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 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.

WNT3A signalling pathway in buffalo (Bubalus bubalis) embryonic stem cells

2014 ◽  
Vol 26 (4) ◽  
pp. 551 ◽  
Author(s):  
Mohammad Zandi ◽  
Musharifa Muzaffar ◽  
Syed Mohmad Shah ◽  
Ramakant Kaushik ◽  
Manoj Kumar Singh ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Polymerase Chain Reaction Analysis ◽  
Bubalus Bubalis ◽  
Embryoid Bodies ◽  
Signalling Pathway ◽  
Leukaemia Inhibitory Factor ◽  
In Vitro Fertilisation ◽  
Canonical Wnt

The aim of this study was to investigate the transcriptional profile and role of WNT3A signalling in maintaining buffalo embryonic stem (ES) cells in a pluripotent state and in the induction of their differentiation. ES cells were derived from embryos produced by in vitro fertilisation (iESC), parthenogenesis (pESC) and hand-made cloning (cESC). The expression of WNT3A, its receptors and intermediate signalling pathways were found to be conserved in ES cells derived from the three different sources. WNT3A was expressed in ES cells but not in embryoid bodies derived from iESC or in buffalo fetal fibroblast cells. It was revealed by real-time polymerase chain reaction analysis that following supplementation of culture medium with WNT3A (100, 200 or 400 ng mL–1) a significant increase (P < 0.05) was observed in the expression level of β-CATENIN, which indicated the activation of the canonical WNT pathway. WNT3A, in combination with exogenous fibroblast growth factor-2 and leukaemia inhibitory factor, induced proliferation of undifferentiated ES cells. Differentiation studies showed that WNT3A caused formation of scaffold-like structures and inhibition of differentiation into neuron-like cells. In conclusion, the WNT3A signalling pathway is necessary both for maintaining undifferentiated buffalo ES cells as well as for directing their differentiation.

Specific Effect of 5-Fluorouracil on α-Fetoprotein Gene Expression During the In Vitro Mouse Embryonic Stem Cell Differentiation

2010 ◽  
Vol 29 (3) ◽  
pp. 297-304 ◽  
Author(s):  
David Pamies ◽  
Néstor Vicente-Salar ◽  
Miguel A. Sogorb ◽  
Enrique Roche ◽  
Juan A. Reig
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryoid Bodies ◽  
Screening Methods ◽  
Embryonic Stem Cell Differentiation ◽  
Visceral Endoderm ◽  
Gene Markers ◽  
Wide Range

Embryonic stem (ES) cells are considered an important alternative to develop in vitro screening methods for embryotoxicity. Mouse ES cells can be cultured as cell suspension aggregates termed “embryoid bodies” (EBs) in which cells start to differentiate. We have studied the expression of several genes in the presence of a wide range of concentrations of 5-fluorouracil (5-FU). This well-established embryotoxic compound completely inhibited cell viability at 200 nmol/L in monolayer cultures. At lower concentrations, 5-FU led to decrease in the expression of the α-fetoprotein gene, a marker of the visceral endoderm, in the EBs. However, the expression of several mesodermal gene markers was not significantly affected at these concentrations. These results suggest a high sensitivity of the visceral endoderm differentiation to 5-FU. Therefore, the quantification of the α-fetoprotein gene after exposure to potential embryotoxicants should be considered an additional end point in future embryotoxicity assays in vitro with ES cells.

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 Evidence for Hepatocyte Differentiation from Embryonic Stem Cells In Vitro

2002 ◽  
Vol 11 (5) ◽  
pp. 429-434 ◽  
Author(s):  
Hitoshi Miyashita ◽  
Atsushi Suzuki ◽  
Katashi Fukao ◽  
Hiromitsu Nakauchi ◽  
Hideki Taniguchi
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Pcr Analysis ◽  
Liver Development ◽  
Hepatic Gene Expression ◽  
Hepatocyte Differentiation ◽  
Cell Based Therapy

We confirmed hepatocyte differentiation from embryonic stem (ES) cells in vitro. RT-PCR analysis revealed that a broad range of hepatic gene expression was observed in ES cells differentiated through formation of embryoid bodies (EBs) and its attachment culture. Quantitative PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of ES cells. The presence of albumin-producing cells in the peripheral region of attached EBs was confirmed by immunocytochemical analysis. Future experiments will reveal the molecules that induce hepatocyte differentiation from ES cells in vitro. This research will provide systems for the investigation of mechanisms in liver development and establish a method of ES cell-based therapy for liver diseases.

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.

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.

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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