Lecture 3 (Adaptation of the Cell Cycle in Stem Cells) Flashcards
Describe the cell cycle kinetics of a fibroblast.
Fibroblasts spend between 20 and 24 hours per cell cycle, with no time spent in quiescence.
Describe the cell cycle kinetics of an embryonic stem cell.
Embryonic stem cells spend between 10 and 12 hours per cell cycle, with no time spent in quiescence. Embryonic stem cells spend the majority of the cell cycle in the S phase, with roughly equal amounts of time spent in the G1 and G2 phases.
Describe the cell cycle kinetics of a foetal haemopoeitic stem cell.
The foetal haemopoeitic stem cell spends 14.5 hours per cell cycle, with no time spent in quiescence.
Describe the cell cycle kinetics of an adult haematopoietic stem cell.
The adult haematopoietic stem cell spends 14.5 hours per cell cycle; if the cell is a long-term haematopoietic stem cell, and therefore capable of self-renewal, then it will spend over 145 days in quiescence, whilst if the cell is a short-term haematopoietic stem cell, and therefore incapable of self-renewal, then it will only be in a quiescent state for approximately 35 days.
Describe the cell cycle kinetics of an adult neural stem cell.
The adult neural stem cell spends between 20-24 hours in each cell cycle, with 50 days in quiescence.
Describe the constitutive CDK activity in embryonic stem cells.
Embryonic stem cells have constitutively high levels of cyclin A, D and E expression; this constitutive cyclin expression maintains Rb in its inactive, hyperphosphorylated state. The inhibition of Rb means that E2F is constitutively active, further enhancing CDK activity through the feedback loop. This accounts for the shortened G1 length seen in the embryonic stem cell cycle.
What is the effect of Rb inactivation on the length of the cell cycle?
High kinase activity maintains Rb in an inactive hyperphosphorylated form, which reduces the requirements for mitogens. This is typical of cells such as embryonic stem cells, which utilise the cell autonomous replication program, which reduces the length of the G1 phase. However, in adult tissues and stem cells, Rb is active and able to maintain control over cell division.
Summarise the differences between a generalised somatic cell cycle, and that of an embryonic stem cell.
In the somatic cell cycle, the action of Rb and CDKIs ensure that G1 progression is mitogen-dependent and therefore tightly regulated, however, in the embryonic stem cell, a lack of CDKI leads to high CDK activity, which inactivates Rb and shortens the G1 phase as a result. The maintenance of the restriction point, which requires sustained mitogen stimulation in order to bypass, in the somatic cell enables cell cycle exit into a quiescent state, therefore maintaining stem cell pools. By contrast, the inactivation of the restriction point in the embryonic stem cell cycle prevents the cell from entering quiescence; instead the cell continuously cycles.
What is the role of quiescence in adult stem cell function?
Quiescence plays a key role in adult stem cell function. Stem cells divide in order to self-renew and produce progenitor cells, however, stem cells are typically quiescent in stem cell tissues. Quiescence helps maintain the stem cell phenotype, as over-division leads to the loss of self-renewal properties.
Describe the functional restriction point seen in adult stem cells.
The functional restriction point of adult stem cells is due to the presence of functional CDKI and Rb within the cells; these active proteins enable the exit of the cell from the cell cycle. Stem cells remain in quiescence for an extended period of time, ensuring that there is always a pool of functional stem cells.
What is the purpose of varying cell cycle length?
Stems cells have a finite replicative capacity i.e. they are limited in the number of cell divisions that they are able to perform. Uncontrolled cellular division can lead to a deterioration in the stem cell population, so adult stem cells are often in quiescence (G0) in order to maintain the stem cell pool. The regulation of self-renewal in stem cells maintains a viable stem cell population throughout our lifetime, with mutations that prevent entry into quiescence leading to the loss of stem cells from tissues.
Describe the experimental evidence for the importance of Rb in the maintenance of quiescence.
The conditional deletion of Rb family proteins (Rb, p107, p130) in mice prevents cell cycle exit and the entry into a quiescent state. This loss of Rb leads to a failure in stem cell self-renewal, with subsequent increase in progenitor cells and loss of haematopoietic stem cells. This causes the onset of a myeloproliferative phenotype and the mouse dies within 1-3 months due to its inability to maintain homeostasis.
How are long-term haematopoietic stem cells maintained in quiescence?
Haematopoietic stem cells respond to both mitogens and anti-mitogens. Anti-mitogens such as thrombopoietin and TGF-β enhance the expression of p57, a CDKI, which prevents cyclin D from entering the nucleus, therefore rendering CDK4/6 inactive. The lack of kinase activity means that Rb remains in an unphosphorylated state and is able to continue to repress E2F-mediated transcription and block cyclin E expression. This sequence of events means that the haematopoietic stem cell cannot bypass the restriction point whilst cellular interaction with anti-mitogens continues.
How are long-term haematopoietic stem cells activated from quiescence?
The sustained prescience of mitogens, such as stem cell factor or interferons, inhibit the expression of the CDKI p57. This means that cyclin D is no longer inhibited, and can translocate to the nucleus and form a complex with CDK4/6. The resulting cyclin D-CDK4/6 can now phosphorylate and prevent the repression of E2F-mediated transcription, which in turn instigates cyclin E expression and G1 exit.
Describe the role of the niche in the regulation of haematopoietic stem cells.
The microenvironment, or niche, in which a particular stem cell is located contributes to its regulation, as direct contacts with proximal cells play a key role. Cells such as CAR (CXCL12 abundant reticular) cells secrete Wnt mitogens, whilst osteoblasts secrete bone morphogenic protein, or BMP, which is an anti-mitogen. The balance between these positive and negative signals determine whether the stem cell divides, and the level of exposure is determined by the location of the cell within the bone marrow. Quiescent haematopoietic stem cells are located within the endosteal region, where the composition of cells promotes anti-mitogenic signalling. However, active haematopoietic stem cells are located in the central marrow region, which lacks osteoblasts, and therefore lacks the anti-mitogenic BMPs which they produce. This region is therefore pro-mitogenic and can be described as a stimulatory zone.
