During development cells become committed and differentiate from one cell into many distinct cell types. Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of the blastocysts of an early embryo. In contrast to committed or differentiated cells, pluripotent cells can differentiate into any fetal or adult cell type and are capable of self-renewal and unlimited proliferation [39
]. These have tremendous potential in medicine, as ES cells could be differentiated into any cell type or even tissue of the body and be used for potential cell replacement therapies.
ES cells are characterized by a particular pattern of gene expression. For example, various genes are upregulated in ES cells and are frequently used as pluripotency markers. Oct4 seems to be an important regulator of pluripotency and differentiation [40
]. It represses or activates expression of different genes, which occurs either directly by binding to promoter regions or indirectly by neutralising transcription activators [41
]. Oct4, also known as Oct3, is a member of the POU transcription family [42
]. These are transcription factors that bind via an octameric sequence to an AGTCAAAT consensus sequence [41
]. The gene is expressed in early mammalian embryos, during gametogenesis, in ES cells [43
], and occasionally in tumours [44
]. After gastrulation, Oct4 becomes silent in mouse and human mammalian somatic cells [45
]. In mouse oocytes, Oct4 mRNA is present as a maternal transcript [46
] and it is downregulated when development proceeds [47
]. It is essential, but not sufficient to maintain cells in an undifferentiated state [48
]. During embryonic development, Oct4 is expressed in early blastomeres. Then, it becomes restricted to the inner cell mass, and is down regulated in the trophectoderm and primitive endoderm [47
]. Oct4 is widely conserved. Homologues even exist in early amphibian development, where they also act as suppressors of cell fate commitment. Even though so far ES cells have not been derived from amphibians, the Xenopus laevis
version of Oct4, Pou91, was able to fully support mouse ES cell self-renewal [49
]. This suggests a similar function for Pou91 in pluripotency.
Pluripotency also requires other factors, for example, the leukaemia inhibitory factor (LIF). LIF is a key molecule required for self-renewal and pluripotency in mouse ES cells [50
], but not for monkey or human ES cells [52
]. It is known to bind to the heterodimer LIF receptor—gp130 and to activate the transcription factor STAT3 by phosphorylation [53
]. Interestingly, overexpression of the gene Nanog can bypass the requirement for LIF in mouse ES cells [54
]. Nanog is also required for maintaining the undifferentiated state of early postimplantation embryos and ES cells [54
], making Nanog an important regulator of pluripotency. There are also other components required, such as bone morphogenic proteins (BMP) that activate the inhibitor of differentiation (Id), which represses differentiation [56
]. Another important regulator is Sox2, which cooperatively binds the Oct4 protein and activates genes promoting pluripotency [57
], but represses its inhibitors [58
Despite obtaining the ES cells from blastocysts, ES or ES-cell-like cells can be obtained by nuclear reprogramming, a term introduced to describe the restoration of the embryonic pattern of gene expression [59
]. Nuclear reprogramming was first demonstrated in nuclear transfer experiments. Xenopus laevis
nuclei of differentiated cells were transplanted into enucleated frog eggs. This gave rise to normal fertile adult frogs, illustrating that differentiated cells can become reprogrammed and give rise to an entire new organism [60
]. Another way to reprogram nuclei was achieved when cells were fused to each other [62
]. Cell fusions with ES cells rejuvenated somatic cells that could differentiate into many different cell types. In these hybrids the silent gene Oct4 was reactivated [64
]. Fusion experiments with an increased expression of the pluripotency gene Nanog increased nuclear reprogramming efficiency by 200-fold [65
]. Nowadays, the most common way somatic cells are reprogrammed to an embryonic-like pattern of gene expression is by overexpressing different factors, such as Oct4, Sox2, c-Myc, and Klf4 under ES cell culture conditions [66
]. Surprisingly, Nanog was not required, even though it seemed to promote nuclear reprogramming in cell fusion experiments [65
]. These ES-like cells had normal ES cell morphology, a gene expression pattern typical for normal ES cells and could differentiate into all three germ layers. They were named iPS cells, induced pluripotent stem cells [66
]. Even though the generation of iPS cells is a very convenient way to generate ES cells, this approach does not reveal the mechanism underlying nuclear reprogramming. Also, it does not identify novel factors that are involved in this process.
To better understand the process of nuclear reprogramming, nuclear transfer experiments of somatic cells into Xenopus oocytes were carried out. It was found that even human or mouse nuclei could be reprogrammed by frog oocytes and induce an ES cell or ES cell-like pattern of gene expression [67
]. For example, genes such as Oct4, Nanog, and Sox2 became transcriptionally active upon nuclear transfer [67
]. Using this system, novel molecules were isolated that interact with the promoter region of Oct4. One of these molecules was TCTP. Further functional assays revealed that it in fact TCTP changed the transcriptional level of Oct4 and even Nanog in human nuclei, genes essential for successful nuclear reprogramming [68
]. A similar effect of TCTP was found in bovine oocytes, suggesting a conserved function of TCTP in activating pluripotency [69
]. TCTP knockout mice have an abnormal number of cells in the epiblast [22
]. The epiblast is formed from the inner cell mass of the blastocyst, from which ES cells can be obtained. Since TCTP activates the pluripotency genes Oct4 and Nanog, it is possible that, in the TCTP knockout mice, the epiblast does not develop normally due to misregulation of pluripotency genes such as Oct4 and Nanog.
It would be interesting to determine if TCTP activates also other pluripotency genes such as Sox2 and Klf4. TCTP might promote pluripotency in two different ways, namely, by (1) activating pluripotency genes and (2) inhibiting somatic gene expression. Genomewide studies in the absence of TCTP could help to determine what other genes TCTP regulates. Another important question is whether TCTP is sufficient for nuclear reprogramming and if its overexpression in somatic cells could replace the four reprogramming factors used to make iPS cells. Even if it does not replace these four factors, it could increase the generation of iPS cells, a currently very inefficient process.
Nuclear actin polymerization has been reported to be required for Oct4 activation in Xenopus laevis
]. Since TCTP has been found to contain an actin-binding site [17
], it is possible that it might interfere with pluripotency gene regulation by interfering with actin. Testing actin polymerization in the absence and presence of TCTP, as well as the effect on Oct4, would help to understand any possible interactions required to induce pluripotency. These experiments could also be analyzed Genomewide, which will greatly help to elucidate the underlying network required to establish pluripotency. Using TCTP as bait to pull down interaction partners together with Genomewide Chromatin Immunoprecipitation analysis of TCTP and its interaction partners will also contribute towards understanding how pluripotency is established.
Another protein that has been found to interact with TCTP in Xenopus oocytes is nucleoplasmin Npm1 [71
]. Similar to TCTP knockout mice, mice deficient in Npm1 are embryonic lethal and have smaller embryo sizes [72
]. Npm1 is a very abundant protein. In fertilized Xenopus
eggs, it is involved in the decondensation and hence transcriptional activation of the paternal genome provided after normal fertilization by the sperm (reviewed in [73
]). It is possible that TCTP not only activates pluripotency genes, but also that it has a role in paternal gene activation by interacting with Npm1. Disturbing the interaction of TCTP and Npm1 could show if TCTP is also involved in this process. But it is possible that pluripotency and paternal and maternal genome activation is actually not that different. After all, when the genome becomes transcriptionally active, it is set as such, so that it can proliferate and differentiate into an entire organism. Hence, zygotic genome activation could be regarded as nuclear reprogramming that occurs naturally in nature, without the need of nuclear transplantation, cell fusion experiments, or overexpression of a few transcription factors.