Oct-4 Is Crucial for Murine Embryonic Stem Cell Secretion of Cytokines/Growth Modulators That Enhance Cell Survival/Anti-Apoptosis of Murine ES Cells and Bone Marrow Hematopoietic Progenitor Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1359-1359
Author(s):  
Ying Guo ◽  
Barbara Graham-Evans ◽  
Charlie R. Mantal ◽  
Robert A. Hromas ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Caspase 3 ◽  
Es Cells ◽  
Embryonic Stem ◽  
Conditional Knockout ◽  
Es Cell ◽  
Wild Type ◽  
Murine Bone Marrow

Abstract Murine embryonic stem (ES) cells may be of potential use for cell replacement and gene therapy. Maintenance of ES cells in an undifferentiated and proliferative state depends on cytokines either secreted by ES cells and/or added to the medium. By understanding the production and release of cytokines in ES cell culture, it may be possible to enhance use of ES cells for clinical usage. Our previous studies indicated that SDF-1/CXCL12, secreted by ES cells, enhances survival, chemotaxis, and hematopoietic differentiation of murine ES cells (Guo et al, Stem cells, in press, 2005). To evaluate whether other cytokines were produced by murine ES cells, we generated conditioned medium (CM) from these cells in the presence of LIF, while the ES cells were in an undifferentiated Oct-4 expressing state, and assayed the CM for cytokines, chemokines, and other growth modulatory factors. ES cell CM enhanced survival in vitro of ES cells subjected to delayed addition of serum to ES cell cultures. Without serum, ES cells didn’t grow in low cell density. However, with CM, ES cells formed colonies at about 63% of the growth of the ES cells in the presence of serum. ES cell CM also enhanced survival of normal murine bone marrow myeloid progenitors (CFU-GM) subjected to delayed growth factor addition in vitro and decreased the rate of apoptosis in murine bone marrow c-kit+lin− cells as assessed by Annexin V assay. Our data showed ES cell CM contained IL-1α, IL-10, IL-11, M-CSF, OSM, SCF, VEGF, as well as a number of chemokines and other proteins. For a number of these proteins, we have already verified that the mRNA for them is expressed in the ES cells. This indicates that ES cells produce and secrete these cytokines. Some of these cytokines are known to have an enhanced survival/antiapoptosis effect on progenitors. IL-6, FGF-9, and TNF-a, which were not detected prior to irradiation of the ES cells, were seen after ES cells were irradiated. Irradiation of the ES cells enhanced release of some proteins and decreased release of others. ES cell CM also stimulated CFU-GM colony formation. Thus, undifferentiated murine ES cells growing in the presence of LIF produce/release a number of biologically active interleukins, CSFs, chemokines, and other growth modulatory proteins. Oct-4 is a marker for undifferentiated ES cells. We wondered if Oct-4 might be a key player for cytokines released from ES cells which supported CFU-GM survival and antiapoptosis. Oct-4 conditional knockout cell line ZHBtc4, received from Dr Austin Smith, was used. CM from the wild type ES cell line enhanced survival of CFU-GM similar to that of other ES cell lines, while the Oct-4 knockout ES cell line didn’t. These results indicate that release of proteins involved in survival enhancement may be related to Oct-4 expression. We also found that the wild type cell line which expressed Oct-4 didn’t initiate caspase 3 dependent apoptosis after mitotic stress. However ZHBTc4, the Oct-4 deleted cell line demonstrated caspase 3 dependent apoptosis. These results may be of physiological significance, although this has not yet been proven, and suggest the possibility of potential future applicability for use of irradiated ES cells as accessory cells for growth modulation in vitro and in vivo.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Unique Primitive Erythropoiesis Defect In Rpl5-Deficient Murine Embryonic Stem Cell Model of Diamond Blackfan Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 877-877
Author(s):  
Tracie A. Goldberg ◽  
Sharon Singh ◽  
Adrianna Henson ◽  
Abdallah Nihrane ◽  
Jeffrey Michael Lipton ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Es Cells ◽  
Mutant Cell ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Es Cell ◽  
Wild Type ◽  
Mutant Cell Line ◽  
Primitive Erythropoiesis

Abstract Abstract 877 Background: Diamond Blackfan anemia (DBA), a rare inherited bone marrow failure syndrome, is characterized mainly by erythroid hypoplasia but is also associated with congenital anomalies, short stature and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS17, RPS19, RPS24, RPL5, RPL11, RPL35a), which renders erythroid precursors highly sensitive to death by apoptosis. The ontogeny and basis of the hematopoietic defect are unclear. The typical presentation of anemia occurs at 2–3 months of age, although there are rare cases of hydrops fetalis. Marked phenotypic variations exist among members of the same family and also between subsets of patients with different mutations. Methods: We studied in vitro hematopoietic differentiation of two murine embryonic stem (ES) cell lines: YHC074, Rps19 mutant with the pGT0Lxf gene trap vector inserted in intron 3 of Rps19, and D050B12, Rpl5 mutant with the FlipRosaβgeo gene trap vector inserted in intron 3 of Rpl5. Wild-type parental cell lines were used as controls. For primary differentiation and generation of embryoid bodies (EBs), ES cells were cultured in serum-supplemented methylcellulose medium containing stem cell factor (SCF). After 7 days, the cultures were fed with medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 12 for hematopoietic percentage. For secondary differentiation into definitive hematopoietic colonies, day 10 EBs were disrupted, and individual cells were suspended in serum-supplemented methylcellulose medium containing SCF, IL-3, Il-6 and epo. Definitive hematopoietic colonies were counted on day 10. Primitive erythropoiesis differentiation assays were performed by disruption of day 4 EBs, followed by suspension of cells in methylcellulose medium containing plasma-derived serum and epo. Primitive erythropoiesis colonies were counted on day 7. Results: We confirmed haploinsufficient expression (∼50% wild type) of Rps19 in YHC074 and Rpl5 protein in D050B12 by Western blot analysis. By polysome analysis, we found a selective reduction in the 40S subunit peak in the Rps19 mutant cell line and in the 60S subunit peak in the Rpl5 mutant cell line. Both types of mutants produced a significantly decreased number of EBs, particularly hematopoietic EBs, compared to parental cell lines. EB size was not compromised in the Rps19 mutant cell line, while Rpl5 mutant ES cells produced significantly smaller EBs, compared to its parental cells. Upon differentiation of cells to definitive hematopoietic colonies, both Rps19 and Rpl5 mutants showed a similar reduction in the erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation ratio. Primitive erythropoiesis was conserved in the Rps19 mutant (Figure 1. 1, top panel). By contrast, the Rpl5 mutant demonstrated a severe primitive erythropoiesis defect (Figure 1. 1, bottom panel). For confirmation of these results in an isogenic background, we stably transfected YHC074 ES cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones expressed Rps19 at the wild-type level. Upon differentiation of a chosen clone, we demonstrated correction of the EB defect and the definitive erythropoiesis defect, suggesting that the hematopoietic differentiation defects seen are directly related to levels of Rps19 protein. We are currently working on correction of the D050B12 ES cells in a similar manner. Conclusion: Murine ES cell lines with Rps19 and Rpl5 mutations exhibit ribosomal protein haploinsufficiency, demonstrate respective ribosome assembly defects, and recapitulate the major DBA hematopoietic differentiation defect. In addition, a unique defect in primitive erythropoiesis in the Rpl5 mutant ES cell line suggests that the Rpl5 mutation in this mouse strain affects early-stage embryogenesis, a finding which may offer insight into the ontogeny of DBA hematopoiesis and may offer an explanation for phenotypic variations seen in patients (such as hydrops fetalis). Disclosures: No relevant conflicts of interest to declare.

Biological Analysis of AML1/RUNX1 Arginine-Mutants by Means of Hematopoietic Rescue Experiments of Runx1-deficient Mouse Embryonic Stem Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3382-3382
Author(s):  
Shinsuke Mizutani ◽  
Masafumi Taniwaki ◽  
Tsukasa Okuda
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Biological Significance ◽  
Deficient Mouse ◽  
Es Cell ◽  
Wild Type ◽  
Post Translational Modification ◽  
Cell Clones ◽  
Arginine Residues

Abstract Abstract 3382 Runt-Related Transcription Factor 1 (RUNX1; also called as Acute Myeloid Leukemia 1: AML1) is one of the most frequently mutated genes associated with human acute leukemia, and encodes DNA binding subunit of the Core-Binding Factor (CBF) transcription complex whose activity is essential for the development of definitive hematopoiesis. RUNX1 serves as a transcriptional activator as well as a repressor to its target genes, depending on the cellular context, mediated through its interaction with co-factors. Increasing evidence obtained these days suggests that post-translational modification of RUNX1, including phosphorylation, methylation, or acetylation on its target amino acid residues, is important for proper and fine tuning of this RUNX1-function, likely by altering its association with functional cofactors. However, biological significance of these modifications has not yet been examined in detail. As an initial effort towards systematic comprehension how these modifications influence RUNX1 function, we tried to evaluate RUNX1 methylation in vitro in this study. Arginine residues just douwnstream to the Runt-domain of RUNX1 were recently reported to be methylated to inhibit corepressor-binding thus enhances its trans-activating activity. In order to elucidate the biological effects of this post-translational modification, we manufactured arginine-to-lysine substitutions at the sites within the mouse cDNA. When these arginine-mutants were exogenously expressed in mammalian cell lines, they showed reduced trans-activating activity detected by a dual-luciferase assay on known reporter constructs in comparison to the wild-type Runx1, confirming previous reports. We then introduced the mutant cDNA into Runx1-deficient mouse embryonic stem (ES) cells by means of a knock-in strategy at the disrupted Runx1 gene locus. These ES cell clones were subjected to the in vitro differentiation to hematopoietic lineages. Wild-type ES cells are known to differentiate into hematopoietic cell lineages via embryoid body formation in a semi-solid culture system, whereas ES cells of Runx1-deficient genotype lose the ability to undergo hematopoietic differentiation. This phenomenon is recognized to be an in vitro phenocopy of the Runx1-deficient mice that suffer from embryonic death due to complete block of fetal liver hematopoiesis. Initial study so far showed that the Runx1-deficient ES cell clones restored the ability to develop hematopoietic cells including macrophages in culture when the arginine-mutant cDNA was re-expressed from the knock-in allele, as is the case for the control Runx1-deficient ES cells with the knocked-in wild-type Runx1. These results suggest that this arginine-to-lysine mutation is dispensable, at least, for the in vitro hematopoietic function of wild-type Runx1 although its trans-activating activity is somewhat impaired. We are currently focusing on introducing this mutation into mouse germ line, and the resultant genome-modified mice should show us the biological significance of the methylation-modification to this important molecule in the context of an entire animal. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Erythroid-cell-specific properties of transcription factor GATA-1 revealed by phenotypic rescue of a gene-targeted cell line.

1997 ◽  
Vol 17 (3) ◽  
pp. 1642-1651 ◽  
Author(s):  
M J Weiss ◽  
C Yu ◽  
S H Orkin
Keyword(s):  
Transcription Factor ◽  
Cell Line ◽  
Zinc Finger ◽  
Es Cells ◽  
Embryonic Stem ◽  
Erythroid Cell ◽  
Transactivation Domain ◽  
Stage Of Development ◽  
Erythroid Maturation

The zinc finger transcription factor GATA-1 is essential for erythropoiesis. In its absence, committed erythroid precursors arrest at the proerythroblast stage of development and undergo apoptosis. To study the function of GATA-1 in an erythroid cell environment, we generated an erythroid cell line from in vitro-differentiated GATA-1- murine embryonic stem (ES) cells. These cells, termed G1E for GATA-1- erythroid, proliferate as immature erythroblasts yet complete differentiation upon restoration of GATA-1 function. We used rescue of terminal erythroid maturation in G1E cells as a stringent cellular assay system in which to evaluate the functional relevance of domains of GATA-1 previously characterized in nonhematopoietic cells. At least two major differences were established between domains required in G1E cells and those required in nonhematopoietic cells. First, an obligatory transactivation domain defined in conventional nonhematopoietic cell transfection assays is dispensable for terminal erythroid maturation. Second, the amino (N) zinc finger, which is nonessential for binding to the vast majority of GATA DNA motifs, is strictly required for GATA-1-mediated erythroid differentiation. Our data lead us to propose a model in which a nuclear cofactor(s) interacting with the N-finger facilitates transcriptional action by GATA-1 in erythroid cells. More generally, our experimental approach highlights critical differences in the action of cell-specific transcription proteins in different cellular environments and the power of cell lines derived from genetically modified ES cells to elucidate gene function.

Human embryonic stem cells: challenges and opportunities

2006 ◽  
Vol 18 (8) ◽  
pp. 839 ◽  
Author(s):  
Steven L. Stice ◽  
Nolan L. Boyd ◽  
Sujoy K. Dhara ◽  
Brian A. Gerwe ◽  
David W. Machacek ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Cell Line ◽  
Cell Fate ◽  
Neural Development ◽  
Es Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Es Cell ◽  
Cell Therapies

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

scholarly journals Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1265-1275 ◽  
Author(s):  
Abby L. Olsen ◽  
David L. Stachura ◽  
Mitchell J. Weiss
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Patient Specific ◽  
Es Cell ◽  
Blood Disorders ◽  
Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

Altered imprinted gene methylation and expression in completely ES cell-derived mouse fetuses: association with aberrant phenotypes

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2273-2282 ◽  
Author(s):  
W. Dean ◽  
L. Bowden ◽  
A. Aitchison ◽  
J. Klose ◽  
T. Moore ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Es Cells ◽  
Embryonic Stem ◽  
Upstream Region ◽  
Imprinted Genes ◽  
Epigenetic Alterations ◽  
Es Cell ◽  
Biallelic Expression

In vitro manipulation of preimplantation mammalian embryos can influence differentiation and growth at later stages of development. In the mouse, culture of embryonic stem (ES) cells affects their totipotency and may give rise to fetal abnormalities. To investigate whether this is associated with epigenetic alterations in imprinted genes, we analysed two maternally expressed genes (Igf2r, H19) and two paternally expressed genes (Igf2, U2af1-rs1) in ES cells and in completely ES cell-derived fetuses. Altered allelic methylation patterns were detected in all four genes, and these were consistently associated with allelic changes in gene expression. All the methylation changes that had arisen in the ES cells persisted on in vivo differentiation to fetal stages. Alterations included loss of methylation with biallelic expression of U2af1-rs1, maternal methylation and predominantly maternal expression of Igf2, and biallelic methylation and expression of Igf2r. In many of the ES fetuses, the levels of H19 expression were strongly reduced, and this biallelic repression was associated with biallellic methylation of the H19 upstream region. Surprisingly, biallelic H19 repression was not associated with equal levels of Igf2 expression from both parental chromosomes, but rather with a strong activation of the maternal Igf2 allele. ES fetuses derived from two of the four ES lines appeared developmentally compromised, with polyhydramnios, poor mandible development and interstitial bleeding and, in chimeric fetuses, the degree of chimerism correlated with increased fetal mass. Our study establishes a model for how early embryonic epigenetic alterations in imprinted genes persist to later developmental stages, and are associated with aberrant phenotypes.

scholarly journals A Shortened Life Span of EKLF−/− Adult Erythrocytes, Due to a Deficiency of β-Globin Chains, Is Ameliorated by Human γ-Globin Chains

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1291-1299 ◽  
Author(s):  
Sai-Kiang Lim ◽  
James J. Bieker ◽  
Chyuan-Sheng Lin ◽  
Frank Costantini
Keyword(s):  
Life Span ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Es Cell ◽  
Wild Type ◽  
Rt Pcr ◽  
Mature Erythrocyte ◽  
Heinz Bodies ◽  
Globin Chains

Abstract Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF−/− ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF−/− colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of βh1-globin, which continued to be expressed at a very low level. The ratio of adult β-globin/α-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF−/− cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF−/− erythrocytes in adult animals must be short-lived, apparently due to the imbalance of β-versus α-globin chains, leading to the precipitation of excess α-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF−/− ES cells of a human LCR/γ-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.

Development of a Testing Strategy for Detecting Embryotoxic Hazards of Chemicals In Vitro by using Embryonic Stem Cell Models

2002 ◽  
Vol 30 (2_suppl) ◽  
pp. 107-109 ◽  
Author(s):  
Susanne Bremer ◽  
Cristian Pellizzer ◽  
Sarah Adler ◽  
Martin Paparella ◽  
Jan de Lange
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Tests ◽  
Es Cell ◽  
Cell Models ◽  
Testing Strategy ◽  
Chemical Effects ◽  
Cell Test ◽  
Stem Cell Models

The importance of developing in vitro tests for embryotoxicity is discussed, and ECVAM's work with its collaborators is summarised. Studies are in progress to find new endpoints for use in the scientifically validated embryonic stem (ES) cell test, so that the potential for chemical effects on endodermal, mesodermal and/or ectodermal differentiation can be identified. This involves, inter alia, the use of genetically modified ES cells.

290 A CYNOMOLGUS MONKEY EMBRYONIC STEM CELL LINE DERIVED FROM A SINGLE BLASTOMERE

2008 ◽  
Vol 20 (1) ◽  
pp. 224
Author(s):  
J. Okahara-Narita ◽  
J. Yamasaki ◽  
C. Iwatani ◽  
H. Tsuchiya ◽  
K. Wakimoto ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cynomolgus Monkey ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Cell Stage ◽  
Vitro Assay ◽  
Single Blastomere ◽  
Cell Colony

The establishment of most embryonic stem (ES) cell lines requires the destruction of embryos. Some ES cell lines in mice and humans are currently derived from a single blastomere, so that remaining blastomeres can still develop into fetuses. However, the procedures currently in use for establishing these lines are very complicated, and other ES cell lines from the same species are needed (Chung et al. 2006 Nature 439, 216–219; Klimanskaya et al. 2006 Nature 444, 481–485). The objective of this study was to devise a method simpler than those previously described for establishing ES cell lines from a single blastomere in the cynomolgus monkey. Controlled ovarian stimulation and oocyte recovery have been described previously by Torii et al. (2000 Primates 41, 39–47). Cumulus-free mature oocytes were fertilized by intracytoplasmic sperm injection (ICSI), and then cultured at 38�C in 5% CO2, 5% O2 for 2 days. The zona pellucida of 4- to 5-cell-stage embryos was disrupted using acidic Tyrode's solution, and individual blastomeres were separated from the denuded embryos using trypsin. These blastomeres were cultured on mitomycin-C-treated mouse embryonic fibroblasts and ES medium containing adrenocorticotropic hormone (ACTH) (Ogawa et al. 2004 Genes to Cells 9, 471–477). After the formation of initial outgrowths, half of the medium was changed every other day until the outgrowths reached approximately 100 cells. Passage of putative monkey ES cells was performed by mechanical dispersion of the colonies and transfer to fresh feeders every 3–4 days until there were enough cells for enzymatic dispersion. One stable ES cell line was obtained from two 4- or 5-cell-stage embryos using ES medium containing ACTH. The morphology of this ES cell colony was consistent with the monkey ES cell colony previous described by Suemori et al. (2001 Dev. Dynamics 222, 273–279). The ES cell line was passaged more than 17 times, and the morphology of the ES cell colony did not differ between the first and seventeenth passages. The ES cells showed normal karyotype and retained pluripotency markers for primate ES cells including octamer-binding protein 4 (Oct-4), stage-specific embryonic antigen (SSEA)-4, tumor-rejection antigen (TRA)-1-60, and TRA-1-81. We are presently confirming whether this ES cell line possesses potencies to differentiate in all three embryonic germ layers using both an in vitro assay and teratoma formation. Here we showed that cynomolgus monkey ES cells can be derived from a single blastomere, without co-culturing another ES cell line, as has been done in previous studies on mice and humans. This method allows the establishment of ES cell lines from a single blastomere, leaving the other blastomeres available for embryo transfer. Thus, the method described here is simpler than previously described methods and alleviates some ethical concerns.

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