Embryonic stem (ES) cells have shown great promise in the creation of replacement cells and tissues to treat a number of diseases. They can be harnessed to develop into specialized cells that are not normally replenished by the body (e.g., neurons which make up the bulk of the brain) and may help treat conditions like stroke in which brain cells die and are not normally replaced. As stem cells can be grown in culture and transformed into specialized cells, their enormous potential for medical applications has been recognized.
Much of the excitement over ES cells springs from their robust capabilities in two competing processes: proliferation and pluripotency. ES cells can divide and self-renew indefinitely. They also have the capability of becoming any type of cell in the body, a characteristic referred to as pluripotency. However, once an ES cell differentiates into a specialized cell, it loses its pluripotency. The exact mechanism by which these cells maintain their pluripotent state (i.e., how they remain as stem cells instead of becoming specialized cells) is not fully understood. The underlying processes that drive stem cells to remain pluripotent or differentiate need to be understood in detail in order to fully utilize the power of stem cells.
An NIBIB-funded Quantum Grant is investigating these underlying processes. Dr. Karen Hirschi from Baylor College of Medicine, with multiple partners, has put together an international team of researchers from Baylor, Rice University, London’s National Institute of Medical Research, King’s College of London, and Edinburgh University to map and regenerate the stem cell niche of the brain regions that promote generation of new neurons.
Identification of Ronin, the Masterless Samurai Protein
Scientists have identified three proteins – Oct4, Nanog, and Sox2 – that play critical roles in maintaining the self-renewing state of embryonic stem cells. These pluripotency proteins are transcription factors, meaning they regulate the copying of DNA into RNA – the first step in making a protein. However, there may be other proteins that contribute to this grand scheme of self-renewal and differentiation. "Pluripotency is very complex," explains Thomas Zwaka, Assistant Professor at Baylor College of Medicine, "Oct4, Nanog, and Sox2 are not the end of the story. More proteins and pathways need to be involved in maintaining this complex state." Zwaka and his colleagues and collaborators recently found another protein that plays a role in maintaining pluripotency.
The researchers previously discovered that caspase-3 – one of several enzymes that regulate a form of cell death known as apoptosis – also plays a role in differentiation. A closer look showed that caspase-3 splits Nanog, one of the three master proteins involved in maintaining the stem cells in their pluripotent (i.e., self-renewing) state. When Nanog is split, the stem cells stop self-renewal and begin to differentiate. "The overlap of apoptosis and differentiation pathways came as a surprise," Zwaka says. "However, philosophically, cell death may be viewed as a specialized form of differentiation because the cells in the stem cell pool can exit the pool either by differentiation or cell death."
Zwaka reasoned that caspase-3 might also regulate other proteins involved in maintaining stem cells in an undifferentiated state. To investigate this hypothesis, he and his colleagues searched for proteins that interact with caspase-3. After a series of complicated screening experiments, they identified a new pluripotency factor that acts independently of the three master regulators (the aforementioned Oct4, Nanog, and Sox2). "After years of work, I was so excited to see that we discovered a major new role player in pluripotency," said Marion Dejosez, a scientist in Zwaka’s laboratory. Because the newly identified protein does not appear to work with any of the three known master regulators of pluripotency, the investigators named it Ronin after Japanese samurai warriors who do not have a master.
Inhibition of Differentiation and Potential Medical Applications
The investigators conducted a number of studies to characterize Ronin. When Ronin was overexpressed (i.e., made in super-abundant quantities), ES cells failed to differentiate and remained pluripotent stem cells, even under conditions that normally promote differentiation.
Ronin is expressed only in egg cells, developing embryos, and some regions of the brain. ES cells can cause tumors (teratocarcinomas) when injected into adult tissues. As expected for a pluripotent factor, when overexpressed, Ronin caused ES cells to be more tumorigenic than control ES cells, presumably by triggering expansion of the stem cell pool. Putting the two together, "It is possible that modulating or blocking the expression of Ronin in brain cells may help control tumor growth," Zwaka surmises.
Ronin is essential for the survival of embryos and embryonic stem cells, which cannot survive if the Ronin gene is inactivated (knocked out). Although Ronin is a transcriptional regulator, its effect on gene expression is probably more general than Oct4, Nanog, and Sox2, which act by suppressing specific genes required for pluripotency or differentiation. Ronin, on the other hand, represses transcription of multiple genes that are directly or indirectly involved in differentiation. Zwaka and Dejosez are investigating whether Ronin silences gene expression by modifying histones, the proteins that pack DNA and play a role in gene regulation. "As Ronin is a transcriptional regulator, it may be a good therapeutic target," Dejosez adds. "Ronin is also a polyglutamine protein, the kind of proteins that play a role in neurological diseases, so we are currently investigating that aspect of Ronin as well."
The discovery of Ronin reveals a new pathway distinct from Oct4, Nanog, and Sox2 that preserves the pluripotency of ES cells. In the future, the NIBIB-supported projects responsible for this breakthrough, as well as other projects, may utilize this knowledge about Ronin as well as other pluripotency and transcription factors in reprogramming stem cells to become specific cell types, such as leukocytes, to treat leukemia and other dreaded diseases.
Dejosez M, Krumenacker JS, Zitur LJ, Passeri M, Chu LF, Songyang Z, Thomson JA, Zwaka TP. Ronin is essential for embryogenesis and the pluripotency of mouse embryonic stem cells. Cell. 2008 Jun 27;133(7):1162–74.
Fujita J, Crane AM, Souza MK, Dejosez M, Kyba M, Flavell RA, Thomson JA, Zwaka TP. Caspase activity mediates the differentiation of embryonic stem cells. Cell Stem Cell. 2008 Jun 5;2(6):595–601.
Zwaka TP. Ronin and caspases in embryonic stem cells: a new perspective on regulation of the pluripotent state. Cold Spring Harb Symp Quant Biol. 2008 (In Press).