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1.
J Biomol Tech ; 22(3): 90-4, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21966256

ABSTRACT

MALDI-TOF mass spectrometry is used here to differentiate different glycoisoforms of normal and variant hemoglobins (Hbs) in nonenzymatic in vitro glycation. Single, double, and/or multiple glycation of the α-globin, ß-globin, and/or γ-globin is observed. Different glycation rates are observed for various Hbs, and the normal Hb A has the slowest rate. Although the Hb A is relatively stable upon condensation with glucose at 37°C, the variants Hb C, Hb E, Hb F, Hb Leiden, and Hb San Diego are less stable. In addition, data reveal that the number of glucose attached/Hb molecule (state of glycation) increases with longer incubation time, higher glucose concentration, and higher temperature. The pH dependence of the state of glycation is more complex and varies for different Hbs. Although pH has little effect on the state of glycation for Hb C, Hb E, and Hb Leiden, it increases for Hb A and Hb F upon changing the pH of the solution from phosphate buffer saline (pH 7.4) to carbonate buffer (pH 10). Results obtained in this study could lead to the inference that the linkage of Hbs with glucose occurs in diabetic conditions in vivo (37°C, ∼neutral pH, ∼0.007 M glucose), and the state of glycation is more severe in the individuals who carry abnormal Hbs.


Subject(s)
Hemoglobins/chemistry , Diabetes Mellitus/blood , Glucose/chemistry , Glycosylation , Hemoglobins/isolation & purification , Humans , Hydrogen-Ion Concentration , Molecular Weight , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
2.
Hemoglobin ; 34(2): 145-50, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20353349

ABSTRACT

Under culture conditions that promote hematopoietic differentiation, human embryonic stem cells (huESC) give rise to primitive erythroid cells that closely resemble the nucleated erythrocytes of early-stage human embryos. The globin chain distribution of these cells is similar to that seen during the embryonic and fetal stages of development. Here we show that huESC-derived erythroid cells produce substantial quantities of homotetrameric hemoglobin (Hb) composed exclusively of gamma-globin-containing subunits. The globin synthesis of these erythroid cells was also significantly unbalanced, with a substantial decrease of alpha-like globin chain synthesis in relation to that of their beta-like globins, a pattern characteristically associated with alpha-thalassemia (alpha-thal). This pattern of unbalanced globin synthesis appears to be an inherent feature of human erythroid cells that synthesize predominantly embryonic-stage globins.


Subject(s)
Embryonic Stem Cells/cytology , Erythroblasts/metabolism , Erythropoiesis/genetics , Gene Expression Regulation, Developmental , alpha-Globins/biosynthesis , alpha-Thalassemia/genetics , beta-Globins/biosynthesis , gamma-Globins/biosynthesis , Cells, Cultured/cytology , Cells, Cultured/metabolism , Hemoglobins, Abnormal/biosynthesis , Hemoglobins, Abnormal/genetics , Humans , alpha-Globins/genetics , beta-Globins/genetics , gamma-Globins/genetics , zeta-Globins/biosynthesis , zeta-Globins/genetics
3.
Stem Cells ; 28(4): 704-12, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20155819

ABSTRACT

Human induced pluripotent stem cells (hiPSC) have been shown to differentiate into a variety of replacement cell types. Detailed evaluation and comparison with their human embryonic stem cell (hESC) counterparts is critical for assessment of their therapeutic potential. Using established methods, we demonstrate here that hiPSCs are capable of generating hemangioblasts/blast cells (BCs), endothelial cells, and hematopoietic cells with phenotypic and morphologic characteristics similar to those derived from hESCs, but with a dramatic decreased efficiency. Furthermore, in distinct contrast with the hESC derivatives, functional differences were observed in BCs derived from hiPSCs, including significantly increased apoptosis, severely limited growth and expansion capability, and a substantially decreased hematopoietic colony-forming capability. After further differentiation into erythroid cells, >1,000-fold difference in expansion capability was observed in hiPSC-BCs versus hESC-BCs. Although endothelial cells derived from hiPSCs were capable of taking up acetylated low-density lipoprotein and forming capillary-vascular-like structures on Matrigel, these cells also demonstrated early cellular senescence (most of the endothelial cells senesced after one passage). Similarly, retinal pigmented epithelium cells derived from hiPSCs began senescing in the first passage. Before clinical application, it will be necessary to determine the cause and extent of such abnormalities and whether they also occur in hiPSCs generated using different reprogramming methods.


Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Cellular Senescence , Hemangioblasts/cytology , Induced Pluripotent Stem Cells/cytology , Cell Line , Cell Proliferation , Humans , Phenotype , Time Factors
4.
Blood ; 112(12): 4475-84, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-18713948

ABSTRACT

Human erythropoiesis is a complex multistep process that involves the differentiation of early erythroid progenitors to mature erythrocytes. Here we show that it is feasible to differentiate and mature human embryonic stem cells (hESCs) into functional oxygen-carrying erythrocytes on a large scale (10(10)-10(11) cells/6-well plate hESCs). We also show for the first time that the oxygen equilibrium curves of the hESC-derived cells are comparable with normal red blood cells and respond to changes in pH and 2,3-diphosphoglyerate. Although these cells mainly expressed fetal and embryonic globins, they also possessed the capacity to express the adult beta-globin chain on further maturation in vitro. Polymerase chain reaction and globin chain specific immunofluorescent analysis showed that the cells increased expression of beta-globin (from 0% to > 16%) after in vitro culture. Importantly, the cells underwent multiple maturation events, including a progressive decrease in size, increase in glycophorin A expression, and chromatin and nuclear condensation. This process resulted in extrusion of the pycnotic nuclei in up to more than 60% of the cells generating red blood cells with a diameter of approximately 6 to 8 mum. The results show that it is feasible to differentiate and mature hESCs into functional oxygen-carrying erythrocytes on a large scale.


Subject(s)
Cell Nucleus/physiology , Embryonic Stem Cells/physiology , Erythrocytes/physiology , Animals , Cell Differentiation/physiology , Cell Fractionation , Cells, Cultured , Embryonic Stem Cells/cytology , Erythrocytes/cytology , Erythrocytes/metabolism , Erythroid Cells/cytology , Erythroid Cells/metabolism , Flow Cytometry , Humans , Mice , Rh-Hr Blood-Group System/metabolism , Tissue Engineering/methods
5.
Stem Cells Dev ; 16(4): 547-59, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17784829

ABSTRACT

Enforced expression of the HoxB4 gene promotes expansion of hematopoietic stem cells (HSCs) and enhances hematopoietic development of both murine and human embryonic stem (ES) cells. HoxB4- expanded HSCs have also been shown to retain their normal potential for differentiation and longterm self-renewal in vivo without the development of leukemia, suggesting that manipulation of HoxB4 expression might represent an effective way to expand functional HSCs for use in transplantation medicine. However, the genetic modification of cells poses clinical concerns, including a potentially increased risk of tumor genicity. Constitutive high-level ectopic viral expression of HoxB4 can also produce perturbations in the lineage differentiation of HSCs, an indication that uncontrolled HoxB4 manipulation may not be a satisfactory therapeutic strategy. Here we demonstrate that recombinant HoxB4 protein fused with a triple protein transduction domain (tPTD) promotes hematopoietic development of hES cells. The tPTD-HoxB4 protein enhanced the development of erythroid, myeloid, and multipotential progenitors in both early- and late-stage embryoid bodies (EBs). This effect varied considerably between different hES cell lines. Addition of the tPTD-HoxB4 protein did not alter the globin gene expression pattern; progeny derived from hES cells expressed high levels of embryonic (epsilon) and fetal (gamma) globin genes with or without tPTD-HoxB4 treatment. CD34+ cells derived from hES cells engrafted in bone marrow when transplanted into fetal CD1 mice, although supplementation of the differentiation medium with tPTD-HoxB4 protein did not result in increased repopulating capacity. This suggests that other gene(s), together with HoxB4, are required for generating more competitive HSCs. In summary, our study demonstrates that the tPTD-HoxB4 protein can be used with other recombinant proteins to efficiently generate transplantable HSCs from human ES cells.


Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/physiology , Hematopoiesis/physiology , Homeodomain Proteins/genetics , Transcription Factors/genetics , Base Sequence , DNA Primers , DNA, Antisense , Embryonic Stem Cells/cytology , Homeodomain Proteins/metabolism , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Promoter Regions, Genetic , Recombinant Fusion Proteins/metabolism , Transcription Factors/metabolism
6.
Methods Enzymol ; 418: 243-51, 2006.
Article in English | MEDLINE | ID: mdl-17141039

ABSTRACT

Embryonic stem (ES) cells, derived from early stage embryos, are pluripotent precursors of all of the tissues and organs of the body. ES cells from the mouse have been shown to undergo differentiation in vitro to form a variety of different cell types, including the differentiated progeny of hematopoietic precursors. These hematopoietic cells, however, exhibit numerous differences from those of human cells, and it has become increasingly clear that mouse ES cell differentiation has significant limitations as a model of human developmental biology. The more recent isolation and characterization of nonhuman primate ES cell lines have made available an experimental model with characteristics considerably more close to human biology. We have developed experimental conditions that promote efficient differentiation of these cells to produce progeny cells with considerable similarity to hematopoietic precursors harvested from bone marrow of adult animals.


Subject(s)
Embryonic Stem Cells/cytology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/physiology , Animals , Cell Culture Techniques/methods , Cell Differentiation , Cell Line , Embryonic Stem Cells/physiology , Fibroblasts/cytology , Fibroblasts/physiology , Hematopoietic Stem Cell Transplantation , Humans , Macaca mulatta , Mice , Primates , Stem Cell Transplantation
7.
Blood ; 103(11): 4134-41, 2004 Jun 01.
Article in English | MEDLINE | ID: mdl-14962900

ABSTRACT

Gene expression patterns of CD34(+)CD38(-) cells derived from human embryonic stem cells (ESCs) were compared with those of cells isolated from adult human bone marrow (BM) using microarrays; 1692 and 1494 genes were expressed at levels at least 3-fold above background in cells from BM and ESCs, respectively. Of these, 494 showed similar levels of expression in cells from both sources, 791 genes were overexpressed in cells from BM (BM versus ESCs, at least 2-fold), and 803 genes were preferentially expressed in cells from ESCs (ESCs versus BM, at least 2-fold). The message of the flt-3 gene was markedly decreased in cells from ESCs, whereas there was substantial flt-3 expression in cells from BM. High levels of embryonic epsilon-globin expression were observed-but no adult beta-globin message-in CD34(+)CD38(-) cells from ESCs, whereas high levels of beta-globin expression-but no embryonic epsilon-globin message-could be detected in cells from BM. Furthermore, high levels of major histocompatibility complex (MHC) gene expression were demonstrated in cells from BM but very low levels of MHC message in corresponding cells from ESCs. These observations demonstrate that CD34(+)CD38(-) cells derived from ESCs correspond consistently to an early developmental stage at which the yolk sac and fetal liver are the primary sites of hematopoiesis.


Subject(s)
ADP-ribosyl Cyclase/metabolism , Antigens, CD34/metabolism , Antigens, CD/metabolism , Gene Expression Regulation, Developmental , Hematopoietic Stem Cells/physiology , Oligonucleotide Array Sequence Analysis , ADP-ribosyl Cyclase 1 , Cells, Cultured , Cytokines/genetics , Hematopoietic Stem Cells/cytology , Hemoglobins/genetics , Histocompatibility Antigens/genetics , Humans , Membrane Glycoproteins , Mitogen-Activated Protein Kinases/genetics
9.
Stem Cells ; 20(5): 428-37, 2002.
Article in English | MEDLINE | ID: mdl-12351813

ABSTRACT

Rhesus monkey embryonic stem (ES) cells undergo differentiation in vitro to generate hematopoietic progenitor cells. Our previous studies demonstrated a high degree of similarity in the expression of genes associated with hematopoietic differentiation, homing, and engraftment in CD34(+) and CD34(+)CD38(-) cells from rhesus monkey ES cells and from fresh or cultured bone marrow (BM). In the present study, we compared the expression patterns of cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors (CDIs) in these cells. The expression of genes for cyclins, CDKs, and CDIs was similar among the hematopoietic progenitor cells of different origins, with only minor differences. Differentially expressed genes were also analyzed in CD34(+) lineage-negative cells derived from mouse ES cells and from BM. No difference or totally divergent results were obtained with the latter system, suggesting that this variation may be species specific. We observed, however, that CD34(+) and CD34(+)CD38(-) cells derived from ES cells expressed embryonic epsilon and zeta as well as alpha, beta, and gamma globin genes, whereas no expression of embryonic globins could be detected in the cell preparations from BM. Moreover, erythroblast-enriched CD34(-) cells derived from 4- or 5-week ES cell differentiation cultures also expressed embryonic, fetal, and adult globin genes, with greater beta gene expression, but otherwise were identical to those of the more primitive CD34(+) cells derived from 2-week ES cultures. These latter observations may reflect the presence of heterogeneous cell populations within the cell fractions that were compared, or they may represent variability among ES-cell-derived hematopoietic stem cells.


Subject(s)
Cell Cycle Proteins/genetics , Cell Differentiation/genetics , Embryo, Mammalian/immunology , Gene Expression Regulation, Developmental/genetics , Hematopoietic Stem Cells/immunology , Neoplasm Proteins , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Antigens, CD34/genetics , Antigens, CD34/immunology , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p18 , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/genetics , Cyclins/genetics , Embryo, Mammalian/cytology , Enzyme Inhibitors , Globins/genetics , HSP40 Heat-Shock Proteins , Hematopoietic Stem Cells/cytology , Homeodomain Proteins/genetics , Humans , Macaca mulatta , Mice , Repressor Proteins/genetics , Transcription Factors/genetics , Tumor Suppressor Proteins/genetics
10.
Exp Hematol ; 30(1): 58-66, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11823038

ABSTRACT

OBJECTIVE: The aim of this study was to characterize at the molecular level the hematopoietic progenitor cells derived from rhesus monkey embryonic stem (ES) cell differentiation. MATERIALS AND METHODS: We purified CD34(+) and CD34(+)CD38(-) cells from rhesus monkey ES cell cultures and examined the expression of a variety of genes associated with hematopoietic development, by semiquantitative polymerase chain reaction analysis. For comparison, we examined cell preparations from fresh or cultured rhesus monkey bone marrow (BM) and from mouse ES cells and BM. RESULTS: We observed a high degree of similarity in the expression patterns of these genes, with only a few exceptions. Most notably, the message of the flt3 gene was undetectable in rhesus monkey ES cell-derived CD34(+) and CD34(+)CD38(-) cells, whereas substantial flt3 expression was observed in the corresponding cells from fresh BM and in CD34(+) cells from cultured BM. The integrin alphaL and interleukin-6 (IL-6) receptor genes also were expressed in CD34(+)CD38(-) cells from BM, but there was little or no expression of these genes in CD34(+)CD38(-) cells derived from ES cells. Parallel analyses, using CD34(+)Lin(-) cells derived from murine ES cell cultures, showed no apparent expression of flt3, integrin alphaL, or IL-6 receptor, whereas corresponding cell preparations isolated from mouse BM expressed high levels of all of these genes. CONCLUSIONS: ES cell-derived hematopoietic progenitors, both from the rhesus monkey and from the mouse, exhibited the same alterations in gene expression compared with BM-derived cells from these animals. These observations could reflect the presence of different subpopulations in the cell fractions that were compared, or they may represent altered biologic properties of ES cell-derived hematopoietic stem cells.


Subject(s)
Antigens, CD , Cell Differentiation/genetics , Hematopoietic Stem Cells/physiology , Stem Cells/physiology , ADP-ribosyl Cyclase , ADP-ribosyl Cyclase 1 , Animals , Antigens, CD34 , Antigens, Differentiation , Cell Lineage/genetics , Gene Expression Profiling , Gene Expression Regulation , Macaca mulatta , NAD+ Nucleosidase , Polymerase Chain Reaction
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