Genotoxic Treatment Eliminates Pluripotent Stem Cells, Enables Safe Transplantation of Differentiated Cells

A recent paper described the development of a treatment regimen designed to enable transplantation of differentiated cells derived from pluripotent stem cells while avoiding the potential formation of teratomas or other cancers.

Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products. However, the risk of residual undifferentiated stem cells within a differentiated progenitor population demands a targeted approach to eliminate contaminating cells prior to delivery. The exploitation of the therapeutic capacity of stem cells while minimizing risk of uncontrolled growth has been an unsolved problem.

Investigators at the Mayo Clinic (Rochester, MN, USA) tried a different approach to eliminate undesirable pluripotent cells from populations of differentiated cells. They developed a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. For this purpose, they worked with a mouse stem cell model to validate the use of the genotoxic drug etoposide.

Etoposide forms a ternary complex with DNA and the topoisomerase II enzyme (which aids in DNA unwinding), prevents re-ligation of the DNA strands, and by doing so causes DNA strands to break. Cancer cells rely on this enzyme more than healthy cells, since they divide more rapidly. Thus, the drug causes errors in DNA synthesis and promotes apoptosis of the cancer cell.

Results published in the September 27, 2012, online edition of the journal Stem Cells Translational Medicine revealed that when compared to somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by etoposide. Hypersensitivity in pluripotent stem cells was stage-specific and was consistently lost upon in vitro differentiation. In other words, the drug eliminated pluripotent cells with little or no damage to differentiated cells.

The investigators used quantitative polymerase chain reaction (q-PCR) and Western blotting techniques to demonstrate that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. PUMA (p53 upregulated modulator of apoptosis) also known as Bcl-2-binding component 3 (BBC3), is a proapoptotic protein, member of the Bcl-2 protein family. PUMA expression is regulated by the tumor suppressor p53 and is involved in p53-dependent and -independent apoptosis induced by a variety of signals. After activation, PUMA interacts with antiapoptotic Bcl-2 family members, thus freeing the proteins Bax and/or Bak, which are then able to signal apoptosis to the mitochondria. Following mitochondrial dysfunction, the caspase cascade is activated ultimately leading to cell death.

“Strategies to improve the safety of stem cell therapy have generally focused on separating or depleting damaged cells after the cells have differentiated. However, while this method was able to diminish the number of tumors formed as well as significantly reduce their size, the technical burdens, and cost of specialized reagents and equipment needed to do so remain a challenge for widespread clinical applications,” said senior author Dr. Timothy J. Nelson, assistant professor of medicine and pharmacology at the Mayo Clinic.

“The results showed that not only did the contaminated cells die off, at the same time, it did not affect the remaining healthy cells’ capability to differentiate nor did it have any negative consequence on their genomic stability,” said Dr. Nelson. “And it worked on stem cells derived from both natural and bioengineered sources. This novel strategy, based on innate mechanisms of pluripotent stem cells, is primed for high-throughput and cost-effective clinical translation.”

Related Links:
Mayo Clinic