Scientific considerations relating to the ethics of the use of human embryonic stem cells in research and medicine

2001 ◽  
Vol 13 (1) ◽  
pp. 23 ◽  
Author(s):  
Martin F. Pera
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Es Cells ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
The Body ◽  
Es Cell ◽  
Developmental Potential ◽  
Wide Range ◽  
Human Es Cells

The recent development of embryonic stem (ES) cells from human blastocysts has the potential to revolutionize many of our approaches to human biology and medicine. Continued objection to the use of human ES cells on ethical grounds may inhibit progress or defer this opportunity indefinitely. It is essential that the ethical discussion proceed on a sound scientific basis. The ethical controversy surrounding human ES cells concerns their origin from human blastocysts and the perception of their developmental potential. It is likely that the worldwide requirement for human ES cells will be met by the development of a small number of cell lines, as has been the case in the mouse; current rates of success for human ES cell establishment suggest that only a modest number of embryos will be required to achieve this goal. It is in the public interest that human ES cell lines be derived under circumstances that will enable their widespread distribution with minimum encumbrances to academic researchers throughout the world. In considering the developmental potential of ES cells, an important distinction exists between pluripotentiality, or the ability to develop into a wide range of somatic and extraembryonic tissues, and totipotentiality, the ability of a cell or collection of cells to give rise to a new individual given adequate maternal support. There is no evidence that ES cells from any species can give rise to a new individual except when combined with cells which are the immediate progeny of a zygote. These developmental limitations of ES cells appear to relate to their inability to undergo axis formation and to generate the body plan. Alternatives to blastocyst-derived ES cells include embryonic germ cells, adult tissue stem cells, transdetermination of committed somatic cells, and therapeutic cloning. These research areas are complimentary and synergistic to ES cell research and it is premature and counterproductive to suggest that one avenue should be pursued in preference to another. The combination of cloning and ES cell technology has the potential to address many important issues in transplantation medicine and research, but a better understanding of the reprogramming of somatic cells is required before we can regard ES cells derived from normal nd nuclear transfer blastocysts as equivalent.

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

Human embryonic stem cells

2000 ◽  
Vol 113 (1) ◽  
pp. 5-10 ◽  
Author(s):  
M.F. Pera ◽  
B. Reubinoff ◽  
A. Trounson
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Embryonal Carcinoma ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Carcinoma Cells ◽  
Mouse Es Cells ◽  
Undifferentiated State ◽  
Human Es Cells

Embryonic stem (ES) cells are cells derived from the early embryo that can be propagated indefinitely in the primitive undifferentiated state while remaining pluripotent; they share these properties with embryonic germ (EG) cells. Candidate ES and EG cell lines from the human blastocyst and embryonic gonad can differentiate into multiple types of somatic cell. The phenotype of the blastocyst-derived cell lines is very similar to that of monkey ES cells and pluripotent human embryonal carcinoma cells, but differs from that of mouse ES cells or the human germ-cell-derived stem cells. Although our understanding of the control of growth and differentiation of human ES cells is quite limited, it is clear that the development of these cell lines will have a widespread impact on biomedical research.

scholarly journals Human embryonic stem cells for brain repair?

2007 ◽  
Vol 363 (1489) ◽  
pp. 87-99 ◽  
Author(s):  
Su-Chun Zhang ◽  
Xue-Jun Li ◽  
M Austin Johnson ◽  
Matthew T Pankratz
Keyword(s):  
Stem Cells ◽  
Motor Neurons ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dopamine Neurons ◽  
Neural Cells ◽  
Es Cell ◽  
Human Es Cells

Cell therapy has been perceived as the main or ultimate goal of human embryonic stem (ES) cell research. Where are we now and how are we going to get there? There has been rapid success in devising in vitro protocols for differentiating human ES cells to neuroepithelial cells. Progress has also been made to guide these neural precursors further to more specialized neural cells such as spinal motor neurons and dopamine-producing neurons. However, some of the in vitro produced neuronal types such as dopamine neurons do not possess all the phenotypes of their in vivo counterparts, which may contribute to the limited success of these cells in repairing injured or diseased brain and spinal cord in animal models. Hence, efficient generation of neural subtypes with correct phenotypes remains a challenge, although major hurdles still lie ahead in applying the human ES cell-derived neural cells clinically. We propose that careful studies on neural differentiation from human ES cells may provide more immediate answers to clinically relevant problems, such as drug discovery, mechanisms of disease and stimulation of endogenous stem cells.

Derivation characteristics and perspectives for mammalian pluripotential stem cells

2005 ◽  
Vol 17 (2) ◽  
pp. 135 ◽  
Author(s):  
Alan Trounson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Embryonic Stem ◽  
The Body ◽  
Preimplantation Embryos ◽  
Specific Cell ◽  
Wide Range

Pluripotential stem cells have been derived in mice and primates from preimplantation embryos, postimplantation embryos and bone marrow stroma. Embryonic stem cells established from the inner cell mass of the mouse and human blastocyst can be maintained in an undifferentiated state for a long time by continuous passage on embryonic fibroblasts or in the presence of specific inhibitors of differentiation. Pluripotential stem cells can be induced to differentiate into all the tissues of the body and are able to colonise tissues of interest after transplantation. In mouse models of disease, there are numerous examples of improved tissue function and correction of pathological phenotype. Embryonic stem cells can be derived by nuclear transfer to establish genome-specific cell lines and, in mice, it has been shown that embryonic stem cells are more successfully reprogrammed for development by nuclear transfer than somatic cells. Pluripotential stem cells are a very valuable research resource for the analysis of differentiation pathways, functional genomics, tissue engineering and drug screening. Clinical applications may include both cell therapy and gene therapy for a wide range of tissue injury and degeneration. There is considerable interest in the development of pluripotential stem cell lines in many mammalian species for similar research interests and applications.

Efficient Non-Viral Transfection of Mouse and Human Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5267-5267
Author(s):  
Zwi N. Berneman ◽  
Jeremy P. Brown ◽  
Sjaak Van der Sar ◽  
Dave Van den Plas ◽  
Lena Van den Eeden ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Reporter Genes ◽  
Cell Phenotype ◽  
Es Cell ◽  
Short Term ◽  
Human Es Cells ◽  
Mrna Electroporation ◽  
Mouse Es Cell

Abstract BACKGROUND: Development of efficient non-viral gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study we investigated mRNA electroporation as a tool for short-term gene transfer in both mouse and human ES cells. METHODS: Culture and mRNA electroporation conditions for feeder-free cultured mouse and human ES cells were optimized on three mouse ES cell lines (E14, R1 and HM-1) and one human ES cell line (H9). After electroporation with EGFP mRNA, transfected ES cell populations were analyzed by FACS for EGFP expression, viability and phenotype. Also, stably-transfected mouse ES cell lines containing Lox-P or FRT-flanked reporter genes were electroporated with mRNA encoding Cre- or FLPe-recombinase proteins. Monitoring recombination efficiency was done based on the appearance and/or disappearance of fluorescent reporter genes, as determined by FACS analysis. ES cells that underwent recombination were further analyzed for potential to differentiate towards the neural lineage and differentiated cells were analyzed by FACS for expression of neural markers. RESULTS: (A) Electroporation of EGFP mRNA in mouse ES cells resulted in high level transgene expression (>90% EGFP positive cells) combined with low electroporation-induced cell mortality (>90% viable cells). Moreover, the electroporation procedure did not influence ES cell phenotype and further cell culture of undifferentiated ES cell populations. Electroporation of mRNA encoding Cre- or FLPe-recombinase proteins in stably-transfected mouse ES cell lines containing LoxP- or FRT-flanked reporter genes resulted in a recombination efficiency of respectively 75% and 90%. Moreover, these recombination events did not have influence on ES cell phenotype, viability, growth potential, and their ability to differentiate towards neural cell types upon retinoic acid stimulation. (B) Although human ES cells are much more sensitive as compared to mouse ES cells, we were able to develop improved culture and electroporation conditions for feeder-free maintained H9 human ES cells, which resulted in high level transgene expression (>90% EGFP+ cells) combined with high cell viability (>90% viable cells) after EGFP mRNA electroporation. CONCLUSIONS: RNA electroporation is a highly efficient method for short-term genetic loading of both mouse and human ES cells. Ongoing research now focuses on either short-term (via direct mRNA electroporation) or sustained (via mRNA-based FLPe-recombination) expression of transcription factors in ES cells and their influence on cell-fate within in vitro cultured embryoid bodies.

scholarly journals Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758 ◽  
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy

2005 ◽  
Vol 85 (2) ◽  
pp. 635-678 ◽  
Author(s):  
Anna M. Wobus ◽  
Kenneth R. Boheler
Keyword(s):  
Developmental Biology ◽  
Cell Lines ◽  
Tissue Regeneration ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Populations ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Human Es Cells

Stem cells represent natural units of embryonic development and tissue regeneration. Embryonic stem (ES) cells, in particular, possess a nearly unlimited self-renewal capacity and developmental potential to differentiate into virtually any cell type of an organism. Mouse ES cells, which are established as permanent cell lines from early embryos, can be regarded as a versatile biological system that has led to major advances in cell and developmental biology. Human ES cell lines, which have recently been derived, may additionally serve as an unlimited source of cells for regenerative medicine. Before therapeutic applications can be realized, important problems must be resolved. Ethical issues surround the derivation of human ES cells from in vitro fertilized blastocysts. Current techniques for directed differentiation into somatic cell populations remain inefficient and yield heterogeneous cell populations. Transplanted ES cell progeny may not function normally in organs, might retain tumorigenic potential, and could be rejected immunologically. The number of human ES cell lines available for research may also be insufficient to adequately determine their therapeutic potential. Recent molecular and cellular advances with mouse ES cells, however, portend the successful use of these cells in therapeutics. This review therefore focuses both on mouse and human ES cells with respect to in vitro propagation and differentiation as well as their use in basic cell and developmental biology and toxicology and presents prospects for human ES cells in tissue regeneration and transplantation.

scholarly journals ERas is Expressed in Primate Embryonic Stem Cells but not Related to Tumorigenesis

2009 ◽  
Vol 18 (4) ◽  
pp. 381-389 ◽  
Author(s):  
Yujiro Tanaka ◽  
Tamako Ikeda ◽  
Yukiko Kishi ◽  
Shigeo Masuda ◽  
Hiroaki Shibata ◽  
...  
Keyword(s):  
Stem Cells ◽  
Nonhuman Primate ◽  
Stromal Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Human Es Cells ◽  
Nog Mice ◽  
Human Orthologue

The ERas gene promotes the proliferation of and formation of teratomas by mouse embryonic stem (ES) cells. However, its human orthologue is not expressed in human ES cells. This implies that the behavior of transplanted mouse ES cells would not accurately reflect the behavior of transplanted human ES cells and that the use of nonhuman primate models might be more appropriate to demonstrate the safety of human ES cell-based therapies. However, the expression of the ERas gene has not been examined in nonhuman primate ES cells. In this study, we cloned the cynomolgus homologue and showed that the ERas gene is expressed in cynomolgus ES cells. Notably, it is also expressed in cynomolgus ES cell-derived differentiated progeny as well as cynomolgus adult tissues. The ERas protein is detectable in various cynomolgus tissues as assessed by immunohistochemisty. Cynomolgus ES cell-derived teratoma cells, which also expressed the ERas gene at higher levels than the undifferentiated cynomolgus ES cells, did not develop tumors in NOD/Shi- scid, IL-2Rγnull (NOG) mice. Even when the ERas gene was overexpressed in cynomolgus stromal cells, only the plating efficiency was improved and the proliferation was not promoted. Thus, it is unlikely that ERas contributes to the tumorigenicity of cynomolgus cells. Therefore, cynomolgus ES cells are more similar to human than mouse ES cells despite that ERas is expressed in cynomolgus and mouse ES cells but not in human ES cells.

226 CELL LINE-DEPENDENT GENE EXPRESSION PROFILES IN MOUSE EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 229
Author(s):  
S. Mamo ◽  
J. Kobolak ◽  
S. Becker ◽  
M. Horsch ◽  
J. Beckers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Molecular Markers ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Gene Expression Profiles ◽  
Es Cell

Molecular phenotyping studies carried out so far on different embryonic stem cells (ESC) have focused mainly on the identification of molecular markers responsible for pluripotency. Unlike these, the goals of our study were to compare and functionally characterize the gene expression profiles of R1 ESC established from F1 (129X1/SvJ � 129S1) blastocysts (Nagy et al. 1993 PNAS 90, 8424–8428) and HM-1 ESC established from an inbred strain (Selfridge et al. 1992 Somat. Cell Mol. Genet. 18, 325–336), as our earlier study showed performance variations between these cells. ES cells were cultured on a mitomycin C-treated mouse embryonic fibroblast feeder cell layer. Cells were grown in standard ES cell medium changed daily [high glucose DMEM (GIBCO-Invitrogen, Carlsbad, CA, USA) supplemented with Na pyruvate (0.11% w/w; GIBCO-Invitrogen), 0.1 mM 2-mercaptoethanol (Sigma-Aldrich, St Louis, MO, USA), fetal bovine serum (15% v/v; HyClone, Logan, UT, USA), 1000 U mL-1 murine-LIF (Chemicon International, Temecula, CA, USA), and antibiotics (penicillin: 50 U mL-1, streptomycin: 50 �g mL-1; Sigma-Aldrich)]. Total RNA was isolated from aliquots of R1 ES cells at passage 13 and HM-1 ES cells at passage 23 using RNeasy Midi kit (Qiagen, D�sseldorf, Germany) procedures. Fifteen �g of total RNA each from the contrasting samples were used for reverse transcription and labeling with either Cy3 or Cy5 dyes (Amersham, Buckinghamshire, UK). The labeled samples were dissolved in hybridization buffer, added to the cDNA arrays (custom produced at GSF) containing over 21 000 sequences, and hybridized for 17 h at 42�C. The microarray results of 4 independent hybridizations were analyzed, and differentially regulated genes were identified. Finally, the results of 4 randomly selected genes were verified by real-time PCR analysis. The analysis revealed 55 transcripts that showed significant variation (P < 0.01) between the 2 ES cell lines. Of these, 8 transcripts were up-regulated and the rest down-regulated in the HM-1 ES cells. Most of these genes were over-represented in important biological processes such as growth and development (21%), cell organization and biogenesis (11%), regulatory roles (21%), and organogenesis (14%). Moreover, the verification analysis using real-time PCR has confirmed the results of microarray. Thus, based on the detailed analysis, and confirmation of the results with independent analysis, it is possible to conclude that the expression profile reflected the true molecular variations between the 2 ES cell lines, and the identified transcripts can serve as molecular markers that explain biological differences between the 2 ES cell lines. This work was supported by EU FP6 (MEXT-CT-2003-509582 and 518240), Wellcome Trust (Grant No.070246), and Hungarian National Science Fund (OTKA T046171).

297 DERIVATION AND CHARACTERIZATION OF THE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER-DERIVED BOVINE EMBRYONIC STEM CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 246
Author(s):  
S. H. Jeong ◽  
H. S. Kim ◽  
H. Lee ◽  
K. J. Uh ◽  
S. H. Hyun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Lines ◽  
Mass Culture ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Es Cell ◽  
Trophoblast Cells

Bovine transgenic embryonic stem (ES) cells have not been reported yet because it seems that the derivation methods and the culture conditions for the inner cell mass are neither consistent nor optimized. Isolation of inner cell mass and primary culture of ES colonies is a critical step toward the establishment of authentic bovine ES cell lines. Herein, we reconstructed somatic cell nuclear transferred (SCNT) bovine blastocysts carrying a vector expressing the human INF-α gene, and isolated inner cell masses to derive transgenic bovine embryonic stem cells. In addition, we added 2 inhibitors, inhibition (2i system) of the mitogen-activated protein kinase (Erk1/2) cascade, PD0325901(3 Î1/4M), and of glycogen synthase kinase 3, CHIR99021 (1 Î1/4M), in the inner cell mass primary culture to check reliability of the 2i system for bovine ES culture. The 2 inhibitors made the morphology of colonies more intact, and primary colonies were better maintained in early passages. However, there were no significant effects on the attachment rate and maintenance in late passages (percent of percent over 3 passages: 2i system, 21/38 (55.3%); control, 22/42 (33.3%); P < 0.05). Inner cell masses were isolated mechanically and subcultured by an enzymatic in primary inner cell mass culture. Massive growth of trophoblast cells appears to inhibit inner cell mass growth, so hatching and hatched blastocysts were cut with a needle to remove trophoblast cells. Poor quality blastocysts were attached by the whole seeding method, and the margin trophoblast cells were consecutively removed in early passages. Established bovine ES cells express alkaline phosphatase, Oct-4, SSEA1, SSEA4, Tra-1–60, and Tra-1–81. We confirmed pluripotent gene expression of bovine ES like cells; Oct-4, SSEA1, and Rex 1 were positive, but trophoblast marker CDX2 was negative. This study shows that the 2i system is a reasonable method for use during inner cell mass culture in early passages. We established 6 transgenic nuclear transfer bovine ES cell lines with the 2i system and 4 in vitro fertilized bovine ES cell lines (all were over 10 passages).

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