scholarly journals 168 ESTABLISHMENT AND MOLECULAR CHARACTERIZATION OF PIG PARTHENOGENETIC EMBRYONIC STEM CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 235 ◽  
Author(s):  
T.A.L. Brevini ◽  
F. Cillo ◽  
F. Gandolfi
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Embryoid Bodies ◽  
Morphological Examination ◽  
Differentiation Markers ◽  
Differentiated Cells

Parthenogenetic embryonic stem cells have been obtained in mouse and in primates. However, it would be desirable to have an alternative experimental model that could be used to investigate the therapeutic potential of these cells. For this purpose, we generated parthenogenetic pig blastocysts from in vitro-matured oocytes activated by sequential exposure to 10 μM ionomycin for 5 min and 2 mM 6-DMAP for 3 h. Inner cell masses were isolated by immunosurgery and plated on mitotically inactivated STO fibroblast feeder layers in 4-well dishes. Cells were incubated in 5% CO2 at 37°C in low glucose DMEM/F10 medium supplemented with 1000 IU/mL of mouse recombinant LIF, 10% Knockout serum replacer (Gibco, Italy), and 5% FBS. Within 3 days, circular colonies with distinct margins of small round cells were observed on both substrates. When a colony enlarged enough to cover half or more of the well surface, cells were trypsinized in clumps never reaching single-cell suspension and passaged to a newly prepared well. The expression of a gene panel was examined by RT-PCR on a portion of the cells at each passage. Oct-4 and nanog were used as markers of pluripotency. Interferon-τ, α-Amilase, Bone Morphogenetic Protein-4, and Neurofilament were used as markers of trophectoderm, endoderm, mesoderm, and ectoderm differentiation respectively. After 4 passages, three colonies expressed Oct-4 and nanog and were negative for all four differentiation markers. Two colonies at the 5th and 7th passages maintained nanog but not Oct-4 expression, while remaining negative to all of the other genes. To induce the formation of embryoid bodies (EBs), cells were cultured in 50-μL droplets of medium without LIF. Initiation of differentiation of EBs was confirmed through both morphological examination and molecular analysis; mesodermal, ectodermal, and endodermal markers were all expressed by Day 9 of culture and Oct-4 and nanog expression was completely down-regulated. Interestingly, when EBs were returned to adherent culture conditions patches of differentiated cells tended to form, spontaneously differentiating into mesodermal, endodermal, or neuroectodermal cell monolayers. The present data suggest that it is possible to establish putative embryonic stem cells from pig parthenotes. Further studies are in progress to determine their ability to stably maintain the undifferentiated state. This work was supported by MIUR COFIN 20022074357 and Fondazione CARIPLO.

scholarly journals Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway

2020 ◽  
Vol 21 (23) ◽  
pp. 8945
Author(s):  
Mercedes Fernández ◽  
Micaela Pannella ◽  
Vito Antonio Baldassarro ◽  
Alessandra Flagelli ◽  
Giuseppe Alastra ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Embryonic Stem Cells ◽  
Thyroid Hormone ◽  
Nuclear Receptors ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Postnatal Life ◽  
Gene And Protein Expression

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRβ), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.

278 EFFECT OF XENOESTROGENS ON THE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

2009 ◽  
Vol 21 (1) ◽  
pp. 236
Author(s):  
E.-M. Jeung ◽  
K.-C. Choi ◽  
E.-B. Jeung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Embryoid Bodies ◽  
Time Dependent ◽  
Dependent Manner ◽  
Cardiomyocyte Differentiation ◽  
Differentiation Markers ◽  
Oct4 Expression

Endocrine disruptors (ED) may have adverse impacts on reproductive and immune systems in human and wild animals. It has been shown that octyl-phenol (OP) and nonyl-phenol (NP) have estrogenicity in estrogen-responding cells or tissues. In this study, we further investigated the effect(s) of OP and NP on the expression of undifferentiation and differentiation markers in mouse embryonic stem cells (ESC), which function as an important factor in the differentiation of ESC into cardiomyocytes. Mouse ESC were cultured in hanging drops to form embryoid bodies (EB). The medium was replaced with phenol red-free DMEM/F-12 supplemented with 5% charcoal-dextran-stripped FBS. The ESC were treated with OP, NP (1Ã-10-6 and 1Ã-10-7 M) or 17β-estradiol (E2; 1Ã-10-8 and 1Ã-10-9 M) in a time-dependent manner (1, 2 and 3 days), and EB were treated with identical concentrations for 4 and 8 days, respectively. High increasing doses of OP and NP were employed in this study because a binding affinity of ED to estrogen receptors (ER) is about 1000 less than that of E2. We determined the mRNA expression of undifferentiation markers (Oct4, Sox2 and Zfp206) and cardiomyocyte differentiation markers (cardiac alpha-MHC, beta-MHC and myosin light chain isoform-2V) using real-time PCR. In ESC, undifferentiation markers were identified. It is of interest that treatment with OP, NP or E2 induced a significant increase (1.4 5.5-fold) in Oct4 expression at the transcription levels according to a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2 and Zfp206 genes in ESC, suggesting that OP and NP may play a role as an Oct4 enhancer in ESC. In addition, both undifferentiation and cardiomyocyte differentiation markers were identified in EB. Treatment with OP and NP induced a significant increase in the expression of Oct4, Sox2 and Zfp206 genes at the transcription levels in a dose-dependent manner for 4 days, whereas Oct4 expression was only induced at these doses for 8 days. In contrast, cardiomyocyte differentiation markers were reduced by these ED in EB. Taken together, these results suggest that OP and NP play a role as a positive regulator in the undifferentiation process of ESC and EB, and maintenance and differentiation of mouse ESC.

Induction of Cardiomyocyte Differentiation From Mouse Embryonic Stem Cells in a Confined Microfluidic Environment

2009 ◽  
Author(s):  
Chen-rei Wan ◽  
Seok Chung ◽  
Ryo Sudo ◽  
Roger D. Kamm
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mechanical Stretch ◽  
Embryoid Bodies ◽  
Negative Regulator ◽  
Differentiation Process ◽  
Cardiomyocyte Differentiation

Embryonic stem cell derived cardiomyocytes are deemed an attractive treatment option for myocardial infarction. Their clinical efficacy, however, has not been unequivocally demonstrated. There is a need for better understanding and characterization of the cardiogenesis process. A microfluidic platform in vitro is used to dissect and better understand the differentiation process. Through this study, we find that while embryoid bodies (EBs) flatten out in a well plate system, differentiated EBs self-assemble into complex 3D structures. The beating regions of EBs are also different. Most beating areas are observed in a ring pattern on 2D well plates around the center, self-assembled beating large 3D aggregates are found in microfluidic devices. Furthermore, inspired by the natural mechanical environment of the heart, we applied uniaxial cyclic mechanical stretch to EBs. Results suggest that prolonged mechanical stimulation acts as a negative regulator of cardiogenesis. From this study, we conclude that the culture environments can influence differentiation of embryonic stem cells into cardiomycytes, and that the use of microfluidic systems can provide new insights into the differentiation process.

scholarly journals Morphogen And Community Effects Determine Cell Fates In Response To BMP4 Signaling In Human Embryonic Stem Cells

2017 ◽  
Author(s):  
Anastasiia Nemashkalo ◽  
Albert Ruzo ◽  
Idse Heemskerk ◽  
Aryeh Warmflash
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Community Effects ◽  
Cell Fates ◽  
Standard Culture

AbstractParacrine signals maintain developmental states and create cell-fate patterns in vivo, and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro. Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells (“μColonies”) to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in μColonies and standard culture conditions and find that in μColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions, BMP4 acts as morphogen, but this effect requires secondary signals and particular cell densities. We further find that a “community effect” enforces a common fate within μColonies both in the state of pluripotency and when cells are differentiated, and that this effect allows more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation.Summary StatementWe quantitatively examined signaling and differentiation in hESC colonies of varying size treated with BMP4. We show that secondary signals result in morphogen and community effects that determine cell fates.

scholarly journals Defining totipotency using criteria of increasing stringency

2020 ◽  
Author(s):  
Eszter Posfai ◽  
John Paul Schell ◽  
Adrian Janiszewski ◽  
Isidora Rovic ◽  
Alexander Murray ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiated Cells ◽  
Totipotent Cell

AbstractTotipotency is the ability of a single cell to give rise to all the differentiated cells that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies upon a variety of assays of variable stringency. Here we describe criteria to define totipotency. We illustrate how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in the mouse, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbor increased totipotent potential relative to conventional embryonic stem cells under in vivo conditions.

Hematopoietic Differentiation of Human Embryonic Stem Cells in Vitro : Comparison of Three Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4787-4787
Author(s):  
Marion Brenot ◽  
Annelise Bennaceur-Griscelli ◽  
Marc Peschanski ◽  
Maria Teresa Mitjavila-Garcia
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

Abstract Human embryonic stem cells (hES) isolated from the inner cell mass of a blastocyst have the ability to self renew indefinitely while maintaining their pluripotency to differentiate into multiple cell lineages. Therefore, hES represent an important source of cells for perspective cell therapies and serve as an essential tool for fundamental research, specifically for understanding pathophysiological mechanisms of human diseases for the development of novel pharmacological drugs. The generation of hematopoietic stem cells from hES may serve as an alternative source of cells for hematopoietic reconstitution following bone marrow transplantation and an interesting approach to understand early stages of hematopoietic development which are difficult to study in human embryos. Using two different methods, we have differentiated three hES cell lines (SA01, H1 and H9) into hematopoietic cells by generating embryoid bodies and co-culturing on the murine Op9 cell line. In both experimental approaches, we obtain cells expressing CD34 and when cultured in hematopoietic conditions, SA01 and H1 cell lines differentiate into various hematopoietic lineages as demonstrated by BFU-E, CFU-GM and CFU-GEMM colony formation, whereas H9 have almost exclusively granulo-macrophage differentiation. Cells composing these hematopoietic colonies express CD45, CD11b, CD31, CD41 and CD235 and staining with May Grundwald-Giemsa demonstrate neutrophil and erythrocyte morphology. These results demonstrate the capacity of hES to differentiate into mature hematopoietic cells in vitro. Nevertheless, there exist some quantitative and qualitative differences about hematopoietic differentiation between the hES cell lines used. However, we still have to evaluate their capacity to reconstitute hematopoiesis in vivo in an immune deficient mouse model. We will also be interested in developing in vitro methods to expand these hematopoietic precursor cells derived from hES which may be used as a viable source for future cell therapy.

scholarly journals Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells

2008 ◽  
Vol 205 (8) ◽  
pp. 1917-1927 ◽  
Author(s):  
Hidekazu Nishikii ◽  
Koji Eto ◽  
Noriko Tamura ◽  
Koichi Hattori ◽  
Beate Heissig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Blood Platelets ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Normal Platelet ◽  
Metalloproteinase Inhibitors ◽  
Flow Cytometric Sorting

Embryonic stem cells (ESCs) could potentially compensate for the lack of blood platelets available for use in transfusions. Here, we describe a new method for generating mouse ESC-derived platelets (ESPs) that can contribute to hemostasis in vivo. Flow cytometric sorting of cells from embryoid bodies on day 6 demonstrated that c-Kit+ integrin αIIb (αIIb)+ cells, but not CD31+ cells or vascular endothelial cadherin+ cells, are capable of megakaryopoiesis and the release of platelet-like structures by day 12. αIIbβ3-expressing ESPs exhibited ectodomain shedding of glycoprotein (GP)Ibα, GPV, and GPVI, but not αIIbβ3 or GPIbβ. ESPs showed impaired αIIbβ3 activation and integrin-mediated actin reorganization, critical events for normal platelet function. However, the administration of metalloproteinase inhibitors GM6001 or TAPI-1 during differentiation increased the expression of GPIbα, improving both thrombogenesis in vitro and posttransfusion recovery in vivo. Thus, the regulation of metalloproteinases in culture could be useful for obtaining high-quality, efficacious ESPs as an alternative platelet source for transfusions.

Expression of Imprinted Genes Kcnq1 and Cdkn1c During the Course of Differentiation from Mouse Embryonic Stem Cells into Islet-like Cells in vitro

2017 ◽  
Vol 126 (04) ◽  
pp. 249-254
Author(s):  
Feng Liu ◽  
Peng yu-huan ◽  
Li Qiang ◽  
Liu Chanchan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Islet Cell ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Imprinted Genes ◽  
Rt Pcr ◽  
Differentiated Cells ◽  
Pcr Rflp

AbstractTo study the effects of inducement on the expression of mouse embryonic stem cells SF1-G imprinted genes, Kcnq1 and Cdkn1c during the course of differentiation into islet-like cells in vitro. Mouse embryonic fibroblasts (MEFs) were isolated from pregnant mice embryos and fibroblast feeder cells were prepared by treating 3–5th generations MEFs with Mitomycin C. Moreover, mouse embryonic stem cells were induced to differentiate into islet-like cells directly. RT-PCR and Immunofluorescence staining were used to test the expression of islet cell-specific markers. Cells were collected at various stages throughout the differentiation process and the imprinted genes Kcnq1 and Cdkn1c were tested by reverse transcription-polymerase chain reaction fragment length polymorphism (RT-PCR/RFLP). In the present study, we found that cells appear islet cell-specific gene expression. Furthermore, immunofluorescence shows us that the islet cell-specific hormone protein can be measured at stage, which confirms that the embryonic stem cells can be successfully induced into islet-like cells in vitro. RT-PCR/RFLP analysis showsthat imprinted genes Kcnq1 and Cdkn1c are biallelic expression in the differentiated cells, suggestive of loss of imprinting (LOI), while these genes demonstrate maternal monoallelic expression in the undifferentiated cells’ continued subculture; this marks the maintenance of imprinting (MOI). Our data indicate that mouse embryonic stem cells are induced into islet-like cells in vitro. The gene imprinting status of Kcnq1 and Cdkn1c may be changed in differentiated cells during the induction in vitro.

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