Spontaneous Human Adult Stem Cell Transformation

Daniel Rubio, Javier Garcia-Castro, Mario C. Martin, Ricardo de la Fuente, Juan C. Cigudosa, Allison C. Lloyd, and Antonio Bernad. Spontaneous Human Adult Stem Cell Transformation. Cancer Research 65, 3035-3039, April 15, 2005.

Summary: Both cancer stem cells and regular stem cells can self-renew and differentiate. The objective of the researchers’ study was to investigate whether human adult stem cells can spontaneously transform into cancer stem cells.

Human mesenchymal stem cells were isolated from human adipose tissue. They were plated on tissue culture plastic and passaged everytime they reached 85% confluence. The MSC’s entered the senescence phase at around 2 months after isolation. During the senescence phase, there is moderate telomerase shortening and cell cycle arrest. The senescence phase lasted from 1-8 weeks. The MSC’s then entered a post-senescence phase where they showed an accelerated cell cycle rate compared to pre-senescence MSC. Karyotype analysis was performed on pre-senescence and post-senescence MSC samples. Although all cells had a normal karyotype after isolation, at least 30% of the post-senescence cells had trisomy of chromosome 8. Both pre-senescence and post-senescence MSC’s were injected into immunodeficient mice. When the mice were sacrificed 1-4 months later, there were no signs of tumor formation.

In vitro, post-senescence MSC cells continued to grow until they reached the crisis phase which typically takes place after the senescence phase. During the crisis phase, the telemeres get critically short, leading to chromosome instability and cell apoptosis. Cell cycle analysis of the crisis MSC’s showed generalized chromosome instability. 50% of the samples tested survived the crisis phase and continued to proliferate. The morphology of the post-crisis MSC’s changed from an elongated spindle shape to a small and rounder shape. Using cell cycle analysis, the researchers determined that post-crisis cultures had a higher percentage of S-phase cells with shorter duplication time compared to the pre-crisis cultures. Post-crisis cells down-regulated membrane markers CD34, CD90, CD105, leading to loss of contact inhibition. The post-crisis cells could also grow in semi-solid agar which is characteristic of tumor cells; thus, the researchers named these cells TMC for tumor mesenchymal cells.

Karyotype analysis of the TMC samples showed non-random mutations, such as a translocation between chromosomes 8 and 11 and intrachromosomal rearrangement of chromosome 5, and an occasional isochromosome 8. From the karyotype analysis, it was suggested that the overexpression of c-myc, an oncogene on chromosome 8 which affects cell cycle progression and gene expression modifications, may play a role in senescence bypass and transformation to TMC. Telomerase activity was also turned on in the TMC, indicating that telomerase is critical to crisis bypass and immortalization.

When the TMC were injected into immunodeficient mice, they led to tumor growth and illness. In contrast, the mice who had been injected with pre-senescence and post-senescence MSC had no tumors or illness as described earlier.

In conclusion, this study shows that human MSC can spontaneously immortalize with prolonged in vitro expansion as 50% of the samples underwent spontaneous transformation. Because MSC are being tested in clinical trials for tissue engineering, this study has safety implications for MSC maintained in prolonged culture.

Critique: I think the researchers could have done a better job writing the article. The methods section was hard to understand until I realized they were describing the procedures for the various techniques that were used instead of describing their experiment in chronological order. Their use of the word “bypass” to describe cells passing the senescence phase caused confusion and should be replaced with the word “pass” because bypass means skipping a phase altogether. Also, the researchers should have stated the time it took for cells to reach the crisis phase and the duration of the phase.

As for the experimental techniques, the researchers had used mouse anti-human mitochondrial monoclonal antibody to stain the mouse organ sections for immunohistochemical analysis. The secondary antibody was goat anti-mouse antibody. Because the organ sections came from mice, there could be non-specific binding of the secondary goat anti-mouse antibody. I would use a non-mouse primary antibody to avoid this problem.