Genetic regulation of stem cell pluripotency
Stem cells would not be interesting for developmental biology and clinical medicine if they did not have pluripotency and would not be able to start development again. The development of conditions for the cultivation of cell lines originating from embryos, the search for ways to perpetuate the pluripotency of stem cells is a necessary basis that will ensure their use in the mainstream of regenerative medicine for the restoration of impaired structures and functions of organs and tissues and the treatment of a wide range of diseases. A deep understanding of molecular programs aimed at maintaining the pluripotency of stem cells and regulating their differentiation is necessary in order to be able to manage their potential.
Studies of various lines of embryonic stem cells have made it possible to identify and clarify the organization of gene networks that control the early stages of development. Based on these studies, the idea of the existence of so-called master genes and slave genes arose. Mammalian embryo development is controlled by key regulatory genes that affect the transcription of other genes. These regulators activate or repress the expression of genes that mediate phenotypic changes during stem cell differentiation. They include cascades of structural genes and thereby determine organ-tissue specialization. Among such important master genes are various families of transcription factors. For example, the POU family can activate the expression of target genes by binding to a specific nucleotide sequence on DNA - this is the AGTCAAAT octamer.
At the molecular level, pluripotency is determined by transcription factors, the expression of which apparently determines whether the cell will be pluripotent or not. Thus, recent studies have revealed the OCT4 transcription factor belonging to the above-mentioned POU family. Studies on mouse embryonic stem cell lines have shown that OCT4 is almost completely expressed only in embryonic cells: at the initial stage – in all blastomeres, then – limited, only in the internal cell mass. Targeted destruction of OCT4 leads to the formation of embryos without pluripotent cell mass, which indicates its crucial role in the control of pluripotency and differentiation. The target genes that are actually responsible for executing OCT4 decisions are only partially known.
Further studies have found other transcription factors that determine the state of pluripotency. These include SOX2. It belongs to the family of DNA-binding proteins that are involved in the regulation of transcription and chromatin structure. When studying mutants for this gene, an autonomous need for its expression for cells of the early stage of development was revealed. A similar role has been identified for other genes that are expressed in the population of the founding cells of embryos - FOXD3. This gene belongs to another family of transcription factors.
Researchers at the Institute of Stem Cell Research at the University of Edinburgh and the Nara Institute of Japan have discovered another gene that is also considered a marker of pluripotency. This gene was named NANOG, after the country of eternal youth Tir Nan Og from Celtic mythology. The function of NANOG in germ cells progressively decreases as the embryo develops. NANOG appears to be one of several factors that are expressed in pluripotent cells and are suppressed at the beginning of differentiation. This gene was discovered during the study of mouse embryonic stem cell lines and is currently not yet included in the group of 532 genes that have been identified as specific to human embryonic stem cells.
Blocking the differentiation of embryonic stem cells both in vitro and in vivo was found for another protein - Pem. The PEM gene regulates the transition between undifferentiated and differentiated cells in early mouse embryos.
Thus, to date, a number of genes have been identified whose expression controls the early stages of embryonic development, when many embryonic stem cells have the property of totipotency or pluripotency.
In addition, in order to realize and maintain the state of pluripotence, it is necessary to have certain signals and a system for their transmission. Such signals include the factor LIF (leukaemia inhibitory factor), which is necessary to maintain the undifferentiated state of mouse embryonic stem cells. LIF performs its effect by binding to the corresponding receptor. Subsequent signal transmission to the nucleus occurs by means of STA3, which functions as a transcription factor. It is noteworthy that this system does not prevent the differentiation of human embryonic stem cells. Other signals include BMP4 (bone morphogenetic protein-4), which in the presence of LIF enhances the self-renewal and pluripotency of embryonic stem cells through activation of the SMAD gene, which also encodes a transcription factor, and subsequent activation of the Id (inhibitor of differentiation) gene family. Recent studies have revealed the importance of the participation of WNT proteins in the signal transmission system, as well as the PTEN gene that controls phosphatase and is at the stage of signal transmission completion.
These regulatory elements function in a certain sequence, there are established hierarchical relationships between them, which determines the final effect. In particular, in mouse embryonic stem cells, cytokine activation of the LIF receptor–mediated pathway and maintenance of Oct4 and Nanog expression are key elements of the genetic program that allows embryonic cells to be stem cells - the state of 'stemness'. Other paths and factors are overlapping key elements. All this increases the combinatorial complexity of regulation.
Many mechanisms of regulation of pluripotency of embryonic stem cells are still far from being fully understood. In addition, the results obtained for mouse stem cells are not always applicable to human stem cells. However, this kind of research is necessary to develop optimal characteristics of stem cell lines and conditions for their maintenance, as well as to formulate standards for research work with human stem cells.