scholarly journals 187EFFECTS OF OXYGEN TENSION ON ESTABLISHMENT, LDH ISOZYMES, AND MRNA EXPRESSION OF MURINE EMBRYONIC STEM CELLS

2004 ◽  
Vol 16 (2) ◽  
pp. 215
Author(s):  
J. Gibbons ◽  
E. Hewitt ◽  
D.K. Gardner
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Mrna Expression ◽  
Cell Lines ◽  
Embryonic Stem ◽  
High O2 ◽  
Culture Environment ◽  
Stem Cell Lines ◽  
Ldh Isozymes

Orchestrated differentiation of embryonic stem cells into specific tissues or cells will be invaluable for xenotransplantation, biomedicine, and pharmacology. However, the lack of a standardized culture environment for establishment and maintenance of cell lines has hindered the application of this technology. In other cell types, O2 concentration in the culture environment can have a profound effect on proliferation and differentiation. This study, therefore, tested the hypothesis that establishment dynamics, LDH isoforms, and mRNA expression patterns of the resulting cell lines would be affected by the O2 tension in the culture environment. Blastocysts recovered from mice (C57BL/6) uteri on Day 4 (post coitis) were placed in Speciality Medium (4500mgL−1 glucose) DMEM (plus 0.01μgmL−1 LIF) and cultured in a gas environment of 5% CO2 and either ∼20% or 5% O2. More (P<0.05) blastocysts hatched and produced outgrowths (Day 4 of DMEM culture) in the low (76.7±0.1%) compared to the high (58.3±0.1%) O2 group. Although the number of cells per outgrowth was similar between groups (low=58.1±5.2, high=58.8±5.7), a smaller number of colonies in the high O2 group (9/15; 64.3%) stained positive for alkaline phosphatase relative to the low O2 group (14/15; 93.3%). Oxygen treatment had no effect on the patterns of activity of the oxioreductase, lactate dehydrogenase (LDH), in either outgrowths or established stem cell lines. Interestingly, the stem cell lines (both O2 groups) displayed multiple isoforms (Isoforms 3, 4, and 5) of LDH, whereas the outgrowths displayed only Isoform 5. In contrast, two-cell embryos and blastocysts displayed only Isoform 1, and fibroblasts displayed Isoforms 4 and 5. Expression (mRNA) profiles were developed from blastocyst outgrowths, stem cell colonies, and established stem cell lines cultured under either high or low O2 tension, using a RT-PCR for LDH (Isozymes α and β) and a key regulatory enzyme of glycolysis, phosphofructokinase-2 (PFK2; Isozyme 1 and 2). There were no differences between the high and low O2 groups in mRNA expression of LDHα in the outgrowths, or established stem cells. Expression of LDHβ was at a very low level regardless of O2 treatment or cell type. Outgrowths from the low O2 group expressed Isozyme 1 of PFK2 whereas the outgrowths from the high O2 group did not. Enhanced expression of PFK2 is suggestive of increased glycolytic capacity. Reduced O2 environment during the peri-hatching period had significant effects on the resulting embryonic stem cells, supporting the hypothesis that O2 tension can affect stem cell establishment and maintenance.

scholarly journals Embryos, Clones, and Stem Cells: A Scientific Primer

2004 ◽  
Vol 4 ◽  
pp. 662-715 ◽  
Author(s):  
Kenyon S. Tweedell
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Adult Stem Cells ◽  
Embryonic Stem ◽  
Normal Function ◽  
Cell Recovery ◽  
Stem Cell Lines

This article is intended to give the nonspecialist an insight into the nuances of “clones”, cloning, and stem cells. It distinguishes embryonic and adult stem cells, their normal function in the organism, their origin, and how they are recovered to produce stem cell lines in culture. As background, the fundamental processes of embryo development are reviewed and defined, since the manipulation of stem cell lines into desired specialized cells employs many of the same events. Stem cells are defined and characterized and shown how they function in the intact organism during early development and later during cell regeneration in the adult. The complexity of stem cell recovery and their manipulation into specific cells and tissue is illustrated by reviewing current experimentation on both embryonic and adult stem cells in animals and limited research on human stem cell lines. The current and projected use of stem cells for human diseases and repair, along with the expanding methodology for the recovery of human embryonic stem cells, is described. An assessment on the use of human embryonic stem cells is considered from ethical, legal, religious, and political viewpoints.

scholarly journals Cytotrophoblast stem cell lines derived from human embryonic stem cells and their capacity to mimic invasive implantation events

2006 ◽  
Vol 21 (6) ◽  
pp. 1349-1358 ◽  
Author(s):  
R. Harun ◽  
L. Ruban ◽  
M. Matin ◽  
J. Draper ◽  
N.M. Jenkins ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Stem Cell Lines

Will Embryonic Stem Cells Change Health Policy?

2010 ◽  
Vol 38 (2) ◽  
pp. 342-351 ◽  
Author(s):  
William M. Sage
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Health Policy ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Stem Cell Lines ◽  
Access And Quality ◽  
Stem Cell Science

Essays on stem cell policy seem to fall into three categories. Some essays in this collection are about logic and principles. Others are about practices and beliefs. The former group draws lines and defends them, a normative project. The latter group attempts to explain the lines that already exist, a descriptive project that may have important normative goals. Still other essays, by scientists, are about growing stem cell lines instead of drawing them.The purpose of this essay is to situate the lines being drawn around stem cell science in the larger landscape of health policy. I am interested in the things that cause health policy to take particular directions and the consequences of those directions for cost, access, and quality — all of which are determined in part by biomedical innovations such as those potentially derived from stem cells.

P–797 A novel method for establishing human embryonic stem cells independent of feeder cells

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
B Cai
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Embryonic Stem Cell ◽  
Success Rate ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Feeder Cells ◽  
Novel Method

Abstract Study question Is there a efficient establishing method of human embryonic stem cells directly from the human blastocysts independent of feeder cells? Summary answer We established a novel method of generating human embryonic stem cells directly from human blastocysts independent of feeder layer cells. What is known already Establishing embryonic stem cells lines mainly needed to coculture ICM clumps with feeder cells (like mouse or human fibroblasts) ,this brought in potential heterogeneous pollution.Although there had be some reports about generating human ESCs independent of feeder cells,but the efficiency was low and conditioned medium were unstable and also had the biological contamination. Study design, size, duration We used ten day5/6 donated human blastocysts from our reproductive center ,most of them were genetically diseased embryos with abnormal PGT diagnosis.After establishing ESCs procedure , all the cell lines were identified with pluripotency and differentiation potential tests.The success rate of system was calculated and compared with the conventional methods. Participants/materials, setting, methods In brief, ICM clumps were separated mechanically by using a micromanipulation system,and then transferred to a 30ul mTESR plus culture media drop pretreated with the geltrex (1:100 dilution) matrix and oxygen concentration was 5%. When cells attached and migrated,we also used laser to destroy the remaining trophoblast cells.About 10 days,the typical ES clone can be mechanically passaged and cells can be cultured in normal oxygen concentrations after passage 2. . Main results and the role of chance Using this method we had successfully established nine embryonic stem cell lines from donated human blastocysts ,the success rate was 90% (9/10). Each cell lines had passed the evaluation test of embryonic stem cell. When compared with the conventional feeder cells dependent method,our novol methods not only eliminated the pollution from heterogeneous cells,but also had higher success rate (90% vs 25%). Limitations, reasons for caution Due to the scarcity of donated human blastocysts, this experiment was a single-center experiment with small samples. Wider implications of the findings: We speculated that the batch differences of culture dishes, matrix and culture medium might affect the establish efficiency , and how to carry out a high level of quality control work might be the key factor to keep the system stable. Trial registration number basic research

P-797 A novel method for establishing human embryonic stem cells independent of feeder cells

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
B Cai
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Embryonic Stem Cell ◽  
Success Rate ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Feeder Cells ◽  
Novel Method

Abstract Study question Is there a efficient establishing method of human embryonic stem cells directly from the human blastocysts independent of feeder cells? Summary answer We established a novel method of generating human embryonic stem cells directly from human blastocysts independent of feeder layer cells. What is known already Establishing embryonic stem cells lines mainly needed to coculture ICM clumps with feeder cells (like mouse or human fibroblasts), this brought in potential heterogeneous pollution. Although there had be some reports about generating human ESCs independent of feeder cells, but the efficiency was low and conditioned medium were unstable and also had the biological contamination. Study design, size, duration We used ten day5/6 donated human blastocysts from our reproductive center, most of them were genetically diseased embryos with abnormal PGT diagnosis. After establishing ESCs procedure, all the cell lines were identified with pluripotency and differentiation potential tests. The success rate of system was calculated and compared with the conventional methods. Participants/materials, setting, methods In brief, ICM clumps were separated mechanically by using a micromanipulation system,and then transferred to a 30ul mTESR plus culture media drop pretreated with the geltrex (1:100 dilution) matrix and oxygen concentration was 5%. When cells attached and migrated,we also used laser to destroy the remaining trophoblast cells. About 10 days,the typical ES clone can be mechanically passaged and cells can be cultured in normal oxygen concentrations after passage 2.. Main results and the role of chance Using this method we had successfully established nine embryonic stem cell lines from donated human blastocysts, the success rate was 90% (9/10). Each cell lines had passed the evaluation test of embryonic stem cell. When compared with the conventional feeder cells dependent method,our novol methods not only eliminated the pollution from heterogeneous cells,but also had higher success rate (90% vs 25%). Limitations, reasons for caution Due to the scarcity of donated human blastocysts, this experiment was a single-center experiment with small samples. Wider implications of the findings We speculated that the batch differences of culture dishes, matrix and culture medium might affect the establish efficiency, and how to carry out a high level of quality control work might be the key factor to keep the system stable. Trial registration number basic research

Hematopoietic progenitor/stem cells immortalized byLhx2 generate functional hematopoietic cells in vivo

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 3939-3946 ◽  
Author(s):  
Perpétua Pinto do Ó ◽  
Karin Richter ◽  
Leif Carlsson
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Progenitor ◽  
Generate Functional ◽  
Hematopoietic Stem

Hematopoietic stem cells (HSCs) are unique in their capacity to maintain blood formation following transplantation into immunocompromised hosts. Expansion of HSCs in vitro is therefore important for many clinical applications but has met with limited success because the mechanisms regulating the self-renewal process are poorly defined. We have previously shown that expression of the LIM-homeobox gene Lhx2 in hematopoietic progenitor cells derived from embryonic stem cells differentiated in vitro generates immortalized multipotent hematopoietic progenitor cell lines. However, HSCs of early embryonic origin, including those derived from differentiated embryonic stem cells, are inefficient in engrafting adult recipients upon transplantation. To address whetherLhx2 can immortalize hematopoietic progenitor/stem cells that can engraft adult recipients, we expressed Lhx2 in hematopoietic progenitor/stem cells derived from adult bone marrow. This approach allowed for the generation of immortalized growth factor–dependent hematopoietic progenitor/stem cell lines that can generate erythroid, myeloid, and lymphoid cells upon transplantation into lethally irradiated mice. When transplanted into stem cell–deficient mice, these cell lines can generate a significant proportion of circulating erythrocytes in primary, secondary, and tertiary recipients for at least 18 months. Thus, Lhx2immortalizes multipotent hematopoietic progenitor/stem cells that can generate functional progeny following transplantation into lethally irradiated hosts and can long-term repopulate stem cell–deficient hosts.

294 NAÏVE STATE-LIKE PLURIPOTENT STEM CELL LINES DERIVED FROM PORCINE EMBRYONIC FIBROBLASTS

2013 ◽  
Vol 25 (1) ◽  
pp. 294
Author(s):  
J.-K. Park ◽  
K.-H. Choi ◽  
D.-C. Son ◽  
J.-I. Oh ◽  
C.-K. Lee
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Pluripotent Stem Cells ◽  
Pluripotent Stem Cell ◽  
Embryonic Stem ◽  
Embryonic Fibroblasts ◽  
Primed State ◽  
Exogenous Factors ◽  
Stem Cell Lines

A recent study has reported that pluripotent stem cells can be categorized according to their pluripotent state. The first is the “naïve” state, which is characterised by small, round or dome-shaped colony morphologies, LIF and BMP4 signalling pathways, and 2 active X chromosomes in females; mouse embryonic stem cells (mESC) represent this type. A second “primed” state has also been described and is possible in mouse epiblast stem cells (mEpiSC) or human embryonic stem cells (hESC). These primed state pluripotent stem cells display flattened monolayer colony morphologies, FGF and nodal/activin signalling pathways, and X chromosome inactivation in females. Meanwhile, a few studies have reported that primed pluripotent stem cell lines could be reverted to a naïve pluripotent state using various exogenous factors including GSK3β and MEK inhibitors (2i), LIF, hypoxic conditions, and upregulation of Oct3 or klf4. Therefore, the purpose of this study was to investigate whether a LIF-dependent naïve pluripotent stem cell line could be derived from porcine embryonic fibroblasts (PEF) via various previously reported factors. We were able to successfully induce PEF into a naïve state-like pluripotent stem cell line by viral infection using FUW-tetO-hOCT4, FUW-tetO-hSOX2, FUW-tetO-hKlf4, FUW-tetO-hMYC, and FUW-M2rtTA obtained from Addgene and addition of 2i and LIF. These naive state-like pluripotent stem cells display mESC-like morphologies, clonogenicity by trypsin, and expression of Oct4, Sox2, Nanog, and SSEA1 using PCR, immunocytochemistry, and fluorescence-activated cell sorting. All cell lines maintained stemness characteristics and stable morphology for more than 30 passages. In addition, naïve state-like pluripotent stem cells could be induced to differentiate to fibroblast-like cells by withdrawal of doxycycline, lif, and 2i. These differentiated cells could be regenerated into naïve state-like pluripotent stem cells by addition of doxycycline, lif, and 2i. We suggest that, as a nonpermissive species, the porcine species undergoes reprogramming into a primed state during the establishment of pluripotent stem cell lines and needs various exogenous factors, including continuous transgene expression, GSK3β and MEK inhibitors (2i), and LIF to be induced into naïve state-like pluripotent stem cells. This work was supported by the BioGreen 21 Program (PJ0081382011), Rural Development Administration, Republic of Korea.

scholarly journals Patentability of the Human Embryonic Stem Cell Lines: A Legal and Ethical Aspect

2020 ◽  
Author(s):  
Tansu Sayar Kanyış ◽  
Ezgi Arslan ◽  
Oğuzhan Kanyış
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Human Embryo ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Point Of View ◽  
Stem Cell Lines

In this study, patentability of the human embryonic stem cell lines has discussed in the legal and ethical perspectives. In vitro human embryonic stem cells can be defined as body parts that are departed from the body. Human embryonic stem cell lines are constituted of differentiated self-renewal pluripotent stem cells, which means they have no characteristics to become a human-being. However, interpreting the terms like human embryo and right to property widely can cause the human embryonic stem cell lines are misunderstood as unpatentable. For our point of view, giving the human embryo the protections of both personal rights of the donor and the right to property of the owner of the invention does not reduce the legal/moral status of the human embryo. Besides, the obligations which these rights imposes to their owners, such as the principle of human dignity and prohibition of financial gain can protect the human embryo in a better way.

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