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.

Neural Stem Cells in Neurospheres, Embryoid Bodies, and Central Nervous System of Human Embryos

2011 ◽  
Vol 17 (4) ◽  
pp. 520-527 ◽  
Author(s):  
A. Henry Sathananthan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neural Stem Cells ◽  
Neural Tube ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Surface Epithelium ◽  
Human Embryos

AbstractThe process of neurogenesis and formation of neural stem cells is reported in human neurospheres (NS) and embryoid bodies (EB) derived from human embryonic stem cells, in vitro, and compared with neural tissue formed in human ectopic embryos in week 4 (stage 9), developed in vivo. This morphological study was done using digital imaging by light microscopy and routine transmission electron microscopy. Both NS and EB form neural rosettes from the surface epithelium much like the process of neural tube formation from ectoderm in the embryo. The rosette is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells and is a radial arrangement of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube. The NS produce all of the major classes of progeny of the neural tube, some of which have been documented here. Specific neural markers expressed in the NS and the clinical implications of this study in cell therapy are also discussed.

229 DERIVATION OF PRESUMPTIVE GONOCYTES IN VITRO FROM PRIMATE EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 231
Author(s):  
T. Teramura ◽  
N. Kawata ◽  
T. Takehara ◽  
N. Fujinami ◽  
M. Takenoshita ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Germ Cells ◽  
Nonhuman Primates ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Embryoid Bodies

Embryonic stem cells (ESCs) of nonhuman primates are important for research into human gametogenesis, because of similarities between the embryos and fetuses of nonhuman primates and those of humans. Recently, the formation of germ cells from mouse ESCs in vitro has been reported. In this study, we established cynomolgus monkey ES (cyES) cell lines and attempted to induce their differentiation into germ cells in order to obtain further information on the development of primate germ cells by observing the transcripts of some markers reported as specific for germ cells. CyES cell lines were established using blastocysts produced by intracytoplasmic sperm injection (ICSI). For inducing superovulation, females were treated with 25 IU kg-1 pregnant mare serum gonadotropin once a day for 9 days, followed by 400 IU kg-1 hCG. Oocytes were collected at 40 h after injection of hCG. After sperm injection, embryos were cultured in mCMRL medium to the blastocyst stage. For cyES cell establishment, inner cell masses (ICMs) were isolated by immunosurgery. The ESC colonies developed at about 10 days after ICM plating, and 3 cell lines were successfully established (3/11; 27.3%). All cell lines expressed Oct3/4, SSEA-4, and ALP activity. These ESCs formed teratomas containing 3 different embryonic layers when injected into SCID mice. And the cells could be passaged over 50 times without losing their original properties. To observe in vitro gametogenesis, we attempted to induce differentiation by non-adherent conditions. When cyES cells differentiated spontaneously, the aggregated structures (i.e. embryoid bodies; EBs) accumulated vasa, the expression of which is restricted to germ cells, and some meiotic markers such as dmc1 and sycp1 that exist only in synaptonemal complexes in meiosis. The existence of these markers was also confirmed by immunocytochemistry on cryosections. Interestingly, these products expressed oct4 and nanog again at Day 16, though the expression of both genes diminished at once with onset of differentiation. In vivo, it is reported that vasa, oct4, and nanog are expressed in migrating PGCs, posibly throughout the development of germ cells into spermatocytes/oocytes. Given the results obtained with the meiotic markers, it is possible that developing germ cells such as PGCs or gonocytes could be formed in cynomolgus EBs as in previous cases with mouse or human EBs. These results demonstrate that cyES cells might contribute to putative germ cells in vitro by differentiating into EBs and could be used as a model for studying mechanisms of germ cell development. This study was supported by a Grant-in-Aid for the 21st Century COE Program of the Japan Mext and by a grant for the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technology Excellence of the JST.

scholarly journals Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo

2015 ◽  
Vol 13 (1) ◽  
pp. 720-730 ◽  
Author(s):  
LIPING OU ◽  
LIAOQIONG FANG ◽  
HEJING TANG ◽  
HAI QIAO ◽  
XIAOMEI ZHANG ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells

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.

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 231
Author(s):  
S. Wang ◽  
X. Tang ◽  
Y. Niu ◽  
H. Chen ◽  
T. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Speed ◽  
Es Cells ◽  
Research Work ◽  
Embryonic Stem ◽  
Morphological Characteristics ◽  
Mouse Escs

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.

In Vitro Differentiation and In Vivo Mineralization of Osteogenic Cells Derived from Human Embryonic Stem Cells

2004 ◽  
Vol 10 (9-10) ◽  
pp. 1518-1525 ◽  
Author(s):  
Robert C. Bielby ◽  
Aldo R. Boccaccini ◽  
Julia M. Polak ◽  
Lee D.K. Buttery
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
In Vitro Differentiation ◽  
Osteogenic Cells
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