scholarly journals Receptor Protein Tyrosine Phosphatase Gamma, Ptpγ, Regulates Hematopoietic Differentiation

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Claudio Sorio ◽  
Paola Melotti ◽  
Daniela D'Arcangelo ◽  
Jeannine Mendrola ◽  
Bruno Calabretta ◽  
...  
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Surface Protein ◽  
Embryoid Bodies ◽  
Receptor Protein ◽  
Hematopoietic Differentiation ◽  
Normal Number ◽  
Embryoid Body Differentiation

Murine embryonic stem (ES) cells have been a useful model system for the study of various aspects of hematopoietic differentiation. Because we had observed a sharp peak of expression of the receptor tyrosine phosphatase gamma (Ptpγ) gene between 14 and 18 days of ES-derived embryoid body differentiation, we investigated the effect of perturbation of expression of the Ptpγ gene on ES cell differentiation, first by analyzing the effect of Ptpγ overexpression. The murine full-length Ptpγ cDNA in an expression vector was transfected into ES-D3 cells and stably transfected clones were isolated. Ptpγ was expressed as an approximately 230-kD cell surface protein, and differentiating ES clones that overexpressed Ptpγ gave rise to a normal number of hematopoietic colonies, approximately 1 CFU per 100 cells. There was, however, a significant increase of expression of early hematopoietic markers in colonies from Ptpγ overexpressing ES cells. To confirm that the pertubation of hematopoietic differentiation was a result of Ptpγ overexpression, we isolated ES stem cell clones expressing Ptpγ antisense constructs and assayed embryoid bodies for the presence of hematopoietic precursors. We observed a complete absence of methylcellulose colonies, indicating absence of hematopoietic lineages. Results of these experiments point to an essential role for Ptpγ in hematopoietic differentiation.

scholarly journals Receptor Protein Tyrosine Phosphatase Gamma, Ptpγ, Regulates Hematopoietic Differentiation

Blood ◽  
1997 ◽  
Vol 90 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Claudio Sorio ◽  
Paola Melotti ◽  
Daniela D'Arcangelo ◽  
Jeannine Mendrola ◽  
Bruno Calabretta ◽  
...  
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Surface Protein ◽  
Embryoid Bodies ◽  
Receptor Protein ◽  
Hematopoietic Differentiation ◽  
Normal Number ◽  
Embryoid Body Differentiation

Abstract Murine embryonic stem (ES) cells have been a useful model system for the study of various aspects of hematopoietic differentiation. Because we had observed a sharp peak of expression of the receptor tyrosine phosphatase gamma (Ptpγ) gene between 14 and 18 days of ES-derived embryoid body differentiation, we investigated the effect of perturbation of expression of the Ptpγ gene on ES cell differentiation, first by analyzing the effect of Ptpγ overexpression. The murine full-length Ptpγ cDNA in an expression vector was transfected into ES-D3 cells and stably transfected clones were isolated. Ptpγ was expressed as an approximately 230-kD cell surface protein, and differentiating ES clones that overexpressed Ptpγ gave rise to a normal number of hematopoietic colonies, approximately 1 CFU per 100 cells. There was, however, a significant increase of expression of early hematopoietic markers in colonies from Ptpγ overexpressing ES cells. To confirm that the pertubation of hematopoietic differentiation was a result of Ptpγ overexpression, we isolated ES stem cell clones expressing Ptpγ antisense constructs and assayed embryoid bodies for the presence of hematopoietic precursors. We observed a complete absence of methylcellulose colonies, indicating absence of hematopoietic lineages. Results of these experiments point to an essential role for Ptpγ in hematopoietic differentiation.

Tyrosine phosphatase SHP-1 acts at different stages of development to regulate hematopoiesis

Blood ◽  
2005 ◽  
Vol 105 (11) ◽  
pp. 4290-4297 ◽  
Author(s):  
Nicholas R. D. Paling ◽  
Melanie J. Welham
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Dominant Negative ◽  
Body Development ◽  
Undifferentiated Cells ◽  
Hematopoietic Precursor ◽  
Multiple Stages

Abstract Mice lacking SHP-1 exhibit a plethora of perturbations in their hematopoietic and immune systems. To reveal the primary effects resulting from SHP-1 deficiency, we used embryonic stem (ES) cells to study the role of SHP-1 in developmental hematopoiesis. We expressed wild-type (WT) and dominant-negative (R459M) forms of SHP-1 in ES cells and used ES/OP-9 coculture and embryoid body development followed by hematopoietic colony assays to demonstrate that SHP-1 acts at multiple stages of hematopoietic differentiation to alter lineage balance. Expression of WT SHP-1 reduced myeloid colony numbers while increasing the numbers of secondary embryoid bodies and mixed hematopoietic colonies obtained. Conversely, expression of R459M SHP-1 resulted in a significant increase in the numbers and sizes of myeloid colonies observed while reducing the numbers of colonies derived from undifferentiated cells or hematopoietic precursor cells. Confining the expression of WT or R459M SHP-1 to the early phases of differentiation decreased and increased progenitor cell numbers, respectively, and influenced colony formation. Overall, our results are consistent with SHP-1 acting during multiple stages of hematopoietic development, and they suggest that the increases in granulocytes and macrophages observed in motheaten mice arise as the result of a cell autonomous effect early during development.

scholarly journals Hepatocyte Differentiation from Human ES Cells using the Simple Embryoid Body Formation Method and the Staged-Additional Cocktail

2009 ◽  
Vol 9 ◽  
pp. 884-890 ◽  
Author(s):  
Katsunori Sasaki ◽  
Hinako Ichikawa ◽  
Shunsuke Takei ◽  
Hee Sung No ◽  
Daihachiro Tomotsune ◽  
...  
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Culture Method ◽  
Polymerase Chain Reaction Analysis ◽  
Embryoid Bodies ◽  
Hepatocyte Differentiation ◽  
Gene Markers ◽  
Transmission Electron ◽  
Hes Cells

To induce hepatocytes from human embryonic stem (hES) cells easily and effectively, a simple suspension culture method that separates ES colonies with a scraper and transfers them into newly developed, nonadherent MPC (2-methacryloyloxyethyl phosphorylcholine) plates, and the staged-additional cocktail method, including growth factors, cytokines, and Lanford serum-free medium, were developed and evaluated mainly by morphological analysis. The formed embryoid bodies (EBs) showed compact cellular agglomeration until day 4 and later formed coeloms in their interior. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis showed that they are gene markers of the three germ layers. Mesenchymal cells with rough endoplasmic reticulum (rER) and extracellular matrix (ECM), and without junctions, were recognized in the interior of the EBs by transmission electron microscopy (TEM) in addition to epithelial cells. When they were stimulated by the staged-additional cocktail, they expressed albumin-positive immunoreactivity, indocyanine green (ICG) uptake, and typical ultrastructures of the hepatocytes, including bile canaliculi. These results indicate that these combined methods promote EB formation and hepatocyte differentiation from hES cells.

scholarly journals Regulated expression of microRNAs-126/126* inhibits erythropoiesis from human embryonic stem cells

Blood ◽  
2011 ◽  
Vol 117 (7) ◽  
pp. 2157-2165 ◽  
Author(s):  
Xinqiang Huang ◽  
Eric Gschweng ◽  
Ben Van Handel ◽  
Donghui Cheng ◽  
Hanna K. A. Mikkola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Protein Tyrosine Phosphatase ◽  
Embryoid Body ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Protein Tyrosine

Abstract MicroRNAs (miRs) play an important role in cell differentiation and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage differentiation. Human embryonic stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34+ EB cells. miRs-126/126* were the most enriched of the 7 miRs that were up-regulated in CD34+ cells, and their expression paralleled the kinetics of hematopoietic transcription factors RUNX1, SCL, and PU.1. To define the role of miRs-126/126* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-126/126* and analyzed their hematopoietic differentiation. Induction of miRs-126/126* during both EB differentiation and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-126/126* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for growth and expansion of erythroid cells, is one target of miR-126. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-126/126*. Our results define an important function of miRs-126/126* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic differentiation of hESCs.

Comparative Effects of Wild Type and T315I Mutated bcr-ABL in Murine Embryonic Stem-Cell Derived Hematopoiesis.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1075-1075
Author(s):  
Michael Melkus ◽  
Maria Theresa Mitjavila-Garcia ◽  
Marie-Laure Bonnet ◽  
Nathalie Sorel ◽  
Jean-Claude Chomel ◽  
...  
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Transduction Efficiency ◽  
Hematopoietic Differentiation ◽  
Packaging Cell Line ◽  
Hematopoietic Stem ◽  
Clonogenic Assays ◽  
Population Doublings

Abstract Recent data suggest that there is a major discrepancy between the expression of BCR-ABL in primitive hematopoietic stem cells as compared to their differentiated counterparts where the expression is much lower. This high expression level predisposes this compartment to a major genetic instability leading to a mutator phenotype. The “gatekeeper” mutation T315I is the most problematic of these mutations leading to a resistance to all three clinically available tyrosine kinase inhibitors (TKI). This mutation is suspected to generate a specific signalling distinct from non-mutated BCR-ABL with high transforming potential but less TK activity. In order to study the effects of both mutated and non-mutated BCR-ABL in a primitive stem cell context, we have transduced retrovirally (Bosc 23 packaging cell line) both BCR-ABL and BCR-ABL T315I as well as an empty GFP control retrovirus, into day+5 embryoid bodies (EB’s) derived from D3 murine embryonic stem (ES) cells. In several experiments, the transduction efficiency was found to vary between 10–45% as evaluated by GFP expression at day+ 3 post-transduction. EB’s were then dissociated and put in hematopoietic differentiation conditions using clonogenic assays and in liquid culture in the presence of OP-9 stroma and hematopoietic growth factors including SCF, IL6 and IL3. Cells were collected weekly and analyzed with regard to their hematopoietic commitment, their amplification potential, their phenotype and their morphology as well as expression of BCR-ABL. In clonogenic assays, BCR-ABL and T315I6transduced cellD induced GFP+ growth-factor-independent colonies. In hematopoietic differentiation conditions, empty vector- and BCR-ABL-transduced cells underwent approximately 8-population doublings in vitro. On the other hand, BCR-ABL T315I mutant-transduced cells underwent a major expansion during this time with 16-Population doublings in 4 weeks. The addition of imatinib mesylate (1 and 2 mM) to cell cultures stimulated cell growth in T315I-BCR-ABL- transduced cells as compared to controls. Interestingly, phenotypic analysis demonstrated the appearance of CD45+ CD34+ cells in BCR-ABL-transduced cells as early as week+2–3 whereas this hematopoietic differentiation appeared to be delayed in BCR-ABL-T315I-transduced cells. Thus, our data suggest that in the context of a primitive hematopoietic stem cell context, T315I BCR-ABL exhibits a higher transforming potential. Current experiments underway are testing the long-term repopulating ability of BCR-ABL and BCR-ABLT315I- expressing cells in NOD/SCID reconstitution assays as well as the occurrence of ABL-kinase mutations in the presence and in the absence of TKI. Thus, the murine ES-cell-derived hematopoiesis could be an important experimental tool to recapitulate the early stages of hematopoiesis and the role of T315I mutation in the rare, primitive stem cell populations.

A definitive role of Shp-2 tyrosine phosphatase in mediating embryonic stem cell differentiation and hematopoiesis

Blood ◽  
2003 ◽  
Vol 102 (6) ◽  
pp. 2074-2080 ◽  
Author(s):  
Rebecca J. Chan ◽  
Scott A. Johnson ◽  
Yanjun Li ◽  
Mervin C. Yoder ◽  
Gen-Sheng Feng
Keyword(s):  
Cell Differentiation ◽  
Embryoid Body ◽  
Es Cells ◽  
Tyrosine Phosphatase ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Initial Step ◽  
Embryonic Stem Cell Differentiation ◽  
Es Cell

Abstract Homozygous mutant (Shp-2Δ46-110) embryonic stem (ES) cells exhibit decreased hematopoiesis; however, the point at which Shp-2 is critical for ES cell differentiation to hematopoietic cells is unknown. We characterized the differentiation defect of Shp-2Δ46-110 ES cells by examining early points of differentiation, conducting leukemia inhibitory factor (LIF)–stimulated biochemical analysis, and performing in vitro reconstitution studies with wild-type (WT) Shp-2. ES cell in vitro differentiation assays were used to compare the differentiation of WT, Shp-2Δ46-110, and reconstituted ES cells to mesoderm, by measuring brachyury expression, to hemangioblasts, by measuring blast colony-forming cell (BL-CFC) formation and flk-1 expression, and to hematopoietic progenitor colony-forming cells, by performing secondary plating assays. LIF-stimulated phospho-Stat3 (known to be critical for ES cell self-renewal and maintenance of an undifferentiated state) and phospho-Erk levels were examined by immunoblotting. ES cell survival, using annexin V staining, and secondary embryoid body (EB) formation were also evaluated. Differentiation to both mesoderm and hemangioblasts was lower in Shp-2Δ46-110 cells compared to WT cells. On reconstitution with WT Shp-2, expression of brachyury and flk-1 and differentiation to hemangioblasts and primitive and definitive hematopoietic progenitors were restored. LIF-stimulated phospho-Stat3 levels were higher, whereas phospho-Erk levels were lower in Shp-2Δ46-110 ES cells than in WT and reconstituted cells. The increased phospho-Stat3 levels correlated with increased Shp-2Δ46-110 ES cell secondary EB formation and survival. We conclude that normal Shp-2 function is critical for the initial step of ES cell differentiation to mesoderm and to hemangioblasts and acts within the LIF-gp130-Stat3 pathway to maintain a proper balance of ES cell differentiation, pluripotency, and apoptosis.

scholarly journals Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2870-2882 ◽  
Author(s):  
Unmesh Jadhav ◽  
J. Larry Jameson
Keyword(s):  
Target Genes ◽  
De Novo ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Steroidogenic Factor 1 ◽  
Lineage Differentiation ◽  
And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

The BMP/BMPR/Smad pathway directs expression of the erythroid-specific EKLF and GATA1 transcription factors during embryoid body differentiation in serum-free media

Development ◽  
2002 ◽  
Vol 129 (2) ◽  
pp. 539-549 ◽  
Author(s):  
Carrie A. Adelman ◽  
Subrata Chattopadhyay ◽  
James J. Bieker
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Dominant Negative ◽  
Erythroid Cell ◽  
Specific Gene ◽  
Smad Pathway ◽  
Cellular Expression ◽  
Novel Approach

Erythroid cell-specific gene regulation during terminal differentiation is controlled by transcriptional regulators, such as EKLF and GATA1, that themselves exhibit tissue-restricted expression patterns. Their early expression, already in evidence within multipotential hematopoietic cell lines, has made it difficult to determine what extracellular effectors and transduction mechanisms might be directing the onset of their own transcription during embryogenesis. To circumvent this problem, we have taken the novel approach of investigating whether the ability of embryonic stem (ES) cells to mimic early developmental patterns of cellular expression during embryoid body (EB) differentiation can address this issue. We first established conditions whereby EBs could form efficiently in the absence of serum. Surprisingly, in addition to mesoderm, these cells expressed hemangioblast and hematopoietic markers. However, they did not express the committed erythroid markers EKLF and GATA1, nor the terminally differentiated β-like globin markers. Using this system, we determined that EB differentiation in BMP4 was necessary and sufficient to recover EKLF and GATA1 expression and could be further stimulated by the inclusion of VEGF, SCF, erythropoietin and thyroid hormone. EBs were competent to respond to BMP4 only until day 4 of differentiation, which coincides with the normal onset of EKLF expression. The direct involvement of the BMP/Smad pathway in this induction process was further verified by showing that erythroid expression of a dominant negative BMP1B receptor or of the inhibitory Smad6 protein prevented induction of EKLF or GATA1 even in the presence of serum. Although Smad1, Smad5 and Smad8 are all expressed in the EBs, BMP4 induction of EKLF and GATA1 transcription is not immediate. These data implicate the BMP/Smad induction system as being a crucial pathway to direct the onset of EKLF and GATA1 expression during hematopoietic differentiation and demonstrate that EB differentiation can be manipulated to study induction of specific genes that are expressed early within a lineage.

scholarly journals Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice

2018 ◽  
Vol 20 (1) ◽  
pp. 19 ◽  
Author(s):  
Yadong Wei ◽  
Krishan Chhiba ◽  
Fengrui Zhang ◽  
Xujun Ye ◽  
Lihui Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mast Cell ◽  
Mast Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Surface Protein ◽  
Cell Types ◽  
Mouse Strains ◽  
Specific Expression

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.

X chromosome retains the memory of its parental origin in murine embryonic stem cells

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 813-821 ◽  
Author(s):  
T. Tada ◽  
M. Tada ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Polar Region ◽  
Biochemical Study ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Parental Origin ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Preferential Inactivation

A cytogenetic and biochemical study of balloon-like cystic embryoid bodies, formed by newly established embryonic stem (ES) cell lines having a cytogenetically or genetically marked X chromosome, revealed that the paternally derived X chromosome was inactivated in the majority of cells in the yolk sac-like mural region consisting of the visceral endoderm and mesoderm. The nonrandomness was less evident in the more solid polar region containing the ectodermal vesicle, mesoderm and visceral endoderm. Since the same was true in embryoid bodies derived from ES cells at the 30th subculture generation, it was concluded that the imprinting responsible for the preferential inactivation of the paternal X chromosome that was limited to non-epiblast cells of the female mouse embryos, was stably maintained in undifferentiated ES cells. Differentiating epiblast cells should be able to erase or avoid responding to the imprint.

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