Are immortalized stem cells poor surrogates?

Deleted gene that immortalizes MSCs may be critical to their function

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JUPITER, Fla.—New research conducted in the lab of Dr. Donald Phinney on the Florida campus of The Scripps Research Institute (TSRI) has identified factors critical to the differentiation of mesenchymal stem cells (MSCs), which are commonly used in stem cell therapies and clinical research. The study, published in the journal Cell Death and Differentiation, suggests a new approach may be needed to how these cell lines should be treated in the lab and studied for clinical purposes.
MSCs are common tools for doctors and researchers looking to stem cell therapies to heal damaged tissue or replace dysfunctional cells. MSCs are popular because they can differentiate into a variety of mature cell types (bone, fat or cartilage, for example), and because they support the formation of blood cells (hematopoiesis) and secrete factors that promote tissue repair.
Researchers, however, oftentimes struggle to predict how primary MSCs from bone marrow will behave. Because these cells are delicate and difficult to keep alive in vitro, many scientists use immortalized MSC lines. Immortalized MSC lines are created by deleting a gene—p53—which controls the normal, programmed cell death (apoptosis) that occurs as a controlled part of an organism's natural growth and development.
Although initially thought to be dispensable for normal cell survival, recent studies have suggested that the gene may act as a sort of master regulator of the cells’ ability to differentiate, and that it influences not only apoptosis but also early development, reproduction, energy metabolism, and hematopoiesis. This finding suggests that the dramatic effects of deleting p53 may make immortalized MSC cell lines an inappropriate surrogate for predicting the cells’ behavior in clinical applications.
“The scientific literature is replete with publications that employ immortalized cell lines as surrogates to study MSC biology,” Phinney tells DDNews. “Our recent data suggest that these lines poorly recapitulate the behavior/function of primary MSCs. I believe the work will have a large impact on those scientists using rodent MSCs. We and others are also striving to delineate critical differences between rodent and human MSCs, which should aide in better translating preclinical data to the clinic.”
Phinney and his team compared cells that came from normal mice against those of mice that did not express the p53 gene, and found that the level of active p53 appeared to be the primary regulating factor that determined how the MSCs differentiated. When p53 was completely deleted, the cells became immortal but developed into bone cells, losing their ability to become other types of cells.
The researchers found that the gene interacts with reactive oxygen species and two transcription factors, TWIST2 and PPARG, to influence cell growth and development. A low level of p53 induced TWIST2, which discouraged any differentiation, keeping the MSCs in a stem state. A moderate level of p53 induced PPARG and reactive oxygen species, which led the cells to differentiate into fat cells rather than bone. At high levels of p53, the cells died.
“A basal level of p53 in cells in the culture is required for them to act as an accurate model for cells in the body,” Dr. Veena Krishnappa, the study’s other lead author, said in a statement announcing the research’s publication.
“Our data argue that basal p53 levels are necessary for MSC self-maintenance,” says Phinney. “We have shown that transient exposure to oxidative stress strongly activates p53, which results in growth arrest, reduced survival and changes in differentiation potential. Prolonged exposure leads to an increase in cellular apoptosis and selection of cells that acquire inactivating mutations in p53, which via immortalization allows rapid and sustained cell growth.”
In addition to the study’s findings of the important role p53 plays in MSC differentiation, the research also suggests that inactivation of p53 may play multiple roles in the progression of bone cancers and other skeletal diseases. Inactivation of the gene may contribute significantly to tumorigenesis and tumor progression by promoting sustained cellular growth, desensitizing the cells to oxidative stress and interfering with pathways that regulate cellular differentiation.
“Mutations in p53 are known to occur at a relatively high frequency in bone cancers, and in about 90 percent of osteosarcoma patients,” Phinney comments. “Efforts are underway to understand how the spectrum of p53 mutations in these tumors impact response rates to therapy, which may lead to different treatment regimens based on the tumor mutation profile. Groups are also examining downstream pathways affected by p53 mutations in the hope of identifying other potential targets for intervention.”
“Our lab is interested in studying how p53 loss-of-function affects the skeletal response to obesity and mechanical unloading as a means to probe pathways that drive skeletal involution in response to these conditions,” he says.

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