scholarly journals Efficient Differentiation of Mouse Embryonic Stem Cells into Insulin-Producing Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Szu-Hsiu Liu ◽  
Lain-Tze Lee
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Definitive Endoderm ◽  
Mouse Es Cells ◽  
Differentiated Cells ◽  
Combined Application ◽  
Insulin Producing Cells ◽  
Differentiation Protocol ◽  
Human Es Cells ◽  
Dose Dependent

Embryonic stem (ES) cells are a potential source of a variety of differentiated cells for cell therapy, drug discovery, and toxicology screening. Here, we present an efficacy strategy for the differentiation of mouse ES cells into insulin-producing cells (IPCs) by a two-step differentiation protocol comprising of (i) the formation of definitive endoderm in monolayer culture by activin A, and (ii) this monolayer endoderm being induced to differentiate into IPCs by nicotinamide, insulin, and laminin. Differentiated cells can be obtained within approximately 7 days. The differentiation IPCs combined application of RT-PCR, ELISA, and immunofluorescence to characterize phenotypic and functional properties. In our study, we demonstrated that IPCs produced pancreatic transcription factors, endocrine progenitor marker, definitive endoderm, pancreaticβ-cell markers, and Langerhansαandδcells. The IPCs released insulin in a manner that was dose dependent upon the amount of glucose added. These techniques may be able to be applied to human ES cells, which would have very important ramifications for treating human disease.

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.

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.

Embryonic Stem Cells: Prospects for Developmental Biology and Cell Therapy

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

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

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

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

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

scholarly journals The Profile of Post-translational Modifications of Histone H1 in Chromatin of Mouse Embryonic Stem Cells

Acta Naturae ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 82-91 ◽  
Author(s):  
T. Yu. Starkova ◽  
T. O. Artamonova ◽  
V. V. Ermakova ◽  
E. V. Chikhirzhina ◽  
M. A. Khodorkovskii ◽  
...  
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone H1 ◽  
Ion Cyclotron Resonance ◽  
Compact Structure ◽  
Post Translational Modifications ◽  
Differentiated Cells ◽  
Eukaryotic Dna ◽  
Nih 3T3

Linker histone H1 is one of the main chromatin proteins which plays an important role in organizing eukaryotic DNA into a compact structure. There is data indicating that cell type-specific post-translational modifications of H1 modulate chromatin activity. Here, we compared histone H1 variants from NIH/3T3, mouse embryonic fibroblasts (MEFs), and mouse embryonic stem (ES) cells using matrix-assisted laser desorption/ ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS). We found significant differences in the nature and positions of the post-translational modifications (PTMs) of H1.3-H1.5 variants in ES cells compared to differentiated cells. For instance, methylation of K75 in the H1.2-1.4 variants; methylation of K108, K148, K151, K152 K154, K155, K160, K161, K179, and K185 in H1.1, as well as of K168 in H1.2; phosphorylation of S129, T146, T149, S159, S163, and S180 in H1.1, T180 in H1.2, and T155 in H1.3 were identified exclusively in ES cells. The H1.0 and H1.2 variants in ES cells were characterized by an enhanced acetylation and overall reduced expression levels. Most of the acetylation sites of the H1.0 and H1.2 variants from ES cells were located within their C-terminal tails known to be involved in the stabilization of the condensed chromatin. These data may be used for further studies aimed at analyzing the functional role played by the revealed histone H1 PTMs in the self-renewal and differentiation of pluripotent stem cells.

2. Embryonic stem cells

2021 ◽  
pp. 21-37
Author(s):  
Jonathan Slack
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Practical Importance ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Human Embryos ◽  
Mouse Es Cells ◽  
Stage Embryo

‘Embryonic stem cells’ focuses on embryonic stem (ES) cells, which are grown in tissue culture from the inner cell mass of a mammalian blastocyst-stage embryo. Human ES cells offer a potential route to making the kinds of cells needed for cell therapy. ES cells were originally prepared from mouse embryos. Although somewhat different, cells grown from inner cell masses of human embryos share many properties with mouse ES cells, such as being able to grow without limit and to generate differentiated cell types. Mouse ES cells have so far been of greater practical importance than those of humans because they have enabled a substantial research industry based on the creation of genetically modified mice.

scholarly journals DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands

2020 ◽  
Vol 6 (35) ◽  
pp. eabb5820 ◽  
Author(s):  
Zhiming Li ◽  
Xu Hua ◽  
Albert Serra-Cardona ◽  
Xiaowei Xu ◽  
Songlin Gan ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Endogenous Retroviruses ◽  
Histone Binding ◽  
Dna Polymerase Α ◽  
Mouse Es Cells ◽  
Dna Strands ◽  
Histone Deposition

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication–linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.

scholarly journals Recql5 and Blm RecQ DNA Helicases Have Nonredundant Roles in Suppressing Crossovers

2005 ◽  
Vol 25 (9) ◽  
pp. 3431-3442 ◽  
Author(s):  
Yiduo Hu ◽  
Xincheng Lu ◽  
Ellen Barnes ◽  
Min Yan ◽  
Hua Lou ◽  
...  
Keyword(s):  
Cancer Susceptibility ◽  
Es Cells ◽  
Embryonic Stem ◽  
Bloom Syndrome ◽  
Embryonic Fibroblasts ◽  
Dna Helicases ◽  
Mouse Es Cells ◽  
Elevated Rate ◽  
Wild Type Control ◽  
Mitotic Cells

ABSTRACT In eukaryotes, crossovers in mitotic cells can have deleterious consequences and therefore must be suppressed. Mutations in BLM give rise to Bloom syndrome, a disease that is characterized by an elevated rate of crossovers and increased cancer susceptibility. However, simple eukaryotes such as Saccharomyces cerevisiae have multiple pathways for suppressing crossovers, suggesting that mammals also have multiple pathways for controlling crossovers in their mitotic cells. We show here that in mouse embryonic stem (ES) cells, mutations in either the Bloom syndrome homologue (Blm) or the Recql5 genes result in a significant increase in the frequency of sister chromatid exchange (SCE), whereas deleting both Blm and Recql5 lead to an even higher frequency of SCE. These data indicate that Blm and Recql5 have nonredundant roles in suppressing crossovers in mouse ES cells. Furthermore, we show that mouse embryonic fibroblasts derived from Recql5 knockout mice also exhibit a significantly increased frequency of SCE compared with the corresponding wild-type control. Thus, this study identifies a previously unknown Recql5-dependent, Blm-independent pathway for suppressing crossovers during mitosis in mice.

182. NOVEL SIGNALLING IN MOUSE EMBRYONIC STEM CELLS GENERATES PRIMITIVE ECTODERM-LIKE CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 100
Author(s):  
M. B. Morris ◽  
N. Hamra ◽  
A. C. Lonic ◽  
F. Felquer
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Leukaemia Inhibitory Factor ◽  
Signalling Pathways ◽  
Specific Cell ◽  
Self Renewal ◽  
Mouse Es Cells ◽  
Homogeneous Production ◽  
Extracellular Molecules

The phenotypic status of embryonic stem (ES) cells is controlled in part by signalling pathways which translate inputs mediated by extracellular molecules. An important extracellular protagonist in mouse ES cells is LIF (leukaemia inhibitory factor) which interacts with the gp130–LIFR receptor complex to activate a number of downstream signalling pathways, including the STAT3, MEK/ERK and PI3K/Akt. These pathways, together with others, interact in complex and sometimes competing ways to generate the well-known characteristics of mouse ES cells of self-renewal, high rates of proliferation, and pluripotence. The addition of a second molecule, L-proline, to the extracellular environment alters the pluripotent status of mouse ES cells, converting them to a second pluripotent population equivalent to the primitive ectoderm of the pre-gastrulating embryo. This conversion, from ES cells to primitive ectoderm-like cells, primes the latter for directed differentiation to specific cell types (1). Here we show, using inhibitor studies and kinome array analysis, that this small molecule appears to work by (i) changing the balance in activity of signalling pathways already stimulated by LIF and (ii) activating additional signalling pathways. Specifically, L-proline rapidly further activates the LIF-stimulated MEK/ERK pathway, tipping the balance in favour of primitive-ectoderm formation and away from ES-cell self-renewal sustained by LIF-mediated activation of the STAT3 pathway. In addition, L-proline rapidly stimulates other pathways including p38, mTOR and PI3K/Akt each of which contributes, to a greater or lesser extent, to the conversion to primitive ectoderm-like cells. These results indicate that (i) L-proline acts in novel ways to stimulate embryo-like developmental progression in ES cells and (ii) through the addition of small, nontoxic activators and inhibitors of signalling pathways, the differentiation of pluripotent ES cells might be controlled sufficiently well for the homogeneous production of specific cell types suitable for use in animal models of human disease.

scholarly journals Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES cells and during differentiation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jie Lan ◽  
Nicholas Rajan ◽  
Martin Bizet ◽  
Audrey Penning ◽  
Nitesh K. Singh ◽  
...  
Keyword(s):  
Es Cells ◽  
Consensus Sequence ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Messenger Rnas ◽  
Related Factors ◽  
Mouse Es Cells ◽  
Mrna Targets ◽  
Tet Enzymes ◽  
And Function

Abstract Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

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