, the migration of erythroid cells in the sinusoid of rat fetal liver occurred through the invasion and migration pores transiently formed at various sites of endothelial cytoplasm, and migrating erythroid cells were mainly nucleated immature types.
Adherens Junctions ; Animals ; Cytoplasm ; Diaphragm ; Emigration and Immigration ; Endothelial Cells ; Endothelium ; Erythroblasts ; Erythroid Cells* ; Intercellular Junctions ; Kupffer Cells ; Liver* ; Pregnancy ; Rats*

OBJECTIVETo construct the ovexpression lentivirus vector of PPP2Cβ, the catalytic subunit of protein phosphatase 2A, so as to obtain high-titer packaged lentivirus particles, and to examine the effect of PPP2Cβ on the erythroid differentiation Methods: The CDS of PPP2Cβ was cloned into the second generation of lentivirus vector FUGW, which should be used to co-transfect HEK 293T cells with the lentiviral expression vector and packaging vectors including pMD2G and pSPAX2. Lentiviruses were harvested at 36 and 48 hours after transfection. Titers of viral stock were determined by using flow cytometric analysis. The Western blot was performed to detect the expression level of PPP2Cβ in K562 cells transinfected with the lentiviruses. Benzidine staining and real-time PCR analysis were used to assess the erythroid differentiation of K562 cells.

RESULTSThe PPP2Cβ overexpressing lentivirus vectors were constructed, the high-titer lentiviral particles were obtained, and then the PPP2Cβ overexpression K562 cell line was established and promote erythroid differentiation of K562 cells.

CONCLUSIONThis study suggests that overexpression PPP2Cβ can promote K562 cell erythroid differentiation.

Myelodysplastic syndrome (MDS) is a heterogeneous disease characterized by dysplasia and ineffective hematopoiesis. The dysplasia is crucial in the diagnosis of MDS, but the morphologic abnormalities of bone marrow cells are not specific for MDS. When the morphological evaluation of marrow dysplasia and cytogenetics can not give enough informations, for diagnosis of MDS, the application of flow cytometry (FCM) for immunophenotyping in MDS will become particularly important. Multiparametric evaluation of myeloid, monocytic maturation and antigen expression pattern contribute to the identification of two or more aberrancies in MDS cases. FCM evaluation of erythroid dysplasia is particularly difficult, because of the limited availability of specific markers. By analyzing the proteins involved in cellular iron metabolism, MDS erythroid cells present an "iron-loaded" phenotype characterized by increased ferritin contents and reduced transferrin receptor, which reflects the degree of dysplasia assessed by morphology. The proportion of CD34(+) cells increased, abnormal expression of surface antigen is also important. The application of flow cytometry in detecting dysplasia of myelodysplastic syndrome is discussed in this article.
Bone Marrow Cells ; pathology ; Erythroid Cells ; metabolism ; Flow Cytometry ; Humans ; Myelodysplastic Syndromes ; blood ; diagnosis ; pathology ; Receptors, Transferrin ; metabolism