Culture of human embryos for studies on the derivation of human pluripotent cells: a preliminary investigation

1998 ◽  
Vol 10 (8) ◽  
pp. 557 ◽  
Author(s):  
M-C. Lavoir ◽  
J. Conaghan ◽  
R. A. Pedersen
Keyword(s):  
High Efficiency ◽  
Preliminary Investigation ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Human Embryos ◽  
Heparin Binding ◽  
Embryonic Cells ◽  
Epidermal Growth ◽  
Serum Substitute

Several different culture conditions were evaluated for culturing grade 4embryos (containing 2–4 blastomeres and with >50%fragmentation) 68 h after fertilization to the blastocyst stage. Embryos wereco-cultured with buffalo rat liver (BRL) cells in Menezo's B2 medium withor without 10% v/v synthetic serum substitute (SSS), co-culturedwith BRL cells in KSOM with or without 10% SSS, or cultured in KSOMwith 100 nM heparin binding epidermal growth factor. The most consistentdevelopment was obtained when embryos were co-cultured with BRL cells in KSOM.Rates of development to the blastocyst stage were between 27% and40%. After reaching the blastocyst stage, continued culture of theseblastocysts was only possible in a medium without serum. In a serum-deprivedmedium cells attached and showed initial outgrowth, but did not survivepassaging. Using another approach, inner cell masses (ICMs), isolated fromblastocysts with high efficiency using immunosurgery, were able to attach to afeeder layer in the presence of serum. Some ICMs differentiated whereas otherscould be succesfully passaged up to four times. The embryonic cells were morphologically different from murine embryonic stem cells. Instead ofwell-defined colonies, the human colonies were characterized by individualcells and colonies without defined borders.

From fibroblasts and stem cells: implications for cell therapies and somatic cloning

2005 ◽  
Vol 17 (2) ◽  
pp. 125 ◽  
Author(s):  
Wilfried A. Kues ◽  
Joseph W. Carnwath ◽  
Heiner Niemann
Keyword(s):  
Stem Cells ◽  
Adult Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Culture Conditions ◽  
Human Embryos ◽  
Somatic Stem Cells ◽  
Primary Fibroblast ◽  
Cell Therapies

Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of somatic stem cells, methods to modify the potency of somatic cells and implications for cell-based therapies.

166 EXPRESSION OF PLURIPOTENCY-DETERMINING FACTORS IN IN VITRO-PRODUCED BUFFALO EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 163
Author(s):  
T. Anand ◽  
D. Kumar ◽  
M. K. Singh ◽  
M. S. Chauhan ◽  
R. S. Manik ◽  
...  
Keyword(s):  
Cell Surface ◽  
Expression Patterns ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Tumor Rejection ◽  
Cell Surface Expression ◽  
Human Embryos ◽  
Strong Expression

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of blastocysts. These are pluripotent cells that retain the ability to differentiate into all cell types. Various cell surface antigens, the expressions of which have been widely used as markers to monitor the pluripotency of ESCs, include Oct-4, stage-specific embryonic antigens (SSEAs) such as SSEA-1, SSEA-3, and SSEA-4, and tumor rejection antigens (TRAs) such as TRA-1-60 and TRA-1-81. In this study, the cell surface expression patterns of these markers were examined in in vitro-produced buffalo embryos at the 2-, 4-, 8- to 16-cell, morula, and blastocyst stages using immunofluorescence microscopy. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF, following which the cleaved embryos were cultured for 9 days for production of embryos at different stages (n = 246). The embryos were fixed in 4% paraformaldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 30 min, permeabilized by treatment with 0.1% Triton X-100 in DPBS for 30 min, and incubated first with the blocking solution (4% normal goat serum) for 30 min and then with the primary antibody (Oct-4: clone 9E3; SSEA-1: MC-480; SSEA-3: MC-631; SSEA-4: MC-813-70; TRA-1-60: clone TRA-1-60; and TRA-1-81: clone TRA-1-81, Chemicon� Inc., Temecula, CA, USA) at a dilution of 1:10 to 1:20 for 1 h. After being washed with DPBS, the embryos were incubated with appropriate FITC-labeled second antibody (anti-rat IgM or anti-mouse IgG or IgM, diluted 1:100 to 1:200) for 1 h and then examined under a fluorescence microscope. Oct-4 expression was detected at all embryonic stages starting from the 2-cell to the blastocyst stage, in which ICM, but not trophectoderm cells, exhibited a strong expression. SSEA-4 signal was found to be strongest at the 2-cell stage, with continued expression at all intermediate stages until the blastocyst stage in which there was a strong expression in ICM cells. In contrast, all of the embryonic stages were found to be negative for SSEA-3 expression. The SSEA-1 signal was present at all of the embryonic stages but was very weak. Expression of TRA-1-60 and TRA-1-81, which was detected only on the inner surface of the zona pellucida and in the perivitelline space in early embryonic stages, was absent in morulae and blastocysts. The results of this study indicate that the pluripotency-determining markers are differentially expressed in buffalo embryos and that the pattern of their expression is distinct from that of murine and human embryos but resembles to some extent that of goat embryos. Comparison of the expression pattern of these markers needs to be done between embryonic cells and ESCs for a better understanding of their developmental regulation.

Production and analysis of ES cell aggregation chimeras

1999 ◽  
Author(s):  
Andras Nagy ◽  
Janet Rossant
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Aggregation ◽  
Blastocyst Stage ◽  
Original Method ◽  
Embryonic Cells ◽  
Es Cell ◽  
Cleavage Stage

Embryonic stem (ES) cells behave like normal embryonic cells when returned to the embryonic environment after injection into a host blastocyst or after aggregation with earlier blastomere stage embryos. In such chimeras, ES cells behave like primitive ectoderm or epiblast cells (1), in that they contribute to all lineages of the resulting fetus itself, as well as to extraembryonic tissues derived from the gastrulating embryo, namely the yolk sac mesoderm, the amnion, and the allantois. However, even when aggregated with preblastocyst stage embryos, ES cells do not contribute to derivatives of the first two lineages to arise in development, namely, the extraembryonic lineages: trophoblast and primitive endoderm (2). The pluripotency of ES cells within the embryonic lineages is critical to their use in introducing new genetic alterations into mice, because truly pluripotent ES cells can contribute to the germline of chimeras, as well as all somatic lineages. However, the ability of ES cells to co-mingle with host embryonic cells, specifically in the embryonic, but not the major extraembryonic lineages, opens up a variety of possibilities for analysing gene function by genetic mosaics rather than by germline mutant analysis alone (3). There are two basic methods for generating pre-implantation chimeras in mice, whether it be embryo ↔ embryo or ES cell ↔ embryo chimeras. Blastocyst injection, in which cells are introduced into the blastocoele cavity using microinjection pipettes and micromanipulators, has been the method of choice for most ES cell chimera work (see Chapter 4). However, the original method for generating chimeras in mice, embryo aggregation, is considerably simpler and cheaper to establish in the laboratory. Aggregation chimeras are made by aggregating cleavage stage embryos together, or inner cell mass (ICM) or ES cells with cleavage stage embryos, growing them in culture to the blastocyst stage, and then transferring them to the uterus of pseudopregnant recipients to complete development. This procedure can be performed very rapidly by hand under the dissecting microscope, thus making possible high throughput production with minimal technical skill (4). In this chapter we describe some of the uses of pre-implantation chimeras, whether made by aggregation or blastocyst injection, but focus on the technical aspects of aggregation chimera generation. We also discuss the advantages and disadvantages of aggregation versus blastocyst injection for chimera production.

277 PLASMID MEDIATED EXPERIMENTAL TELOMERE EXTENSION IN BOVINE EMBRYOS BY ECTOPIC EXPRESSION OF HUMAN TERT

2009 ◽  
Vol 21 (1) ◽  
pp. 235 ◽  
Author(s):  
K. Iqbal ◽  
W. A. Kues ◽  
H. Niemann
Keyword(s):  
Telomere Length ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Mrna Levels ◽  
Bovine Embryos ◽  
Fluid Medium ◽  
Gfp Fluorescence ◽  
Morula Stage

Telomeres are composed of repetitive hexanucleotide sequences, (TTAGGG)n, encompassing several kilobase pairs, and protecting the ends of eukaryotic chromosomes. In somatic cells, the telomeres are eroded with each cell division and may reach a critical length at which viability becomes compromised. In germ cells, expression of the enzyme telomerase leads to restoration of telomere length. During early cleavage and up to the morula stage, telomerase is not active or is found only at low levels, but high telomerase activity is detectable at the blastocyst stage in bovine and human embryos. The goal of this study was to unravel the physiological consequences of an ectopic overexpression of the catalytic subunit of telomerase (TERT) in early bovine embryos. Human TERT (hTERT) has 80% sequence homology with bovine TERT. Oocytes were collected by slicing ovaries obtained from a local abattoir, followed by maturation in TCM-199 supplemented with eCG and hCG. The IVF of matured oocytes was carried out in Fert-TALP supplemented with hypotaurine, heparin, and epinephrine. Fertilized oocytes were used for DNA microinjection experiments; injected zygotes and nontreated controls were cultured in modified synthetic oviduct fluid medium (SOF) in reduced oxygen concentration. Two plasmid encoding CMV promoter-driven sequences of hTERT and green fluorescent protein (GFP) were coinjected in bovine zygotes, and GFP driven by a muscle specific promoter was injected for mock experiments. The hTERT and GFP were co-injected to allow live separation of embryos. A total of 768 bovine embryos were injected; 468 (61%) of the treated embryos showed specific GFP-fluorescence. Of a total of 132 blastocysts (17%), 45 showed GFP fluorescence (34%). The GFP-expressing embryos were selected at various developmental stages and were analyzed for hTERT expression. Both endogenous TERT and ectopic hTERT mRNA levels were assessed by RT-PCR from zygote to blastocyst. The mRNA level of the ectopic hTERT began to increase from the 4- to the 8-cell stage and remained high up to the morula stage. Embryos at the morula and blastocyst stages were spread on slides and analyzed by quantitative fluorescence in situ hybridization (qFISH). A Cy3-labeled 18-mer peptide nucleic acid (PNA) probe was used to hybridize the telomeres. The resulting spot intensities were quantified by using TFL-Telo software and were statistically analyzed. A modest increase in telomere length was observed in hTERT injected [775 ± 69 fluorescence unit (fu)] group compared to uninjected control (679 ± 75 fu) group at blastocyst stage. In conclusion, this study demonstrates that the ectopic expression of hTERT in embryos results in telomere elongation; overexpression of TERT may facilitate the derivation of bovine embryonic stem cells. Supported by DFG and Goyaike SAACIYF.

scholarly journals Embryonic Stem Cell Research and Religion: The Ban on Federal Funding as a Violation of the Establishment Clause

2006 ◽  
Vol 68 (1) ◽  
Author(s):  
Larry J. Pittman
Keyword(s):  
Stem Cells ◽  
Medical Condition ◽  
Embryonic Stem ◽  
Petri Dish ◽  
Medical Personnel ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Vitro Fertilization ◽  
New Treatments

Many Americans die each day from diseases affecting the heart, liver, kidneys, brain and a whole host of other bodily organs. Scientific researchers are constantly trying to develop new treatments for such medical conditions. Presently, the research community is working hard to develop medical treatments using stem cells from human embryos. That process involves extracting stem cells from either excess embryos that are no longer needed for in vitro fertilization or from embryos that are created through therapeutic cloning. At the blastocyst stage, about five days after the beginning of an embryo, researchers extract stem cells from the embryo and place them in a petri dish where the cells divide to produce a line of millions of stem cells. These stem cells are undifferentiated, meaning that they are still capable of transforming themselves into many different types of cells that exist in the human body. The hope is that physicians and other medical personnel will one day be able to inject these stem cells into a patient’s diseased heart, kidney, brain, liver, spinal cord or other organ, and the stem cells willtransform themselves into the same type of cells that comprise the host organ. The expectation is that the stem cells will repair the patient’s heart or other organs by curing diseases and otherwise improving the patient’s medical condition and life expectancy.

scholarly journals Use of Insulin to Increase Epiblast Cell Number: Towards a New Approach for Improving ESC Isolation from Human Embryos

2013 ◽  
Vol 2013 ◽  
pp. 1-7
Author(s):  
Jared M. Campbell ◽  
Michelle Lane ◽  
Ivan Vassiliev ◽  
Mark B. Nottle
Keyword(s):  
Embryonic Stem ◽  
Cell Number ◽  
Poor Quality ◽  
Blastocyst Stage ◽  
Human Embryos ◽  
Cleavage Stage ◽  
Embryo Viability ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Hesc Derivation

Human embryos donated for embryonic stem cell (ESC) derivation have often been cryopreserved for 5–10 years. As a consequence, many of these embryos have been cultured in media now known to affect embryo viability and the number of ESC progenitor epiblast cells. Historically, these conditions supported only low levels of blastocyst development necessitating their transfer or cryopreservation at the 4–8-cell stage. As such, these embryos are donated at the cleavage stage and require further culture to the blastocyst stage before hESC derivation can be attempted. These are generally of poor quality, and, consequently, the efficiency of hESC derivation is low. Recent work using a mouse model has shown that the culture of embryos from the cleavage stage with insulin to day 6 increases the blastocyst epiblast cell number, which in turn increases the number of pluripotent cells in outgrowths following plating, and results in an increased capacity to give rise to ESCs. These findings suggest that culture with insulin may provide a strategy to improve the efficiency with which hESCs are derived from embryos donated at the cleavage stage.

scholarly journals Heparin Binding Epidermal Growth Factor-Like Growth Factor and PD169316 Prevent Apoptosis in Mouse Embryonic Stem Cells

2008 ◽  
Vol 145 (2) ◽  
pp. 177-184 ◽  
Author(s):  
M. Krishnamoorthy ◽  
J. Heimburg-Molinaro ◽  
A. M. Bargo ◽  
R. J. Nash ◽  
R. J. Nash
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Heparin Binding ◽  
Epidermal Growth
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