High-level embryonic globin production with efficient erythroid differentiation from a K562 erythroleukemia cell line.

A reliable cell line capable of robust in vitro erythroid differentiation would be useful to investigate red blood cell (RBC) biology and genetic strategies for RBC diseases. K562 cells are widely utilized for erythroid differentiation; however, current differentiation methods are insufficient to analyze globin proteins. In this study, we sought to improve erythroid differentiation from K562 cells to enable protein-level globin analysis. K562 cells were exposed to a variety of reagents, including hemin, rapamycin, imatinib, and/or decitabine (known erythroid inducers), and cultured in a basic culture medium or erythropoietin-based differentiation medium. All single reagents induced observable erythroid differentiation with higher glycophorin A (GPA) expression but were insufficient to produce detectable globin proteins. We then evaluated various combinations of these reagents and developed a method incorporating imatinib preexposure and an erythropoietin-based differentiation culture containing both rapamycin and decitabine capable of efficient erythroid differentiation, high-level GPA expression (>90%), and high-level globin production at protein levels detectable by hemoglobin electrophoresis and high performance liquid chromatography. In addition, ß-globin gene transfer resulted in detectable adult hemoglobin. In summary, we developed an in vitro K562 erythroid differentiation model with high-level globin production. This model provides a practical evaluation tool for hemoglobin production in human erythroid cells.

Selective amine labeling of cell surface proteins guided by coiled-coil assembly.

Covalent labeling of target proteins in living cells is useful for both fluorescence live-cell imaging and the subsequent biochemical analyses of the proteins. Here, we report an efficient method for the amine labeling of membrane proteins on the cell surface, guided by a noncovalent coiled-coil interaction. A carboxyl sulfosuccinimidyl ester introduced at the C-terminus of the coiled-coil probe reacted with target proteins under mild labeling conditions ([probe] = 150 nM, pH 7.4, 25°C) for 20 min. Various fluorescent moieties with different hydrophobicities are available for covalent labeling with high signal/background labeling ratios. Using this method, oligomeric states of glycophorin A (GpA) were compared in mammalian CHO-K1 cells and sodium dodecyl sulfate (SDS) micelles. In the cell membranes, no significant self-association of GpA was detected, whereas SDS-PAGE suggested partial dimerization of the proteins. Membrane cholesterol was found to be an important factor that suppressed the dimerization of GpA. Thus, the covalent functionality enables direct comparison of the oligomeric state of membrane proteins under various conditions. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 484-490, 2016.

CD34 and CD49f Double-Positive and Lineage Marker-Negative Cells Isolated from Human Myometrium Exhibit Stem Cell-Like Properties Involved in Pregnancy-Induced Uterine Remodeling.

Repeated and dramatic pregnancy-induced uterine enlargement and remodeling throughout reproductive life suggests the existence of uterine smooth muscle stem/progenitor cells. The aim of this study was to isolate and characterize stem/progenitor-like cells from human myometrium through identification of specific surface markers. We here identify CD49f and CD34 as markers to permit selection of the stem/progenitor cell-like population from human myometrium and show that human CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells exhibit stem cell-like properties. These include side population phenotypes, an undifferentiated status, high colony-forming ability, multilineage differentiation into smooth muscle cells, osteoblasts, adipocytes, and chondrocytes, and in vivo myometrial tissue reconstitution following xenotransplantation. Furthermore, CD45(-) CD31(-) glycophorin A(-) and CD49f(+) CD34(+) myometrial cells proliferate under hypoxic conditions in vitro and, compared with the untreated nonpregnant myometrium, show greater expansion in the estrogen-treated nonpregnant myometrium and further in the pregnant myometrium in mice upon xenotransplantation. These results suggest that the newly identified myometrial stem/progenitor-like cells influenced by hypoxia and sex steroids may participate in pregnancy-induced uterine enlargement and remodeling, providing novel insights into human myometrial physiology.

Erythroid and megakaryocytic differentiation of K562 erythroleukemic cells by monochloramine.

The induction of leukemic cell differentiation is a hopeful therapeutic modality. We studied the effects of monochloramine (NH2Cl) on erythroleukemic K562 cell differentiation, and compared the effects observed with those of U0126 and staurosporine, which are known inducers of erythroid and megakaryocytic differentiation, respectively. CD235 (glycophorin) expression, a marker of erythroid differentiation, was significantly increased by NH2Cl and U0126, along with an increase in cd235 mRNA levels. Other erythroid markers such as γ-globin and CD71 (transferrin receptor) were also increased by NH2Cl and U0126. In contrast, CD61 (integrin ß3) and CD42b (GP1bα) expression, markers of megakaryocytic differentiation, was increased by staurosporine, but did not change significantly by NH2Cl and U0126. NH2Cl retarded cell proliferation without a marked loss of viability. When ERK phosphorylation (T202/Y204) and CD235 expression were compared using various chemicals, a strong negative correlation was observed (r = -0.76). Paradoxically, NH2Cl and staurosporine, but not U0126, induced large cells with multiple or lobulated nuclei, which was characteristic to megakaryocytes. NH2Cl increased the mRNA levels of gata1 and scl, decreased that of gata2, and did not change those of pu.1 and klf1. The changes observed in mRNA expression were different from those of U0126 or staurosporine. These results suggest that NH2Cl induces the bidirectional differentiation of K562. Oxidative stress may be effective in inducing leukemic cell differentiation.

In vitro large scale production of human mature red blood cells from hematopoietic stem cells by coculturing with human fetal liver stromal cells.

In vitro models of human erythropoiesis are useful in studying the mechanisms of erythroid differentiation in normal and pathological conditions. Here we describe an erythroid liquid culture system starting from cord blood derived hematopoietic stem cells (HSCs). HSCs were cultured for more than 50 days in erythroid differentiation conditions and resulted in a more than 10(9)-fold expansion within 50 days under optimal conditions. Homogeneous erythroid cells were characterized by cell morphology, flow cytometry, and hematopoietic colony assays. Furthermore, terminal erythroid maturation was improved by cosculturing with human fetal liver stromal cells. Cocultured erythroid cells underwent multiple maturation events, including decrease in size, increase in glycophorin A expression, and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to 80% of the cells. Importantly, they possessed the capacity to express the adult definitive ß -globin chain upon further maturation. We also show that the oxygen equilibrium curves of the cord blood-differentiated red blood cells (RBCs) are comparable to normal RBCs. The large number and purity of erythroid cells and RBCs produced from cord blood make this method useful for fundamental research in erythroid development, and they also provide a basis for future production of available RBCs for transfusion.

Low expression of stem cell antigen-1 on mouse haematopoietic precursors is associated with erythroid differentiation.

Sca1 is a surface marker of haematopoietic stem cell but its role in erythropoiesis is still largely unknown. In this work we evaluated the ability of Sca1⁺ cells to differentiate into cells of the erythrocytic lineage. We performed FACS analysis of complete and purified Sca1⁺ bone marrow cells from C3H/HeNHsd mice and measured the expression of CD71 and Terr119 to evaluate the stages in erythroid development. Definitive erythropoiesis was evident within the complete bone marrow, while only proerythroblasts were found in Sca1⁺ cells, suggesting that Sca1 is a negative regulator of erythropoiesis. We also used FDCP-mix cells and their PU.1 and SCL transfectants. The PU.1 transfectant showed significantly increased expression of Sca1 and was not induced to differentiate into red blood cells, while the SCL transfectant showed significantly lower expression of Sca1 and produced red blood cells. The results of this study suggest that increased Sca1 expression on erythropoietic precursors inhibits erythroid differentiation.

Expression of the Rh/RhAG complex is reduced in Mi.III erythrocytes.

BACKGROUND AND OBJECTIVES: Miltenberger blood group antigen subtype III (Mi.III) is characterized by expression of a glycophorin B-A-B hybrid (Gp.Mur) on the erythrocyte surface. The two alleles of glycophorin B are substituted with the B-A-B hybrid alleles in homozygous Mi.III (Mi.III(+/+)), and thus, Mi.III(+/+) erythrocytes lack glycophorin B (GPB) and express Gp.Mur only. Because GPB is a major component of the Rh complex on RBCs, in this study, we explored how the absence of GPB might affect Rh expression in Mi.III RBCs. MATERIALS AND METHODS: (1) Mi.III+ RBCs were serologically identified and further differentiated their homozygosity or heterozygosity by immunoblot or direct sequencing. (2) RhD and RhCcEe mRNA was cloned, and their sequences analysed. (3) The expression levels of Rh antigen, Rh-associated glycoprotein (RhAG) and the U antigen in MI.III vs. non-Mi.III RBCs were assessed by flow cytometry and Western blot. RESULTS: Compared with the non-Mi.III samples, the surface expression of the Rh antigen was reduced to 76·4% in Mi.III(+/+) RBCs and 93·6% in Mi.III(+/-). RhAG expression was also significantly reduced in Mi.III(+/+), but not in Mi.III(+/-). The U antigen expression in Mi.III(+/-) was only 14·9% relative to the control RBCs, while GPB was half the level of the controls. The mRNA sequences of Rh polypeptides from Mi.III+ samples were identical to the NCBI reference sequences. CONCLUSION: Substitution of GPB with Gp.Mur significantly reduced the expression of Rh antigen and RhAG on the Mi.III(+/+) erythrocyte membrane. The Mi.III phenotype is predicted to induce considerable structural variations within the band 3/Rh-associated macrocomplexes.

Apigetrin induces erythroid differentiation of human leukemia cells K562: proteomics approach.

SCOPE: Induction of cancer-cell differentiation is an alternative approach for cancer chemotherapy. There are numerous studies that diets containing an abundance of fruits and vegetables have protection against cancers, and the main agents thought to provide such protective effect are flavonoids. In this study we used apigetrin as a possible cell differentiation inducer and chronic leukemia cells K562 for their pluripotent differentiating potency. METHODS AND RESULTS: Prolonged treatment with 75 µM apigetrin induced erythroid differentiation of K562 cells with specific marker glycophorin A expression and fetal hemoglobin synthesis in treated cells, which was accompanied with G(2) /M arrest. Proteomics data revealed the downregulation of several proteins expression involved in cell cycle regulation, protein synthesis and nuclear import and export of signaling molecules. CONCLUSION: This is the first evidence that natural compound apigetrin may induce cancer cell differentiation thus could be one of the possible explanations of its antitumor effects.

Brief report: ectopic expression of NUP98-HOXA10 augments erythroid differentiation of human embryonic stem cells.

Hox genes encode highly conserved transcription factors that have been implicated in hematopoietic development and self-renewal of hematopoietic stem cells (HSCs) and hematopoietic development. The potency of NUP98-HOXA10hd (NA10) on adult murine bone marrow HSC self-renewal prompted us to examine its effect on specification and proliferation of hematopoietic cells derived from human embryonic stem cells (hESCs). Here, we demonstrate that expression of NA10 in hESCs influences the hematopoietic differentiation program. The specific effect of NA10 is dependent on the developmental stage of hematopoietic emergence from hESCs. Overexpression of NA10 in either undifferentiated hESCs or early hemogenic precursors augmented the frequency of CD45(-) GlycophorinA(+) cells and erythroid progenitors (blast-forming unit-erythrocyte). In contrast, targeted NA10 expression in primitive CD34+ cells committed to the hematopoietic lineage had no effect on erythropoietic capacity but instead increased hematopoietic progenitor proliferation. Our study reveals a novel neomorphic effect of NA10 in early human erythroid development from pluripotent stem cells.

Functional characterization and modified rescue of novel AE1 mutation R730C associated with overhydrated cation leak stomatocytosis.

We report the novel, heterozygous AE1 mutation R730C associated with dominant, overhydrated, cation leak stomatocytosis and well-compensated anemia. Parallel elevations of red blood cell cation leak and ouabain-sensitive Na(+) efflux (pump activity) were apparently unaccompanied by increased erythroid cation channel-like activity, and defined ouabain-insensitive Na(+) efflux pathways of nystatin-treated cells were reduced. Epitope-tagged AE1 R730C at the Xenopus laevis oocyte surface exhibited severely reduced Cl(-) transport insensitive to rescue by glycophorin A (GPA) coexpression or by methanethiosulfonate (MTS) treatment. AE1 mutant R730K preserved Cl(-) transport activity, but R730 substitution with I, E, or H inactivated Cl(-) transport. AE1 R730C expression substantially increased endogenous oocyte Na(+)-K(+)-ATPase-mediated (86)Rb(+) influx, but ouabain-insensitive flux was minimally increased and GPA-insensitive. The reduced AE1 R730C-mediated sulfate influx did not exhibit the wild-type pattern of stimulation by acidic extracellular pH (pH(o)) and, unexpectedly, was partially rescued by exposure to sodium 2-sulfonatoethyl methanethiosulfonate (MTSES) but not to 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) or 2-(trimethylammonium)ethyl methanethiosulfonate bromide (MTSET). AE1 R730E correspondingly exhibited acid pH(o)-stimulated sulfate uptake at rates exceeding those of wild-type AE1 and AE1 R730K, whereas mutants R730I and R730H were inactive and pH(o) insensitive. MTSES-treated oocytes expressing AE1 R730C and untreated oocytes expressing AE1 R730E also exhibited unprecedented stimulation of Cl(-) influx by acid pH(o). Thus recombinant cation-leak stomatocytosis mutant AE1 R730C exhibits severely reduced anion transport unaccompanied by increased Rb(+) and Li(+) influxes. Selective rescue of acid pH(o)-stimulated sulfate uptake and conferral of acid pH(o)-stimulated Cl(-) influx, by AE1 R730E and MTSES-treated R730C, define residue R730 as critical to selectivity and regulation of anion transport by AE1.

Erythroid progenitor cells expanded from peripheral blood without mobilization or preselection: molecular characteristics and functional competence.

BACKGROUND: Continued development of in-vitro procedures for expansion and differentiation of erythroid progenitor cells (EPC) is essential not only in hematology and stem cell research but also virology, in light of the strict erythrotropism of the clinically important human parvovirus B19. METHODOLOGY/PRINCIPAL FINDINGS: We cultured EPC directly from ordinary blood samples, without ex vivo stem cell mobilization or CD34+ cell in vitro preselection. Profound increase in the absolute cell number and clustering activity were observed during culture. The cells obtained expressed the EPC marker combination CD36, CD71 and glycophorin, but none of the lymphocyte, monocyte or NK markers. The functionality of the generated EPC was examined by an in vitro infection assay with human parvovirus B19, tropic for BFU-E and CFU-E cells. Following infection (i) viral DNA replication and mRNA production were confirmed by quantitative PCR, and (ii) structural and nonstructural proteins were expressed in >50% of the cells. As the overall cell number increased 100-200 fold, and the proportion of competent EPC (CD34+ to CD36+) rose from <0.5% to >50%, the in vitro culture procedure generated the EPC at an efficiency of >10,000-fold. Comparative culturing of unselected PBMC and ex vivo-preselected CD34+ cells produced qualitatively and quantitatively similar yields of EPC. CONCLUSIONS/SIGNIFICANCE: This approach yielding EPC directly from unmanipulated peripheral blood is gratifyingly robust and will facilitate the study of myeloid infectious agents such as the B19 virus, as well as the examination of erythropoiesis and its cellular and molecular mechanisms.

Dynamics of alpha-globin locus chromatin structure and gene expression during erythroid differentiation of human CD34(+) cells in culture.

OBJECTIVE: The aim of the present study has been to establish serum-free culture conditions for ex vivo expansion and differentiation of human CD34(+) cells into erythroid lineage and to study the chromatin structure, gene expression, and transcription factor recruitment at the alpha-globin locus in the developing erythron. MATERIALS AND METHODS: A basal Iscove's modified Dulbecco's medium cell culture medium with 1% bovine serum albumin as a serum replacement and a combination of cytokines and growth factors was used for expansion and differentiation of the CD34(+) cells. Expression patterns of the alpha- and beta-like genes at various stages of erythropoiesis was studied by reverse transcriptase quantitative polymerase chain reaction analysis, profile of key erythroid transcription factors was investigated by Western blotting, and the chromatin structure and transcription factor recruitment at the alpha-globin locus was investigated by chromatin immunoprecipitation quantitative polymerase chain reaction analysis. RESULTS: Human CD34(+) cells in the serum-free medium undergo near synchronous erythroid differentiation to yield large amount of cells at different differentiation stages. We observe distinct patterns of the histone modifications and transcription factor binding at the alpha-globin locus during erythroid differentiation of CD34(+) cells. Nuclear factor erythroid-derived 2 (NF-E2) was present at upstream activator sites even before addition of erythropoietin (EPO), while bound GATA-1 was only detectable after EPO treatment. After 7 days of EPO treatment, H3K4Me2 modification uniformly increases throughout the alpha-globin locus. Acetylation at H3K9 and binding of Pol II, NF-E2, and GATA-1 were restricted to certain hypersensitive sites of the enhancer and theta gene, and were conspicuously low at the alpha-like globin promoters. Rearrangement of the insulator binding factor CTCF took place at and around the alpha-globin locus as CD34(+) cells differentiated into erythroid pathway. CONCLUSION: Our results indicate that remodeling of the upstream elements may be the primary event in activation of alpha-globin gene expression. Activation of alpha-globin genes upon EPO treatment involves initial binding of Pol II, downregulation of pre-existing factors like NF-E2, removal of CTCF from the locus, then rebinding of CTCF in an altered pattern, and concurrent or subsequent binding of transcription factors like GATA-1.

The role of zinc finger protein 521/early hematopoietic zinc finger protein in erythroid cell differentiation.

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

Hypoxia alters progression of the erythroid program.

Hypoxia can induce erythropoiesis through regulated increase of erythropoietin (Epo) production. We investigated the direct influence of oxygen tension (pO(2)) in the physiologic range (2-8%) on erythroid progenitor cell differentiation using cultures of adult human hematopoietic progenitor cells exposed to decreasing (20% to 2%) pO(2) and independent of variation in Epo levels. Decreases in hemoglobin (Hb)-containing cells were observed at the end of the culture period with decreasing pO(2). This is due, in part, to a reduction in cell growth and, at 2% O(2), a marked increase in cell toxicity. Analysis of the kinetics of cell differentiation showed an increase in the proportion of cells with glycophorin-A expression and Hb accumulation at physiologic pO(2). Cells were characterized by an early induction of gamma-globin expression and a delay and reduction in peak levels of beta-globin expression. Overall, fetal Hb and gamma-globin expression were increased at physiologic pO(2), but these increases were reduced at 2% O(2) as cultures become cytotoxic. At reduced pO(2), induction of Epo-receptor (Epo-R) by Epo was decreased and delayed, analogous to the delay in beta-globin induction. The oxygen-dependent reduction of Epo-R can account for the associated cytotoxicity at 2% O(2). Epo induction of erythroid transcription factors, EKLF, GATA-1, and SCL/Tal-1, was also delayed and decreased at reduced pO(2), consistent with lower levels of Epo-R and resultant Epo signaling. These changes in Epo-R and globin gene expression raise the possibility that the early increase of gamma-globin is a consequence of reduced Epo signaling and a delay in induction of erythroid transcription factors.

[Glycophorins of human erythrocytes as receptors for the malaria parasite Plasmodium falciparum]. / Glikoforyny ludzkich erytrocytów jako receptory dla zarodzca sierpowego malarii (Plasmodium falciparum).

Malaria causes an estimated 300-500 million clinical cases in sub-Saharan Africa and Indochina. The most severe form of malaria is caused by Plasmodium falciparum, a parasite responsible for the death of 2 million children annually. Understanding the molecular basis of the parasite's invasion process is important for the development of new drugs and vaccines. Invasion of erythrocytes by the malaria parasite is a multistep process involving several specific interactions between the parasite's ligands and receptors on red blood cells. It was shown that glycophorins A, B, and C, sialoglycoproteins of human erythrocytes, act as receptors for Plasmodium falciparum ligands of the DBL family: EBA-175 and EBA-140 antigens. The binding specificity of EBA-175 is determined by the presence of sialic acid residues of the O-linked oligosaccharide chain clusters of glycphorin A and the amino-acid sequence, which contribute to their proper conformation. Glycophorin B, the next in terms of amount, can take on the role of glycophorin A as the receptor, but the glycophorin B- and sialic acid-dependent invasion of erythrocytes by Plasmodium falciparum involves a different parasite ligand. The third, and minor, glycophorin C appears to be the receptor for the antigen BAEBL, a paralogue of EBA-175. The binding of BAEBL to glycophorin C is dependent on the sialic acid residues of the O- and N-linked oligosaccharide chains and a peptide as well. It seems that the correct receptor site on glycophorin C needs to be elucidated in detail.

Delta-4 Notch ligand promotes erythroid differentiation of human umbilical cord blood CD34+ cells.

OBJECTIVE: Important roles of Notch signaling have been demonstrated in hematopoiesis. In many cases, activation of the Notch pathway leads to the inhibition of differentiation of immature precursors, suggesting a potential role in self-renewal promotion. However, the function of Notch and Notch ligands is not so straightforward because it is considerably dependent on cytokine context. In this study, we analyzed effects of one Notch ligand, Delta-4, whose function is less clear than others, such as Delta-1 and Jagged-1 and -2. METHODS: CD34(+) cells isolated from human umbilical cord blood were cocultured with a Delta-4-expressing murine stromal cell line, SC9-19, and induced to erythroid differentiation by adding stem cell factor and erythropoietin. To examine the involvement of Delta-4, we utilized stromal cell subclones expressing Delta-4 protein at higher or lower level than parental SC9-19 by plasmid transfection. Erythroid maturation was examined by surface phenotype (CD34 and glycophorin A) and cytospin morphology. Recombinant human Delta-4 protein was prepared to analyze direct effects of Delta-4. RESULTS: Under erythroid lineage-inducing conditions, we found that the increase in Delta-4 expression of SC9-19 promoted erythroid differentiation whereas the decrease in Delta-4 expression inhibited it. Morphologic examination as well as colony formation analysis supported this observation. Moreover, the experiment using recombinant Delta-4 provided direct evidence of the Delta-4 activity found in coculture system. CONCLUSIONS: By modifying Delta-4 expression of the stromal cells and using the recombinant protein, we demonstrated that Delta-4 had a differentiation promoting activity for human primitive hematopoietic cells into erythroid lineage.

In vitro dual effect of arsenic trioxide on hemopoiesis: inhibition of erythropoiesis and stimulation of megakaryocytic maturation.

Although the arsenic compounds are now widely utilized in clinics in the treatment of various tumors, their effects on normal hematopoiesis do not seem to have been explored. In the present study, we provide evidence that arsenic trioxide (As(2)O(3)) exerts in vitro a potent inhibitory effect on normal erythropoiesis and a stimulatory action on megakaryocytic differentiation. The effect of As(2)O(3) on erythroid and megakaryocytic differentiation was evaluated on both erythroleukemic cell lines K562 and HEL and on normal hemopoietic progenitor cells (HPCs) induced to selective erythroid or megakaryocytic differentiation. The inhibitory effect of As(2)O(3) on erythropoiesis is related to: (a) the inhibition of Stat5 activation with consequent reduced expression of the target genes Bcl-X(L) and glycophorin-A; (b) the activation of an apoptotic mechanism that leads to the cleavage of the erythroid transcription factors Tal-1 and GATA-1, whose integrity is required for erythroid cell survival and differentiation; (c) the reduced expression of heat shock protein 70, required for GATA-1 integrity. The stimulatory effect of As(2)O(3) on normal megakaryocytopoiesis is seemingly related to upmodulation of GATA-2 expression and to stimulation of MAPK activity. These observations may have implications for the patients undergoing anti-leukemic treatment with this compound.

Identification and differentiation of hepatic stem cells during liver development.

Stem cells responsible for maintenance and repair of tissues are found in a number of organs. The liver's remarkable capacity to regenerate after hepatectomy or chemical-induced injury does not involve proliferation of stem cells. However, recent studies suggest that liver stem cells exist in both embryonic and adult livers. Using fluorescence-activated cell sorting and a culture system in which primitive hepatic progenitor cells form colonies, a novel class of cells with the marker profile c-Met(+)CD49f(+/low)c-Kit(-)CD45(-)TER119(-) was found in the developing liver. This class apparently represents the population of cells that form colonies containing distinct hepatocytes and cholangiocytes. When cells in this class are transplanted into the spleen or liver of mice subjected to liver injury, the cells migrate and differentiate into liver parenchymal cells and cholangiocytes that are morphologically and functionally indistinguishable from their native counterparts. During mid-gestation, hematopoietic cells migrate into the liver from a region bounded by aorta, gonad, and mesonephros and produce oncostatin M (OSM). In combination with glucocorticoid hormones, OSM induces maturation of liver stem and progenitor cells, including those of the c-Met(+)CD49f(+/low)c-Kit(-)CD45(-)TER119(-) class. The ability to manipulate the proliferation and differentiation of liver stem cells in vitro will greatly aid in analyzing mechanisms of liver development and offers promise in stem cell therapy of liver diseases.

Smenospongine, a spongean sesquiterpene aminoquinone, induces erythroid differentiation in K562 cells.

The differentiation of K562 chronic myelogenous leukemia (CML) cells by smenospongine, which is a sesquiterpene aminoquinone isolated from a marine sponge, was examined. Smenospongine increased hemoglobin production in K562 cells at concentrations of 3-15 microM. In addition, flow cytometric analysis of smenospongine-treated K562 cells with FITC-labeled glycophorin A antibody showed an increase of glycophorin A expression, a marker for erythroid differentiation. Cell-cycle analysis showed G1 arrest in K562 cells after treatment with smenospongine for 24 h. The effect on expression of CIP/KIP family cyclin-dependent kinase inhibitors was investigated by Western blotting analysis and the result showed increased expression of p21, which is known to play an important role in differentiation. Furthermore, smenospongine was also found to inhibit the phosphorylation of Crkl, a substrate of Bcr-Abl tyrosine kinase, which is known as a causative protein of CML. In conclusion, our investigation indicated that smenospongine induced the differentiation of K562 cells into erythroblasts along with cell-cycle arrest at G1 phase and the mechanism might be attributed to the increased expression of p21.