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Developmental cell biology

Regulatory networks in embryo-derived pluripotent stem cells

Key Points

  • Embryo-derived stem cells can be considered archetypal stem cells. Among them, embryonic stem cells (ESCs) are derived at an earlier stage of embryo development than others, such as embryonal carcinoma cells (ECCs) and embryonic germ cells (EGCs). Compared to adult or fetal stem cells, embryo-derived stem cells are easier to identify, to isolate and to use as established cell lines.

  • The golden feature of ESCs is their pluripotency, as defined as the ability to give rise to any derivative of the three primary embryonic germ layers and to germ cells. Pluripotency can be maintained indefinitely if ESCs are cultured under correct conditions, and can be demonstrated in mice when ESCs injected into blastocysts become incorporated in the conceptus and participate in normal development. Notably, disorganized proliferation and tumour formation of ESCs might also occur, regarded here as 'the other side' of pluripotency.

  • ESCs behave normally within a blastocyst but give rise to tumours when transplanted ectopically, for example, under the skin, it therefore implies that exogenous cues have a bearing on cell fate. Whether they do so by priming or by selecting ESC states is not known.

  • Most of the extrinsic and intrinsic regulators that are known to operate in mouse ESCs (for example, LIF and BMP4 extracellular ligands; OCT4, SOX2 and Nanog transcription factors) have a dose-dependent effect on pluripotency. Genes that are required to maintain pluripotency in human ESCs are mostly unknown, with the exception of OCT4. Combinatorial interactions and nonlinear responses, which have been described in mice, compound this situation.

  • The current lack of definitive control over stem-cell fate finds an interesting parallel in the lack of definitive control over nuclei of differentiated cells regaining pluripotency after transfer into oocytes (cloning).

  • An understanding of how pluripotency is secured in ESCs is likely to indicate ways to reinstate pluripotency in somatic cells. The oocyte is the common reactor that is used to reinstate pluripotency on somatic nuclei. However, in the future we might not even need oocytes to reinstate pluripotency, as has been shown for somatic cells fused with ESCs and for bone marrow cells that can become multipotent adult progenitor cells (MAP-C cells) in culture.

  • An understanding of the ESC circuitry might reveal how to achieve phenotypic changes without genetic manipulation of Oct4 and Nanog and other pluripotency-associated genes.

Abstract

Mammalian development requires the specification of over 200 cell types from a single totipotent cell. Investigation of the regulatory networks that are responsible for pluripotency in embryo-derived stem cells is fundamental to understanding mammalian development and realizing therapeutic potential. Extracellular signals and second messengers modulate cell-autonomous regulators such as OCT4, SOX2 and Nanog in a combinatorial complexity. Knowledge of this circuitry might reveal how to achieve phenotypic changes without the genetic manipulation of Oct4, Nanog and other toti/pluripotency-associated genes.

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Figure 1: Origin of stem cells in the mammalian embryo.
Figure 2: Combinatorial signalling pathways involved in maintaining mouse ESC pluripotency.
Figure 3: The phenotypic effects of gene-targeting experiments in mice.

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Zixuan Zhao, Xinyi Chen, … Hanry Yu

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Acknowledgements

We regret not being able to cite all the relevant publications due to space constraints. We wish to thank J. Rossant for her valuable comments on the manuscript, and acknowledge the members of our laboratory for helpful discussions and A. Malapetsa for help in the preparation of the manuscript. This work was supported by the Max-Planck Society.

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Correspondence to Hans R. Schöler.

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DATABASES

Entrez Gene

Lif

OCT4

Pem

Swiss-Prot

BMP4

ERK1

ERK2

FOXD3

gp130

LIF

LIFR

mTOR

Nanog

OCT4

PEM

PTEN

SOX2

STAT3

STAT4

FURTHER INFORMATION

The official National Institutes of Health resource for stem cell research: Appendix E

Hans Schöler's laboratory

Michele Boiani's laboratory

Glossary

STEM CELLS

Cells that have the ability to divide for indefinite periods in the undifferentiated state but retain the potential to give rise to specialized cells.

HOMEOSTASIS

A property of cells or organisms to regulate the internal environment in response to external cues to maintain a stable condition inside.

EMBRYO

In the mouse, the embryo is the developing organism from the time of fertilization until E10.5, when it becomes known as a fetus. The stages are: pre-implantation embryo, until E3.5; post-implantation embryo, from E3.5–E10.5; fetus, E10.5 onwards.

PLURIPOTENT

The ability of a single stem cell to develop into all different cell types of the body but not into the extra-embryonic cell types.

EMBRYONIC STEM CELLS

(ESCs). Derivative cells of the ICM of the pre-implantation embryo that have the potential to become all specialized cell types of the adult organism. As established cell lines, ESCs have been cultured under in vitro conditions that allow proliferation without differentiation for months or years.

TROPHOBLAST STEM CELLS

(TSCs). Cells derived from the trophectoderm of the blastocyst or early postimplantation trophoblast that have the potential to form all the cell types of the trophoblast-derived layer of the placenta.

INNER CELL MASS

(ICM). The prominent cluster of cells inside the blastocyst. In vivo, these cells give rise to the fetus. In vitro, the ICM cells are the source for ESCs.

PRIMITIVE ECTODERM

Also known as the epiblast. The upper layer of a group of cells derived from the inner cell mass of the blastocyst; it gives rise to all cells of the fetus.

BLASTOCYST

A pre-implantation mouse embryo of about 40–80 cells at day 3–4 after fertilization (in the case of human blastocysts the pace of development would be slower). The blastocyst consists of a hollow sphere made up of an outer layer of cells (the trophectoderm), a fluid-filled cavity (the blastocyst cavity or 'blastocoel'), and a cluster of cells at one pole on the interior (the inner cell mass).

FEEDER CELLS

Cells used in co-culture to maintain pluripotent stem cells. Feeder cells usually consist of 'mouse embryonic fibroblasts' (MEFs), which are actually fetal mesenchymal cells obtained from E13.5 fetuses. These cells can proliferate for only a few passages in vitro (primary MEFs) or be immortalized (STO (SIM mouse thioguanine- and ouabain-resistant)-SNL (STO, NEO, LIF) cells).

CHIMAERISM

The contribution to embryonic and fetal tissues of exogenous sources of cells with a different genotype, including cells that come from a different organism or species. For example, the injection of cells from one embryo into the blastocyst cavity of another embryo.

MESODERM

Germ layer; the middle layer of a group of cells derived from the primitive ectoderm of the fully expanded blastocyst; in general, it gives rise to bone, muscle, connective tissue and the middle layer of the skin. There is also an extra-embryonic mesoderm.

TROPHECTODERM

The outer cell layer of a blastocyst, made up of a mural and polar component. Gives rise to trophoblast cells upon implantation in the uterine wall.

TRANSCRIPTOME

The full complement of messenger RNA (mRNA) and non-coding RNAs, transcripts in the cell, weighted by their expression levels.

DIAPAUSE

In general, a period of physiologically enforced dormancy between periods of activity. In the pre-implantation-stage embryo, it is a transient/reversible state of reduced vitality during which the blastocyst floats in the uterus without implanting.

SRC-HOMOLOGY-2 (SH2)

Src-homology-2 (SH2) domains are modules of 100 amino acids that bind to specific phosphotyrosine-containing peptide motifs. SH2-domain proteins have numerous roles such as adaptors, scaffolds, kinases, phosphatases, Ras signalling, transcription, ubiquitination, cytoskeletal regulation, signal regulation, phospholipid second-messenger signalling, and yet others.

TERATOMA

Also known as terratocarcinoma. A tumour composed of tissues from the three embryonic germ layers, which is usually found in the ovary and testis. It can be produced experimentally in animals by the injection of pluripotent stem cells, to determine the abilities of the stem cells to differentiate into various types of tissues.

POU DOMAIN

A specific region or amino-acid sequence in a protein that is associated with specific sequences such as the octamer motif (5′-ATGCAAAT-3′) on DNA. It was originally identified in the transcription factors PIT1, OCT1, OCT2 and UNC86.

MULTIPOTENT

Ability of a single stem cell to differentiate along multiple lineages and give rise to different tissues. It has broad potential but less than a pluripotent cell.

TROPHOBLAST

The extra-embryonic, highly proliferative tissue that is responsible for implantation, contributing to the placenta that controls the exchange of oxygen and metabolites between mother and embryo.

SUPPRESSION-SUBTRACTIVE HYBRIDIZATION

A method to identify DNA/RNA that is present in one sample but not in the others.

HOMEODOMAIN

A domain in a protein that is encoded for by a homeobox—it consists of about 60 amino-acid residues and recognizes and binds to specific AT-rich DNA sequences.

EST (EXPRESSED SEQUENCE TAG)

A unique stretch of DNA within a coding region of a gene that is useful for identifying full-length genes and serves as a landmark for gene mapping.

CRE/LOXP RECOMBINATION SYSTEM

A site-specific recombination system derived from Escherichia coli bacteriophage P1. Two short DNA sequences (loxP sites) are engineered to flank the target DNA. Activation of the Cre-recombinase enzyme catalyses recombination between the loxP sites, leading to excision of the intervening sequence.

PRIMITIVE ENDODERM

Also known as the hypoblast. Lower layer of a group of cells derived from the inner cell mass of the blastocyst. It gives rise to visceral (lining the inner cell mass) and parietal extra-embryonic (lining the trophectoderm) endoderm.

NUCLEAR TRANSFER

The procedure of removing the nucleus of a donor cell and transplanting it into another. Typically this involves a somatic cell (donor) and an oocyte (recipient) and is instrumental to the cloning procedure therefore known as SCNT (somatic cell nuclear transfer).

CLONAL (CLONED)

Describes the genetically identical progeny of a single mitotic cell. Also applied to an embryo to indicate its derivation from the splitting of another embryo or by nuclear transfer into a recipient oocyte.

UNIPOTENT

Refers to a cell that can only differentiate in one specific way.

METHYLATION

Covalent modification of the DNA nucleotide cytosine by addition of a methyl (CH3) group.

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Boiani, M., Schöler, H. Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol 6, 872–881 (2005). https://doi.org/10.1038/nrm1744

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