G0 phase

The G₀ phase describes a cellular state outside of the replicative cell cycle. Classically^[when?], cells were thought to enter G₀ primarily due to environmental factors, like nutrient deprivation, that limited the resources necessary for proliferation. Thus it was thought of as a resting phase. G₀ is now known to take different forms and occur for multiple reasons. For example, most adult neuronal cells, among the most metabolically active cells in the body, are fully differentiated and reside in a terminal G₀ phase. Neurons reside in this state, not because of stochastic or limited nutrient supply, but as a part of their developmental program.

G₀ was first suggested as a cell state based on early cell cycle studies. When the first studies defined the four phases of the cell cycle using radioactive labeling techniques, it was discovered that not all cells in a population proliferate at similar rates. A population's "growth fraction" – or the fraction of the population that was growing – was actively proliferating, but other cells existed in a non-proliferative state. Some of these non-proliferating cells could respond to extrinsic stimuli and proliferate by re-entering the cell cycle. Early contrasting views either considered non-proliferating cells to simply be in an extended G₁ phase or in a cell cycle phase distinct from G₁ – termed G₀. Subsequent research pointed to a restriction point (R-point) in G₁ where cells can enter G₀ before the R-point but are committed to mitosis after the R-point. These early studies provided evidence for the existence of a G₀ state to which access is restricted. These cells that do not divide further exit G₁ phase to enter an inactive stage called quiescent stage.

Three G₀ states exist and can be categorized as either reversible (quiescent) or irreversible (senescent and differentiated). Each of these three states can be entered from the G₁ phase before the cell commits to the next round of the cell cycle. Quiescence refers to a reversible G₀ state where subpopulations of cells reside in a 'quiescent' state before entering the cell cycle after activation in response to extrinsic signals. Quiescent cells are often identified by low RNA content, lack of cell proliferation markers, and increased label retention indicating low cell turnover. Senescence is distinct from quiescence because senescence is an irreversible state that cells enter in response to DNA damage or degradation that would make a cell's progeny nonviable. Such DNA damage can occur from telomere shortening over many cell divisions as well as reactive oxygen species (ROS) exposure, oncogene activation, and cell-cell fusion. While senescent cells can no longer replicate, they remain able to perform many normal cellular functions. Senescence is often a biochemical alternative to the self-destruction of such a damaged cell by apoptosis. In contrast to cellular senescence, quiescence is not a reactive event but part of the core programming of several different cell types. Finally, differentiated cells are stem cells that have progressed through a differentiation program to reach a mature – terminally differentiated – state. Differentiated cells continue to stay in G₀ and perform their main functions indefinitely.

The transcriptomes of several types of quiescent stem cells, such as hematopoietic, muscle, and hair follicle, have been characterized through high-throughput techniques, such as microarray and RNA sequencing. Although variations exist in their individual transcriptomes, most quiescent tissue stem cells share a common pattern of gene expression that involves downregulation of cell cycle progression genes, such as cyclin A2, cyclin B1, cyclin E2, and survivin, and upregulation of genes involved in the regulation of transcription and stem cell fate, such as FOXO3 and EZH1. Downregulation of mitochondrial cytochrome C also reflects the low metabolic state of quiescent stem cells.

Many quiescent stem cells, particularly adult stem cells, also share similar epigenetic patterns. For example, H3K4me3 and H3K27me3, are two major histone methylation patterns that form a bivalent domain and are located near transcription initiation sites. These epigenetic markers have been found to regulate lineage decisions in embryonic stem cells as well as control quiescence in hair follicle and muscle stem cells via chromatin modification.

Functional tumor suppressor genes, particularly p53 and Rb gene, are required to maintain stem cell quiescence and prevent exhaustion of the progenitor cell pool through excessive divisions. For example, deletion of all three components of the Rb family of proteins has been shown to halt quiescence in hematopoietic stem cells. Lack of p53 has been shown to prevent differentiation of these stem cells due to the cells' inability to exit the cell cycle into the G₀ phase. In addition to p53 and Rb, cyclin dependent kinase inhibitors (CKIs), such as p21, p27, and p57, are also important for maintaining quiescence. In mouse hematopoietic stem cells, knockout of p57 and p27 leads to G₀ exit through nuclear import of cyclin D1 and subsequent phosphorylation of Rb. Finally, the Notch signaling pathway has been shown to play an important role in maintenance of quiescence.

Post-transcriptional regulation of gene expression via miRNA synthesis has been shown to play an equally important role in the maintenance of stem cell quiescence. miRNA strands bind to the 3′ untranslated region (3′ UTR) of target mRNAs, preventing their translation into functional proteins. The length of the 3′ UTR of a gene determines its ability to bind to miRNA strands, thereby allowing regulation of quiescence. Some examples of miRNA's in stem cells include miR-126, which controls the PI3K/AKT/mTOR pathway in hematopoietic stem cells, miR-489, which suppresses the DEK oncogene in muscle stem cells, and miR-31, which regulates Myf5 in muscle stem cells. miRNA sequestration of mRNA within ribonucleoprotein complexes allows quiescent cells to store the mRNA necessary for quick entry into the G1 phase.

Stem cells that have been quiescent for a long time often face various environmental stressors, such as oxidative stress. However, several mechanisms allow these cells to respond to such stressors. For example, the FOXO transcription factors respond to the presence of reactive oxygen species (ROS) while HIF1A and LKB1 respond to hypoxic conditions. In hematopoietic stem cells, autophagy is induced to respond to metabolic stress.