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.

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

216 EMBRYONIC AND INDUCED PLURIPOTENT STEM CELLS ANALOGOUS TO INNER CELL MASS-DERIVED LIF-DEPENDENT MOUSE EMBRYONIC STEM CELLS ESTABLISHED FROM THE DOMESTIC PIG, SUS SCROFA

2012 ◽  
Vol 24 (1) ◽  
pp. 220
Author(s):  
B. P. Telugu ◽  
T. Ezashi ◽  
A. Alexenko ◽  
S. Lee ◽  
R. S. Prather ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Cell Lines ◽  
Umbilical Cord ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Induced Pluripotent

Authentic embryonic stem cells (ESC) may never have been successfully derived from the inner cell mass (ICM) of pig and other ungulates, despite over 25 years of effort. Recently, porcine induced pluripotent stem cells (piPSC) were generated by reprogramming somatic cells with a combination of four factors OCT4, SOX2, KLF4 and c-MYC (OSKM) delivered by lentiviral transduction. The established piPSC are analogous to FGF2-dependent human (h) ESC and murine “epiblast stem cells,” and are likely to advance swine as a model in biomedical research. Here, we report for the first time, the establishment of LIF-dependent, so called naïve type pluripotent stem cells (1) from the inner cell mass (ICM) of porcine blastocysts by up-regulating the expression of KLF4 and POU5F1; and (2) from umbilical cord mesenchyme (Wharton's jelly) by transduction with OSKM factors and subsequent culture in the presence of LIF-based medium with inhibitors that substitute for low endogenous expression of c-MYC and KLF4 and promote pluripotency. The 2 compounds that have been used in this study are, CHIR99021 (CH), which substitutes c-MYC by inhibiting GSK3B and activating WNT signalling and Kenpaullone (KP), which inhibits both GSK3B and CDK1 and supplants KLF4 function. The lentiviral vectors employed for introducing the re-programming genes were modified for doxycycline-mediated induction of expression (tet-on) and are ‘floxed’ for Cre-mediated recombination and removal of transgenes following complete reprogramming. Two LIF-dependent cell lines have been derived from the ICM cells of late d 5.5 in vitro produced blastocysts and four from umbilical cord mesenchyme recovered from fetuses at d 35 of pregnancy. The derived stem cell lines are alkaline phosphatase-positive, resemble mouse embryonic stem cells in colony morphology, cell cycle interval, transcriptome profile and expression of pluripotent markers, such as POU5F1, SOX2 and surface marker SSEA1. They are dependent on LIF signalling for maintenance of pluripotency, can be cultured over extended passage (>50) with no senescence. Of importance, the ICM-derived lines have been successful in their ability to form teratomas. The cells could be cultured in feeder free conditions on a synthetic matrix in the presence of chemically defined medium and can be coaxed to differentiate under xeno-free conditions. Currently, the piPSC lines are being investigated for their ability to give rise to teratomas and to produce a live offspring by nuclear transfer. Supported by Addgene Innovation Award, MO Life Sciences Board Grant 00022147 and NIH grant HD21896.

scholarly journals Stem Cell and Markers

2019 ◽  
Vol 1 (1) ◽  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Adult Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Stem Cell Markers ◽  
Cell Markers ◽  
Wide Range

Stem cells have the ability to go through various cell divisions and also maintain undifferentiated state. Stem cells are Embryonic (Pluripotent) and adult stem cells. Pluripotent stem cells give rise to all tissues such as ectoderm, mesoderm and endoderm. Embryonic stem cells isolated from inner cell mass of embryo blastocyst. Adult stem cells are also undifferentiated cells present in adult organisms and repair the tissue when damaged occurs but number in less. Adult stem cells are present in bone marrow, adipose tissue, blood and juvenile state umbilical cord and tissue of specific origin like liver, heart, intestine and neural tissue. Embryonic stem cells from blastocyst have the ethical problems and tumorogenecity. These can be identified by flow cytometry. There are wide range of stem cell markers which are useful in identifying them. Most of the pluripotent cell markers are common with tumor cell markers which throws a challenge for certainty.

scholarly journals Human ES Cell Culture Conditions Fail to Preserve the Mouse Epiblast State

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
A. S. Devika ◽  
Anna Montebaur ◽  
S. Saravanan ◽  
Raghu Bhushan ◽  
Frederic Koch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Culture Condition ◽  
Prolonged Exposure ◽  
Inner Cell Mass ◽  
Species Difference ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Preimplantation Embryos

Mouse embryonic stem cells (mESCs) and mouse epiblast stem cells (mEpiSCs) are the pluripotent stem cells (PSCs), derived from the inner cell mass (ICM) of preimplantation embryos at embryonic day 3.5 (E3.5) and postimplantation embryos at E5.5-E7.5, respectively. Depending on their environment, PSCs can exist in the so-called naïve (ESCs) or primed (EpiSCs) states. Exposure to EpiSC or human ESC (hESC) culture condition can convert mESCs towards an EpiSC-like state. Here, we show that the undifferentiated epiblast state is however not stabilized in a sustained manner when exposing mESCs to hESC or EpiSC culture condition. Rather, prolonged exposure to EpiSC condition promotes a transition to a primitive streak- (PS-) like state via an unbiased epiblast-like intermediate. We show that the Brachyury-positive PS-like state is likely promoted by endogenous WNT signaling, highlighting a possible species difference between mouse epiblast-like stem cells and human Embryonic Stem Cells.

scholarly journals BRG1 GovernsNanogTranscription in Early Mouse Embryos and Embryonic Stem Cells via Antagonism of Histone H3 Lysine 9/14 Acetylation

2015 ◽  
Vol 35 (24) ◽  
pp. 4158-4169 ◽  
Author(s):  
Timothy S. Carey ◽  
Zubing Cao ◽  
Inchul Choi ◽  
Avishek Ganguly ◽  
Catherine A. Wilson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Histone Deacetylation ◽  
Preimplantation Embryos ◽  
Nucleosome Remodeling ◽  
Histone Deacetylase 1

During mouse preimplantation development, the generation of the inner cell mass (ICM) and trophoblast lineages comprises upregulation ofNanogexpression in the ICM and its silencing in the trophoblast. However, the underlying epigenetic mechanisms that differentially regulateNanogin the first cell lineages are poorly understood. Here, we report that BRG1 (Brahma-related gene 1) cooperates with histone deacetylase 1 (HDAC1) to regulateNanogexpression. BRG1 depletion in preimplantation embryos andCdx2-inducible embryonic stem cells (ESCs) revealed that BRG1 is necessary forNanogsilencing in the trophoblast lineage. Conversely, in undifferentiated ESCs, loss of BRG1 augmentedNanogexpression. Analysis of histone H3 within theNanogproximal enhancer revealed that H3 lysine 9/14 (H3K9/14) acetylation increased in BRG1-depleted embryos and ESCs. Biochemical studies demonstrated that HDAC1 was present in BRG1-BAF155 complexes and BRG1-HDAC1 interactions were enriched in the trophoblast lineage. HDAC1 inhibition triggered an increase in H3K9/14 acetylation and a corresponding rise inNanogmRNA and protein, phenocopying BRG1 knockdown embryos and ESCs. Lastly, nucleosome-mapping experiments revealed that BRG1 is indispensable for nucleosome remodeling at theNanogenhancer during trophoblast development. In summary, our data suggest that BRG1 governsNanogexpression via a dual mechanism involving histone deacetylation and nucleosome remodeling.

The derivation and potential use of human embryonic stem cells

2001 ◽  
Vol 13 (8) ◽  
pp. 523 ◽  
Author(s):  
Alan O. Trounson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Regenerative Medicine ◽  
Human Embryonic Stem Cells ◽  
High Efficiency ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Cell Types ◽  
Specific Cell

Human embryonic stem cells lines can be derived from human blastocysts at high efficiency (>50%) by immunosurgical isolation of the inner cell mass and culture on embryonic fibroblast cell lines. These cells will spontaneously differentiate into all the primary embryonic lineages in vitro and in vivo, but they are unable to form an integrated embryo or body plan by themselves or when combined with trophectoderm cells. They may be directed into a number of specific cell types and this enrichment process requires specific growth factors, cell-surface molecules, matrix molecules and secreted products of other cell types. Embryonic stem (ES) cells are immortal and represent a major potential for cell therapies for regenerative medicine. Their use in transplantation may depend on the formation of a large bank of suitable human leucocyte antigen (HLA) types or the genetic erasure of their HLA expression. Successful transplantation may also require induction of tolerance in recipients and ongoing immune suppression. Although it is possible to customize ES cells by therapeutic cloning or cytoplasmic transfer, it would appear unlikely that these strategies will be used extensively for producing ES cells compatible for transplantation. Embryonic stem cell research may deliver a new pathway for regenerative medicine.

scholarly journals Depletion of Demethylase KDM6 Enhances Early Neuroectoderm Commitment of Human PSCs

2021 ◽  
Vol 9 ◽  
Author(s):  
Yajing Meng ◽  
Tianzhe Zhang ◽  
Ran Zheng ◽  
Song Ding ◽  
Jie Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Cell Lines ◽  
Histone Methylation ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Neural Induction

Epigenetic modifications play a crucial role in neurogenesis, learning, and memory, but the study of their role in early neuroectoderm commitment from pluripotent inner cell mass is relatively lacking. Here we utilized the system of directed neuroectoderm differentiation from human embryonic stem cells and identified that KDM6B, an enzyme responsible to erase H3K27me3, was the most upregulated enzyme of histone methylation during neuroectoderm differentiation by transcriptome analysis. We then constructed KDM6B-null embryonic stem cells and found strikingly that the pluripotent stem cells with KDM6B knockout exhibited much higher neuroectoderm induction efficiency. Furthermore, we constructed a series of embryonic stem cell lines knocking out the other H3K27 demethylase KDM6A, and depleting both KDM6A and KDM6B, respectively. These cell lines together confirmed that KDM6 impeded early neuroectoderm commitment. By RNA-seq, we found that the expression levels of a panel of WNT genes were significantly affected upon depletion of KDM6. Importantly, the result that WNT agonist and antagonist could abolish the differential neuroectoderm induction due to manipulating KDM6 further demonstrated that WNT was the major downstream of KDM6 during early neural induction. Moreover, we found that the chemical GSK-J1, an inhibitor of KDM6, could enhance neuroectoderm induction from both embryonic stem cells and induced pluripotent stem cells. Taken together, our findings not only illustrated the important role of the histone methylation modifier KDM6 in early neurogenesis, providing insights into the precise epigenetic regulation in cell fate determination, but also showed that the inhibitor of KDM6 could facilitate neuroectoderm differentiation from human pluripotent stem cells.

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).

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.

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