Regulatory circuits underlying pluripotency and reprogramming

doi:10.1016/j.tips.2009.03.003

Trends in Pharmacological Sciences

Volume 30, Issue 6, June 2009, Pages 296-302

https://doi.org/10.1016/j.tips.2009.03.003 Get rights and content

The ability of pluripotent stem cells to differentiate into all cell types of an organism has received widespread attention in basic and clinical research and holds tremendous potential for pharmacologic and medical applications. In this review, we provide an overview of the factors and pathways involved in pluripotency and discuss a possible mechanism underlying genetic reprogramming using defined transcription factors. We specifically address the association between core transcription factors (e.g. Oct4, Sox2 and Nanog) and the cellular machinery (e.g. chromatin remodeling complex, DNA methylation, microRNA and X chromosome inactivation), which has an important role in cell fate determination.

Introduction

The classification of mammalian cells is based on a hierarchical organization of cellular differentiation, which determines the developmental potency of a cell: totipotency, pluripotency, multipotency and unipotency [1] (Figure 1). Totipotency is the ability of a cell to differentiate into all cell types of an entire organism including the trophoblast lineage in a coordinated and highly complex manner. The only totipotent cells are the zygotes and blastomeres of the earliest stages of natural or cloned embryos, which implant into the uterus and subsequently develop into both embryonic and extraembryonic cell lineages.

Pluripotency is the ability of a cell to differentiate into all cell types of an organism excluding the trophoblast lineage. Pluripotent cells are present in early embryos, from which they are derived to establish cell lines in vitro. The five types of pluripotent stem cells known to date are embryonic stem (ES), embryonic germ (EG), embryonic carcinoma (EC), induced pluripotent stem (iPS) and multipotent germline stem (mGS) cells 2, 3, 4. A recently discovered novel blastocyst-derived stem cell, FAB-SCs, might exist in a ‘partially pluripotent’ state, which can gain full pluripotency by the mere addition of growth factors (e.g. leukemia inhibitory factor [LIF] and bone morphogenetic protein [BMP]4) [5]. Although pluripotent cells normally do not develop into extraembryonic tissues (see Glossary), they can contribute to the germline in chimeras with support of extraembryonic tissues from fertilized embryos via morula aggregation or blastocyst injection.

Multipotency is the ability of a cell to differentiate along multiple cell lineages. Somatic stem cells that exhibit this feature are, for example, neural stem cells, which can differentiate into neurons, astrocytes and oligodendrocytes. Unipotency is the ability of a cell to differentiate into only one cell type. Spermatogonial stem cells, for example, are unipotent because they can differentiate only into sperm in vivo [2].

Because pluripotent cells can differentiate into all cell types of an organism including germ cells and can be cultured for an extended period of time without losing their key characteristics, they are considered to be an ideal resource for therapeutic applications in regenerative medicine such as, for example, cell and tissue replacement in the treatment of degenerative diseases. However, little is known about the mechanism underlying cellular pluripotency. Recent studies have shown that factors such as microRNAs (miRNAs), transcription factors, chromatin structure or chromatin modifiers and processes such as signaling pathways and DNA methylation have essential roles in maintaining the pluripotent state of ES cells 6, 7, 8, 9, 10. Here, we review how pluripotency of ES cells is maintained and propose a cellular mechanism by which somatic cells are genetically reprogrammed to the pluripotent state using defined factors.

Section snippets

ES cells are the standard for pluripotent stem cells

To recapitulate, ES, EG, EC, iPS and mGS cells represent the five types of pluripotent stem cells known to date. EG and EC cells show a lower potential for multilineage differentiation and germline contribution; these cells also do not fully meet the most stringent criteria for pluripotency because they do not contribute to the development of whole embryos after tetraploid complementation [11]. Although mGS cells are referred to as ‘multipotent’ germline stem cells, we classify them as

Pluripotency and ‘stemness’

Pluripotent stem cells are defined by the capacity for unlimited self-renewal, a quality usually referred to as ‘stemness’. Speculation has arisen as to whether the self-renewal of pluripotent and somatic stem cells could be controlled by a similar set of ‘stemness genes’. Three independent investigations examining gene-expression profiling in ES cells and two somatic stem cells (neural stem cells and hematopoietic stem cells) have shown that only one gene, Itga6 (encoding integrin α6), is

Transcription factors involved in the pluripotency of mouse ES cells

The transcription factors Oct4, Sox2 and Nanog are the three core transcription factors involved in cellular pluripotency 19, 20, 21. Oct4 and Nanog play an essential part in early development and maintenance of pluripotency, with their expression being restricted to early embryos, germ cells and pluripotent cells. Loss of function of Oct4 and Nanog results in loss of cellular pluripotency and differentiation 2, 20, 22. Oct4 is involved in preventing trophectoderm formation by regulating Cdx2

Pluripotency controlled by DNA chromatin modification and DNA methylation

The chromatin structure of pluripotent ES cells is transcriptionally more permissive and enriched in active histone marks than that found in differentiated cells [45]. Polycomb group (PcG) proteins indirectly act as transcriptional repressors of developmental genes by catalyzing histone H3 Lys27 (H3K27) methylation, resulting in the heterochromatic state of their promoter regions, a mark for gene silencing 23, 46, 47. Although the mechanism underlying the role of PcG protein in regulating

miRNAs and pluripotency

miRNAs are short single-stranded RNAs (18–25 nucleotides), which do not code for protein but regulate gene expression by interacting with target messenger (m)RNA to result in mRNA degradation, deadenylation or translational repression 60, 61, 62. According to the miRNA database, almost 800 miRNA genes have been identified in the mouse to date (http://microrna.sanger.ac.uk/cgi-bin/targets/v5/genome.pl). During development, miRNAs are expressed in a tissue-specific manner, suggesting that they

Implication of pluripotency for active X chromosome (Xa)

A phenotypic characteristic of pluripotent cells is the lack of an inactive X chromosome (Xi), regardless of X chromosome number. Therefore, female ES cells contain two active X chromosomes (XaXa), whereas tetraploid fusion hybrid cells contain 3 (XaXaXaY) or 4 Xas (XaXaXaXa) [69]. However, the Xa of pluripotent cells is different from the Xa of somatic cells because ES cells express low levels of Xist (100- to 1000-fold lower than female somatic cells) [69], which is detected as a pinpoint

Pluripotent cells transfer pluripotency to the somatic cells by cell fusion

Pluripotent cells, ES, EG, EC and iPS cells, can transfer their pluripotent property to the somatic cell genome by cell fusion 69, 70. Notwithstanding that the mechanism remains to be elucidated, it is known that pluripotent cell nuclear factors can reset the somatic cell genome to the pluripotent state [72], which is evidenced by activation of pluripotency markers, inactivation of tissue-specific markers and resetting of epigenetic modifications [73]. Moreover, overexpression of pluripotency

Concluding remarks and perspectives

Although the mechanism by which somatic cells regain a pluripotent phenotype remains largely unknown, we herein propose a model for the cellular mechanism of reprogramming during the induction of iPS cells, which is defined by the introduction of four transcription factors and exhibits the transcriptional regulatory system and the epigenetics of ES cells (Figure 2).

Exogenous factors are directly involved in inducing the expression of their endogenous counterparts, which normally takes ∼7–15

Acknowledgements

We thank Jeanine Müller-Keuker for providing the figure illustrations.

Glossary

Active (Xa) and inactive (Xi) X chromosome: in female mammalian somatic cells, one of the two X chromosomes is randomly inactivated to achieve dosage compensation for X-linked genes. The X chromosome chosen as active or inactive is referred to as Xa or Xi, respectively.
Blastocyst injection: another technique for testing in vivo developmental ability of a certain type of cells. Cells for testing are injected into the blastocoel of normal blastocyst and transferred to the uterus of the foster mother.

References (80)

R. Jaenisch et al.
Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming
Cell
(2008)
M. Kanatsu-Shinohara
Generation of pluripotent stem cells from neonatal mouse testis
Cell
(2004)
Y.F. Chou
The growth factor environment defines distinct pluripotent ground states in novel blastocyst-derived stem cells
Cell
(2008)
B.E. Bernstein
A bivalent chromatin structure marks key developmental genes in embryonic stem cells
Cell
(2006)
M. Bibikova
Unraveling epigenetic regulation in embryonic stem cells
Cell Stem Cell
(2008)
L.A. Boyer
Core transcriptional regulatory circuitry in human embryonic stem cells
Cell
(2005)
Y. Shi
Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds
Cell Stem Cell
(2008)
I. Chambers
Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells
Cell
(2003)
J. Nichols
Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4
Cell
(1998)
K. Mitsui
The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells
Cell
(2003)

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Induced pluripotent stem cell (iPSC) technology that could revert the fate of terminally differentiated somatic cells into embryonic stem cell–like status by using ectopic expression of four transcription factors (e.g., Oct4, Sox2, Klf4, and c-Myc) opened up a new era of stem cell research to study and treat intractable human diseases, including neurodegenerative disorders such as Parkinson’s and Huntington’s diseases. Nevertheless, our current understanding of the reprogramming process is incomplete and requires further investigation into the underlying mechanisms and development of optimal reprogramming techniques. In this chapter, we provide an overview of the current techniques and critical issues for further optimization of high-quality iPSC generation.
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All these three Nanog proteins are defined with distinctive biochemical activities and biological properties (Jeter et al., 2015). Nanog is the head – master for the maintenance of pluripotency and self – renewal of embryonic stem cell and cellular reprogramming (Boyer et al., 2005; Chambers and Tomlinson, 2009; Patra et al., 2018)which is carried out by slightly modified mechanistic pathways (Do and Schöler, 2009). Reports suggest that, even though Nanog has only marginal sequence identity in somatic cells, it still has a small role in reprogramming of somatic cells back into its pluripotent state (Moon et al., 2013; Silva et al., 2009; Theunissen et al., 2011).
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During transition to the pluripotent state, many genes expressed in the differentiated state are silenced and many others are activated (Theunissen and Jaenisch, 2014). The iPS state, in turn, is maintained by feedback loops involving (at least) endogenously encoded OCT4, SOX2, and KLF4 (Boyer et al., 2005; Chew et al., 2005; Do and Schöler, 2009; Jaenisch and Young, 2008; Kim et al., 2008; Loh et al., 2006; Martello and Smith, 2014). The efficiency of reprogramming human neonatal fibroblasts is typically low (0.002%–0.02%), and that for human cells from older donors is even lower (Maherali et al., 2008; Park et al., 2008; Paull et al., 2015; Rohani et al., 2014; Takahashi et al., 2007; Yu et al., 2007).
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The conditions for qPCR were described previously [26]. The expressions of octamer-binding transcription factor 4 (OCT-4; also known as POU5F1 (POU domain, class 5, transcription factor 1)), homeobox protein NANOG, Zinc finger protein 42 (ZFP-42; also known as Rex-1), and alkaline phosphatase (TNAP) were used as markers of the undifferentiated ESC phenotype [13,29–34]. Further, the expressions of Fibroblast growth factor 5 (FGF5), as a marker of early ESC differentiation [33]; Sex-determining region Y-box 1 transcription factor (SOX1) and Paired box protein (PAX6), as markers of neuronal differentiation; Brachyury (BRACH T), as a marker of mesodermal differentiation; GATA binding protein 4 (GATA4) and Alpha-fetoprotein (AFP), as markers of early endodermal differentiation; ETS-domain transcription factor E74-like factor 5 (ELF5) and Caudal type homeobox 2 (CDX2), as markers of trophoblast differentiation; and vascular endothelial growth factor (VEGF) as the HIF-1α target gene were determined [13,29–34].
Hypoxic conditions are suggested to affect the differentiation status of stem cells (SC), including embryonic stem cells (ESC). Hypoxia inducible factor (HIF) is one of the main intracellular molecules responsible for the cellular response to hypoxia. Hypoxia stabilizes HIF by inhibiting the activity of HIF prolyl-hydroxylases (PHD), which are responsible for targeting HIF-alpha subunits for proteosomal degradation. To address the impact of HIF stabilization on the maintenance of the stemness signature of mouse ESC (mESC), we tested the influence of the inhibition of PHDs and hypoxia (1% O₂ and 5% O₂) on spontaneous ESC differentiation triggered by leukemia inhibitory factor withdrawal for 24 and 48 h. The widely used panhydroxylase inhibitor dimethyloxaloylglycine (DMOG) and PHD inhibitor JNJ-42041935 (JNJ) with suggested higher specificity towards PHDs were employed. Both inhibitors and both levels of hypoxia significantly increased HIF-1alpha and HIF-2alpha protein levels and HIF transcriptional activity in spontaneously differentiating mESC. This was accompanied by significant downregulation of cell proliferation manifested by the complete inhibition of DNA synthesis and partial arrest in the S phase after 48 h. Further, HIF stabilization enhanced downregulation of the expressions of some pluripotency markers (OCT-4, NANOG, ZFP-42, TNAP) in spontaneously differentiating mESC. However, at the same time, there was also a significant decrease in the expression of some genes selected as markers of cell differentiation (e.g. SOX1, BRACH T, ELF5). In conclusion, the short term stabilization of HIF mediated by the PHD inhibitors JNJ and DMOG and hypoxia did not prevent the spontaneous loss of pluripotency markers in mESC. However, it significantly downregulated the proliferation of these cells.
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Trends in Pharmacological Sciences

ReviewRegulatory circuits underlying pluripotency and reprogramming

Introduction

Section snippets

ES cells are the standard for pluripotent stem cells

Pluripotency and ‘stemness’

Transcription factors involved in the pluripotency of mouse ES cells

Pluripotency controlled by DNA chromatin modification and DNA methylation

miRNAs and pluripotency

Implication of pluripotency for active X chromosome (Xa)

Pluripotent cells transfer pluripotency to the somatic cells by cell fusion

Concluding remarks and perspectives

Acknowledgements

Glossary

Cell

Cell

Cell

Cell

Cell Stem Cell

Cell

Cell Stem Cell

Cell

Cell

Cell

J. Biol. Chem.

Cell

Cell

Cell

Cell

J. Biol. Chem.

Trends Biochem. Sci.

Cell

Cell

Cell

Cell

Curr. Biol.

J. Biol. Chem.

Cell

Trends Biochem. Sci.

Dev. Cell

Dev. Cell

Cell Stem Cell

Cell Stem Cell

Curr. Biol.

Cell Stem Cell

Curr. Opin. Genet. Dev.

Regulatory networks in embryo-derived pluripotent stem cells

Nat. Rev. Mol. Cell Biol.

Generation of germline-competent induced pluripotent stem cells

Nature

A mammalian microRNA cluster controls DNA methylation and telomere recombination via Rbl2-dependent regulation of DNA methyltransferases

Nat. Struct. Mol. Biol.

MicroRNAs control de novo DNA methylation through regulation of transcriptional repressors in mouse embryonic stem cells

Nat. Struct. Mol. Biol.

Derivation of completely cell culture-derived mice from early-passage embryonic stem cells

Proc. Natl. Acad. Sci. U. S. A.

Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors

Nature

Derivation of pluripotent epiblast stem cells from mammalian embryos

Nature

New cell lines from mouse epiblast share defining features with human embryonic stem cells

Nature

Review
Regulatory circuits underlying pluripotency and reprogramming