Relevance and Safety of Telomerase for Human Tissue Engineering

Relevance and safety of telomerase for human tissue engineering.
Rebecca Y. Klinger, Juliana L. Blum, Bevin Hearn, Benjamin Lebow, and Laura E. Niklason. PNAS (Proceedings of the National Academy of Sciences). Vol 103. No. 8. 21 February, 2006.
*Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved January 23, 2006 (received for review September 19, 2005).

http://www.pnas.org/content/103/8/2500.abstract
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One of the goals of tissue engineering is to be able to engineer tissues for a patient based off that patient’s own cells, so as to reduce rejection of the tissue. Patients are often elderly though, and the telomerase activity in their cells is greatly reduced. Elderly donor vascular cells can’t be cultured normally past 5-10 doublings. However, insertion of the hTERT gene, which expresses telomerase, allows cells to undergo enough doublings to form actual tissue for transplantation.

Smooth muscle cells from eldery patients and one young patient were examined in this paper. The SMC cells were retrovirally transformed with hTERT to ectopically express telomerase. Then, there were two issues investiaged: the impact of donor age on the karyotype, and the formation of tumor-like characteristics due to increased telomerase activity.

Impact of donor age on karyotype:
Both the control cells and the hTERT cells had some instances of aneuploidy. Aneuploidy is where the number of chromosomes is not a multiple of a haploid, i.e. in humans, the number was not 2N, or 46. There was either the loss or a replica of a random chromosome, most often the loss of a Y chromosome. In two elderly patients cells, karyotype abnormalities only existed in the control, and no the hTERT transformed cells; on the other hand, in another elderly patient’s cells, karyotype abnormalities only existed in the hTERT transformed cells. Regardless, hTERT cells, no matter the age, can be grown into vascular tissue for transplantation. However, decreased collagen accumulation and decreased burst strength become a function of age. So regardless of how immortal an eldery patient’s cell line can be (passaged many times), they still show biochemical signs of ageing.

Formation of tumor-like characteristics:
While telomerase activity isn’t considered a main oncogene, reactivation of telomerase is seen in 97% of human cancers. Thus it is important to explore if hTERT transformed cells exhibit tumorigenic characteristics. There were a few measures used in this study: population doubling times, cell cycle regulation, the soft agar assay, and the nude mouse assay. Cancer cells often display shortened population doubling times; however hTERT transformed cells had longer population doubling times, a non-tumorigenic characteristic. Furthermore, hTERT cells grew far past their controls, which were senescent at passage 20, with intact cell cycle regulation. The soft agar assay shows if cells can grow in an anchorage independent manner, a characteristic of cancer cells. The hTERT cells did not grow in soft agar, and thus exhibit another non-tumoritgenic characteristic. In the nude mouse assay, cells are injected into a mouse—if a tumor is produced the cells are tumorigenic. hTERT cells did not form tumors after injection. Despite all these indications that hTERT transformed cells don’t exhibit tumorigenic characteristics, studies show that after many passages, hTERT cells can still spontaneously form malignancys. Researchers recommend <100 doublings.

Significance:
If we want to eventually develop patient-specific tissue engineering, we need to be able to culture a patient’s cells long enough to get tissue from them. hTERT transformation shows a lot of potential to help the cells’ shelf life. However, its effects, especially the tumorigenic ones, need to be explored, as done so introductorily by this paper. On a further note, the researchers recommend andenoviral or nonviral gene transfer for actual therapy. (Retroviral transfer is potentially dangerous for all therapies.)