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.

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.

220 NONHUMAN PRIMATE EMBRYONIC STEM CELLS SIMILAR TO THE BIOLOGICAL PROPERTIES OF MOUSE EMBRYONIC STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 222
Author(s):  
A. Kusanagi ◽  
J. Yamasaki ◽  
C. Iwatani ◽  
H. Tsuchiya ◽  
R. Torii
Keyword(s):  
Nonhuman Primate ◽  
Cynomolgus Monkey ◽  
Es Cells ◽  
Embryonic Stem ◽  
Biological Properties ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Mouse Es Cell

Human and mouse embryonic stem (ES) cells are derived from the inner cell mass of preimplantation blastocysts and human ES cells were long thought to be equivalent to mouse ES cells, despite clear morphological difference and different signalling pathways to maintain their pluripotency between these two ES cell types. Mouse ES cells depend on leukemia inhibitory factor (LIF) and bone morphogenic protein 4 (BMP4) signalling, whereas their human counterparts rely on basic fibroblast growth factor (bFGF) and activin A signalling. The biggest difference of two ES cells is the ability of chimera formation and mouse ES cells can contribute chimera but primate ES cells fails to do that. Monkey ES cells in primates only can be tested for chimera formation in vivo due to the ethical issue and cynomolgus monkey is the most common nonhuman primate to be used for the safety study of drug discoveries. The objective of this study was to develop novel cynomolgus monkey ES cells that have similar biological properties with mouse ES cell and our ultimate goal is to establish germline competent nonhuman primate ES cells. Ovarian stimulation and oocyte collection were carried out for the derivation of ES cells as previously described by Torii et al. Briefly, GnRH (0.9 mg/head) was administered to cynomolgus monkey and two weeks later, a micro infusion pump (iPRECIO™, Primetech Corp) contains FSH was implanted subcutaneously. Follicular aspiration was then performed 40 h after hCG injection and metaphase II oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Cynomolgus monkey ES cells were then established under mouse ES cell conditions such as LIF/STAT signalling and a dome tree-dimensional (3D) morphology nonhuman primate ES cells were selected. On the other hands, ES cells that were established with the presence of basic FGF showed conventional layer-type morphology. Dome-type ES cells express pluripotent transcriptional factors such as Oct-3/4, Nonog and Sox2 as same as layer-type ES cells and both ES lines were capable of multilineage differentiations in vitro after embryoid body formation. Dome-type nonhuman ES cells can also form teratomas and differentiated into all three germ layers when grafted into immunodeficiency mice. For fluorescent gene delivery to nonhuman primate ES cells, feeder-free condition was applied and CAG-GFP vector was transfected into ES cells using Neon electroporation system (Invitrogen Inc.) for the tracing ES cells in the transplantation study. In this study, we have established dome-type ES cell lines that similar to mouse ES cells in morphology and signalling pathway. Dome-type nonhuman primate ES cells express pluripotent gene markers and prove their pluripotency both of in vitro and in vivo, in addition, these modifications would be important to create germline competent ES cells.

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.

Human embryonic stem cells: prototypical pluripotential progenitors

2002 ◽  
Vol 10 (3) ◽  
pp. 187-199 ◽  
Author(s):  
R Mollard ◽  
BJ Conley ◽  
AO Trounson
Keyword(s):  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Germ Layers ◽  
Mouse Es Cells ◽  
Morphological Criteria ◽  
Human Es Cells

Embryonic stem (ES) cells are a primitive cell type derived from the inner cell mass (ICM) of the developing embryo. When cultured for extended periods, ES cells maintain a high telomerase activity, normal karyotype and the pluripotential developmental capacity of their ICM derivatives. Such capacity is best demonstrated by mouse ES cells which can contribute to all tissues of the developing embryo following either their injection into host blastocysts or tetraploid embryo complimentation (for a review see Robertson). For both practical and ethical reasons it is not possible to inject human ES cells into blastocysts for the development of a term fetus. However, when injected beneath the testicular capsule of severe combined immunodeficient (SCID) mice, human ES cells form teratomas comprising tissue representatives of all three embryonic germ layers (ectoderm, mesoderm and endoderm) thus attesting to their pluripotency. Based upon morphological criteria, neuronal, cardiac, bone, squamous epithelium, skeletal muscle, gut and respiratory epithelia are readily identifiable within the human ES-cell-derived teratomas. With the demonstrated capability to isolate and maintain pluripotent human ES cells in vitro, their ability to give rise to tissue representatives of all three embryonic germ layers and the technical advances made possible by research on mouse ES cells, a rapid increase in human ES cell research aimed at drug discovery and human cell and gene therapies has occurred. Indeed in the mouse, dissociated embryoid bodies (EBs) have already been demonstrated capable of repopulating the haematopoietic system of recipient animals (for a review see Keller) and mouse ES cells are currently being used in attempts to repair mouse neural degenerative lesions.

Strategies for the isolation and characterization of bovine embryonic stem cells

1994 ◽  
Vol 6 (5) ◽  
pp. 569 ◽  
Author(s):  
RA Cherny ◽  
TM Stokes ◽  
J Merei ◽  
L Lom ◽  
MR Brandon ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Farm Animals ◽  
Preimplantation Embryos ◽  
Es Cell ◽  
Isolation And Characterization

The practical application of advanced breeding technologies and genetic manipulation of domestic animals is dependent on the efficient and routine isolation of embryonic stem (ES) cell lines from these species. ES cell lines of proven totipotency have thus far been isolated only from the mouse. Murine ES cells can be identified by a number of criteria including morphology and characteristics in culture, the presence of specific markers, differentiative capacity and contribution to chimaeras. Reported cell lines derived from ruminant preimplantation embryos do not stably exhibit these characteristics. As demonstrated for the mouse, primordial germ cells may provide an alternative source for pluripotential cell lines. The isolation, culture and preliminary characterization of bovine primordial germ cell-derived (PGCd) cells are described in this paper. The PGCd cells are capable of differentiation in vitro and display murine ES cell markers including alkaline phosphatase. With farm animals, long generation intervals and small numbers of offspring make it important to develop techniques for evaluating chimaeric embryos in vitro before embarking on expensive in vivo programmes. A method for labelling putative pluripotential cells with a fluorochrome marker to follow the fate of such cells was developed. Labelled PGCd cells were injected into blastocysts and the chimaeric embryos were monitored in vitro. Preliminary results demonstrate that the labelled PGCd cells incorporate preferentially within the inner cell mass of the host blastocyst.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Inhibition of Cardiomyocytes Differentiation of Mouse Embryonic Stem Cells by CD38/cADPR/Ca2+ Signaling Pathway

2012 ◽  
Vol 287 (42) ◽  
pp. 35599-35611 ◽  
Author(s):  
Wen-Jie Wei ◽  
Hai-Ying Sun ◽  
Kai Yiu Ting ◽  
Li-He Zhang ◽  
Hon-Cheung Lee ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Embryoid Body ◽  
Troponin T ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Adp Ribosyl Cyclase

Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca2+ mobilizing messenger that is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). The main ADP-ribosyl cyclase in mammals is CD38, a multi-functional enzyme and a type II membrane protein. Here we explored the role of CD38-cADPR-Ca2+ in the cardiomyogenesis of mouse embryonic stem (ES) cells. We found that the mouse ES cells are responsive to cADPR and possess the key components of the cADPR signaling pathway. In vitro cardiomyocyte (CM) differentiation of mouse ES cells was initiated by embryoid body (EB) formation. Interestingly, beating cells appeared earlier and were more abundant in CD38 knockdown EBs than in control EBs. Real-time RT-PCR and Western blot analyses further showed that the expression of several cardiac markers, including GATA4, MEF2C, NKX2.5, and α-MLC, were increased markedly in CD38 knockdown EBs than those in control EBs. Similarly, FACS analysis showed that more cardiac Troponin T-positive CMs existed in CD38 knockdown or 8-Br-cADPR, a cADPR antagonist, treated EBs compared with that in control EBs. On the other hand, overexpression of CD38 in mouse ES cells significantly inhibited CM differentiation. Moreover, CD38 knockdown ES cell-derived CMs possess the functional properties characteristic of normal ES cell-derived CMs. Last, we showed that the CD38-cADPR pathway negatively modulated the FGF4-Erks1/2 cascade during CM differentiation of ES cells, and transiently inhibition of Erk1/2 blocked the enhanced effects of CD38 knockdown on the differentiation of CM from ES cells. Taken together, our data indicate that the CD38-cADPR-Ca2+ signaling pathway antagonizes the CM differentiation of mouse ES cells.

Genomic imprinting in primate embryos and embryonic stem cells

2006 ◽  
Vol 18 (8) ◽  
pp. 817 ◽  
Author(s):  
Shoukhrat M. Mitalipov
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Paternal Allele ◽  
Aberrant Methylation ◽  
Es Cell ◽  
Biallelic Expression ◽  
Cell Based Therapy

Embryonic stem (ES) cells hold promise for cell and tissue replacement approaches to treating human diseases. However, long-term in vitro culture and manipulations of ES cells may adversely affect their epigenetic integrity including imprinting. Disruption or inappropriate expression of imprinted genes is associated with several clinically significant syndromes and tumorigenesis in humans. We demonstrated aberrant biallelic expression of IGF2 and H19 in several rhesus monkey ES cell lines while SNRPN and NDN were normally imprinted and expressed from the paternal allele. In contrast, expanded blastocyst-stage embryos, from which these ES cells were derived, exhibited normal paternal expression of IGF2 and maternal expression of H19. To test the possibility that aberrant methylation at an imprinting centre (IC) upstream of H19 accounts for the relaxed imprinting of IGF2 and H19, we performed comprehensive methylation analysis by investigating methylation profiles of CpG sites within the IGF2/H19 IC. Our results demonstrate abnormal hypermethylation within the IGF2/H19 IC in all analysed ES cell lines consistent with biallelic expression of these genes. Cellular overproliferation and tumour formation resulting from tissue or cell transplantation are potential problems that must be addressed before clinical trials of ES cell-based therapy are initiated.

scholarly journals Monkey Embryonic Stem Cell Lines Expressing Green Fluorescent Protein

2002 ◽  
Vol 11 (7) ◽  
pp. 631-635 ◽  
Author(s):  
Tatsuyuki Takada ◽  
Yutaka Suzuki ◽  
Yasushi Kondo ◽  
Nae Kadota ◽  
Kinji Kobayashi ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Transplantation ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Green Fluorescent

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

Transcriptional heterogeneity in mouse embryonic stem cells

2009 ◽  
Vol 21 (1) ◽  
pp. 67 ◽  
Author(s):  
Tetsuya S. Tanaka
Keyword(s):  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
The Body ◽  
Cell Subpopulation ◽  
Es Cell ◽  
Differentiation Potential ◽  
Mouse Es Cells

The embryonic stem (ES) cell is a stem cell derived from early embryos that can indefinitely repeat self-renewing cell division cycles as an undifferentiated cell in vitro and give rise to all specialised cell types in the body. However, manipulating ES cell differentiation in vitro is a challenge due to, at least in part, heterogeneous gene induction. Recent experimental evidence has demonstrated that undifferentiated mouse ES cells maintained in culture exhibit heterogeneous expression of Dppa3, Nanog, Rex1, Pecam1 and Zscan4 as well as genes (Brachyury/T, Rhox6/9 and Twist2) normally expressed in specialised cell types. The Nanog-negative, Rex1-negative or T-positive ES cell subpopulation has a unique differentiation potential. Thus, studying the mechanism that generates ES cell subpopulations will improve manipulation of ES cell fate and help our understanding of the nature of embryonic development.

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