scholarly journals Nuclear transfer reprogramming does not improve the low developmental potency of embryonic stem cells induced by long-term culture

Reproduction ◽  
2006 ◽  
Vol 132 (2) ◽  
pp. 257-263 ◽  
Author(s):  
T Amano ◽  
M Gertsenstein ◽  
A Nagy ◽  
H Kurihara ◽  
H Suzuki
Keyword(s):  
Cell Lines ◽  
Nuclear Transfer ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Developmental Potential ◽  
Developmental Capacity ◽  
Developmental Rates

Epigenetic states of embryonic stem (ES) cells are easily altered by long-term cultivation and lose their developmental potential. To rescue this reduced developmental capacity, nuclear transfer (NT) of ES cells was carried out, and original ES and ES cells from cloned blastocysts (ntES) cells established after NT were compared with in vitro differentiation ability and developmental potential by embryoid body formation and tetraploid aggregation respectively. In the establishment of ntES cell lines, the oocytes fused with the ES cell were activated, and further cultured to cloned blastocysts. When in vitro differentiation ability was examined between original and ntES cell lines derived from ES cells with extensive passages (ES-ep), the day of appearance of simple embryoid body, cystic embryoid body, and spontaneous beating was almost similar. The developmental rates of ES-ep cells, that aggregated with tetraploid embryos to term, ranged from 3 to 6%. Moreover, the majority of live pups died soon after birth. In the ntES cell lines derived from ES-ep cells, developmental rates ranged from 0 to 5%. Those pups also died soon after birth, similar to the ES-ep-derived pups. These results suggest that profound epigenetic modifications of ES cells were retained in the re-established cell lines by NT.

scholarly journals Induction of Pluripotency in Adult Equine Fibroblasts without c-MYC

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Khodadad Khodadadi ◽  
Huseyin Sumer ◽  
Maryam Pashaiasl ◽  
Susan Lim ◽  
Mark Williamson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histochemical Staining ◽  
Chromosome Count ◽  
Embryoid Bodies ◽  
Retroviral Transduction

Despite tremendous efforts on isolation of pluripotent equine embryonic stem (ES) cells, to date there are few reports about successful isolation of ESCs and no report ofin vivodifferentiation of this important companion species. We report the induction of pluripotency in adult equine fibroblasts via retroviral transduction with three transcription factors usingOCT4, SOX2, andKLF4in the absence of c-MYC. The cell lines were maintained beyond 27 passages (more than 11 months) and characterized. The equine iPS (EiPS) cells stained positive for alkaline phosphatase by histochemical staining and expressed OCT4, NANOG, SSEA1, and SSEA4. Gene expression analysis of the cells showed the expression ofOCT4, SOX2 NANOG, andSTAT3. The cell lines retained a euploid chromosome count of 64 after long-term culture cryopreservation. The EiPS demonstrated differentiation capacity for the three embryonic germ layers bothin vitroby embryoid bodies (EBs) formation andin vivoby teratoma formation. In conclusion, we report the derivation of iPS cells from equine adult fibroblasts and long-term maintenance using either of the three reprogramming factors.

ES cells have only a limited lymphopoietic potential after adoptive transfer into mouse recipients

Development ◽  
1993 ◽  
Vol 118 (4) ◽  
pp. 1343-1351
Author(s):  
A.M. Muller ◽  
E.A. Dzierzak
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Development ◽  
Hematopoietic Stem ◽  
Developmental Potential

While hematopoietic stem cells from adult and fetal stages of murine development are capable of long term reconstitution of all mature blood lineages in vivo, embryonic hematopoietic stem cell repopulation in vivo has proved difficult. It is thought that there are many fewer hematopoietic stem cells in the embryo than in the fetal/adult stages of mouse development and that these cells possess a different developmental potential. One source of such cells are embryonic stem (ES) cells which can differentiate into most mature blood lineages in vitro. We have therefore used transplantation of differentiated ES cells to assess the hematopoietic potential of embryonic hematopoietic cells in vivo. We demonstrate here that precursors obtained from in vitro cultures of normal ES cells can contribute only to restricted and limited hematopoiesis in a mouse without leading to tumour formation. Repopulation occurs for greater than 6.5 months at levels ranging from 0.1% to 6% in B and T cell lineages in peripheral blood. In contrast to in vitro colony data demonstrating the myeloid lineage developmental potential of ES cells, no donor-derived myeloid repopulation was observed in CFU-S assays and no macrophage and mast cells were found in long term repopulated recipients. Thus, the hematopoietic potential of ES cells in vivo is limited to low levels of repopulation and is restricted to the lymphoid lineage.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

Long-term in vitro culture and characterisation of avian embryonic stem cells with multiple morphogenetic potentialities

Development ◽  
1996 ◽  
Vol 122 (8) ◽  
pp. 2339-2348 ◽  
Author(s):  
B. Pain ◽  
M.E. Clark ◽  
M. Shen ◽  
H. Nakazawa ◽  
M. Sakurai ◽  
...  
Keyword(s):  
Culture System ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Telomerase Activity ◽  
Specific Antibodies ◽  
Typical Morphology ◽  
Germinal Tissue

Petitte, J.N., Clarck, M.E., Verrinder Gibbins, A. M. and R. J. Etches (1990; Development 108, 185–189) demonstrated that chicken early blastoderm contains cells able to contribute to both somatic and germinal tissue when injected into a recipient embryo. However, these cells were neither identified nor maintained in vitro. Here, we show that chicken early blastoderm contains cells characterised as putative avian embryonic stem (ES) cells that can be maintained in vitro for long-term culture. These cells exhibit features similar to those of murine ES cells such as typical morphology, strong reactivity toward specific antibodies, cytokine-dependent extended proliferation and high telomerase activity. These cells also present high capacities to differentiate in vitro into various cell types including cells from ectodermic, mesodermic and endodermic lineages. Production of chimeras after injection of the cultivated cells reinforced the view that our culture system maintains in vitro some avian putative ES cells.

Overexpression of HOX11 Leads to the Immortalization of Embryonic Precursors With Both Primitive and Definitive Hematopoietic Potential

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 877-887 ◽  
Author(s):  
Gordon Keller ◽  
Charles Wall ◽  
Andrew Z.C. Fong ◽  
Teresa S. Hawley ◽  
Robert G. Hawley
Keyword(s):  
Cell Lines ◽  
Transcriptional Control ◽  
Growth Regulation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Embryonic Life ◽  
Cytokine Responsiveness ◽  
American Society

Abstract Primitive and definitive erythropoiesis represent distinct hematopoietic programs that differ with respect to stage of development, transcriptional control, and growth regulation. Although these differences have been recognized for some time, the relationship of the two erythroid lineages to each other is not well established. We have used a model system based on the hematopoietic development of embryonic stem (ES) cells in culture to investigate the origins of the earliest hematopoietic populations. Using ES cells transduced with a retrovirus that overexpresses the HOX11 gene, we have established factor-dependent hematopoietic cell lines that represent novel stages of embryonic hematopoiesis. Analysis of three of these cell lines indicates that they differ with respect to cytokine responsiveness, cell surface markers, and developmental potential. Two of the cell lines, EBHX1 and EBHX11, display the unique capacity to generate both primitive and definitive erythroid progeny as defined by morphology and expression of βH1 and βmajor globin. The third line, EBHX14, has definitive erythroid and myeloid potential, but is unable to generate cells of the primitive erythroid lineage. Analysis of the cytokine responsiveness of the two lines with primitive erythroid potential has indicated that exposure to leukemia inhibitory factor (LIF) results in the upregulation of βH1 and a change in cellular morphology to that of primitive erythrocytes. These findings are the first demonstration of a clonal cell line with primitive and definitive hematopoietic potential and support the interpretation that these lineages may arise from a common precursor in embryonic life. In addition, they suggest that LIF could play a role in the regulation of primitive erythropoiesis. © 1998 by The American Society of Hematology.

scholarly journals Generation of multipotent cell lines from a distinct population of male germ line stem cells

Reproduction ◽  
2008 ◽  
Vol 135 (6) ◽  
pp. 771-784 ◽  
Author(s):  
Fariborz Izadyar ◽  
Francis Pau ◽  
Joel Marh ◽  
Natalia Slepko ◽  
Tracy Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Germ Cell ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Complex Mixture ◽  
Neural Cells ◽  
Differentiation Potential

Spermatogonial stem cells (SSCs) maintain spermatogenesis by self-renewal and generation of spermatogonia committed to differentiation. Under certain in vitro conditions, SSCs from both neonatal and adult mouse testis can reportedly generate multipotent germ cell (mGC) lines that have characteristics and differentiation potential similar to embryonic stem (ES) cells. However, mGCs generated in different laboratories showed different germ cell characteristics, i.e., some retain their SSC properties and some have lost them completely. This raises an important question: whether mGC lines have been generated from different subpopulations in the mouse testes. To unambiguously identify and track germ line stem cells, we utilized a transgenic mouse model expressing green fluorescence protein under the control of a germ cell-specific Pou5f1 (Oct4) promoter. We found two distinct populations among the germ line stem cells with regard to their expression of transcription factor Pou5f1 and c-Kit receptor. Only the POU5F1+/c-Kit+ subset of mouse germ line stem cells, when isolated from either neonatal or adult testes and cultured in a complex mixture of growth factors, generates cell lines that express pluripotent ES markers, i.e., Pou5f1, Nanog, Sox2, Rex1, Dppa5, SSEA-1, and alkaline phosphatase, exhibit high telomerase activity, and differentiate into multiple lineages, including beating cardiomyocytes, neural cells, and chondrocytes. These data clearly show the existence of two distinct populations within germ line stem cells: one destined to become SSC and the other with the ability to generate multipotent cell lines with some pluripotent characteristics. These findings raise interesting questions about the relativity of pluripotency and the plasticity of germ line stem cells.

Overexpression of HOX11 Leads to the Immortalization of Embryonic Precursors With Both Primitive and Definitive Hematopoietic Potential

Blood ◽  
1998 ◽  
Vol 92 (3) ◽  
pp. 877-887 ◽  
Author(s):  
Gordon Keller ◽  
Charles Wall ◽  
Andrew Z.C. Fong ◽  
Teresa S. Hawley ◽  
Robert G. Hawley
Keyword(s):  
Cell Lines ◽  
Transcriptional Control ◽  
Growth Regulation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Embryonic Life ◽  
Cytokine Responsiveness ◽  
American Society

Primitive and definitive erythropoiesis represent distinct hematopoietic programs that differ with respect to stage of development, transcriptional control, and growth regulation. Although these differences have been recognized for some time, the relationship of the two erythroid lineages to each other is not well established. We have used a model system based on the hematopoietic development of embryonic stem (ES) cells in culture to investigate the origins of the earliest hematopoietic populations. Using ES cells transduced with a retrovirus that overexpresses the HOX11 gene, we have established factor-dependent hematopoietic cell lines that represent novel stages of embryonic hematopoiesis. Analysis of three of these cell lines indicates that they differ with respect to cytokine responsiveness, cell surface markers, and developmental potential. Two of the cell lines, EBHX1 and EBHX11, display the unique capacity to generate both primitive and definitive erythroid progeny as defined by morphology and expression of βH1 and βmajor globin. The third line, EBHX14, has definitive erythroid and myeloid potential, but is unable to generate cells of the primitive erythroid lineage. Analysis of the cytokine responsiveness of the two lines with primitive erythroid potential has indicated that exposure to leukemia inhibitory factor (LIF) results in the upregulation of βH1 and a change in cellular morphology to that of primitive erythrocytes. These findings are the first demonstration of a clonal cell line with primitive and definitive hematopoietic potential and support the interpretation that these lineages may arise from a common precursor in embryonic life. In addition, they suggest that LIF could play a role in the regulation of primitive erythropoiesis. © 1998 by The American Society of Hematology.

scholarly journals Full-term development of enucleated mouse oocytes fused with embryonic stem cells from different cell lines

Reproduction ◽  
2001 ◽  
pp. 729-733 ◽  
Author(s):  
T Amano ◽  
Y Kato ◽  
Y Tsunoda
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Clear Correlation ◽  
Developmental Potential ◽  
Mouse Oocytes

The developmental potential of enucleated mouse oocytes receiving embryonic stem cells from ten lines with either the same or different genetic backgrounds using the cell fusion method was examined in vitro and in vivo. The development of nuclear-transferred oocytes into blastocysts was high (34-88%). However, there was no clear correlation between development into blastocysts after nuclear transfer and the chimaera formation rate of embryonic stem cells. The development into live young was low (1-3%) in all cell lines and 14 of 19 young died shortly after birth. Most of the live young had morphological abnormalities. Of the five remaining mice, two died at days 23 and 30 after birth, but the other three mice are still active at days 359 (mouse 1) and 338 (mice 4 and 5) after birth, with normal fertility. However, the reasons for the abnormalities and postnatal death of embryonic stem cell-derived mice are unknown.

Relationship between bovine oocyte morphology and in vitro developmental potential

Zygote ◽  
2006 ◽  
Vol 14 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Masashi Nagano ◽  
Seiji Katagiri ◽  
Yoshiyuki Takahashi
Keyword(s):  
Small Diameter ◽  
Blastocyst Stage ◽  
Cortical Granules ◽  
Developmental Potential ◽  
Developmental Capacity ◽  
Sperm Penetration ◽  
Developmental Rates ◽  
Large Clusters

We investigated the relationship between the morphology of oocytes collected from small antral follicles and their developmental capacity. Immature oocytes were classified into seven groups and cultured in vitro for maturation (IVM), fertilization (IVF) and development to blastocysts (IVC). After IVF, sperm penetration and normal fertilization rates were higher in the oocytes whose cytoplasm appeared brown. The rate of polyspermy was highest in the oocytes whose cytoplasm was black. After IVC, the rates of cleavage and of development to the blastocyst stage were also higher in the brown oocytes. Although the oocytes with dark clusters in a pale cytoplasm showed lower cleavage rates, cleaved zygotes had high developmental rates the same as the oocytes with a brown cytoplasm. Transmission electron microscopy showed that the oocytes with a pale or black cytoplasm had organelles arranged differently from other oocytes before IVM. Most of the oocytes with a brown and homogeneous cytoplasm or small diameter had the characteristics of immature cytoplasm (large clusters of cortical granules) even after IVM. On the other hand, the brown oocytes with a dark zone at the periphery or with dark clusters showed the same arrangement of organelles as in vivo matured oocytes. The oocytes with a pale or black cytoplasm appeared to be degenerating and/or ageing. In conclusion, a dark ooplasm indicates an accumulation of lipids and good developmental potential, while a light-coloured ooplasm indicates a low density of organelles and poor developmental potential. A black ooplasm indicates ageing and low developmental potential.

Human Fetal Liver Derived Stem Cells Can Be Support the Maintenance of Human Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4264-4264
Author(s):  
Jin-Young Baek ◽  
Yun-Hee Rhee ◽  
Kwang-Yul Cha ◽  
Hyung-Min Chung
Keyword(s):  
Stem Cells ◽  
Mononuclear Cells ◽  
Embryoid Body ◽  
Fetal Liver ◽  
Es Cells ◽  
Embryonic Stem ◽  
Human Fetal Liver ◽  
Human Es Cells ◽  
Cells Cultured

Abstract Prolonged propagation of human embryonic stem (ES) cells is currently achieved by co-culture with primary or immortalized mouse embryonic fibroblast (MEF) cells. In order to replace the heterologous with homologous co-culture systems, an attempt was made using mononuclear cells derived from human fetal liver. Human fetal liver-derived mesenchymal-like stem cells (FL-MLSC) can be maintained for the prolonged period of time. They showed the characteristics of mesenchymal stem cells in various aspects. They retained a normal diploid karyotype and growth characteristics over the successive culture. Human ES cells cultured on human FL-MLSC cells up to 8 passages displayed the unique morphology and molecular markers characteristic for undifferentiated human ES cells as cultured on MEF cells. Alkaline phosphatase activity was detected in human ES cells co-cultured on human FL-MLSC. Immunocytochemical analyses showed that expressions of stage-specific embryonic antigen-3, -4 and Oct-4 were not altered on human ES cells cultured on human FLDSC. Reverse-transcriptase PCR analyses showed that similar expressions of Oct-4 and Nanog genes, markers for undifferentiated ES cells, were also observed in human ES cells cultured on both human FL-MLSC and MEF cells. Furthermore, human ES cells cultured on human FL-MLSC retained unique differentiation potentials in culture when allowed to form embryoid body. Results of this study suggest that human FL-MLSC can support the maintenance of human ES cell in vitro.

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