Statement of Benefit to California:
Year 1Human embryonic stem cells (hESCs) originate directly from human embryos, whereas induced pluripotent stem cells (iPSCs) originate from body (somatic) cells that are re-programmed by producing or introducing proteins that control the process making specific RNAs. Together, both these pluripotent cell types are referred to as human pluripotent stem cells (hPSCs). Several reports have observed that in hESCs grown for long times, their genetic material, DNA, is unstable. The stable maintenance of DNA is performed by groups of proteins functioning in different systems globally known as DNA repair pathways. Since the development of aneuploidy is closely linked to cancer and to deficiencies in DNA repair, we have studied the propensity of hPSCs to repair their DNA efficiently by 4 major known DNA repair pathways. In addition, we are also investigating if specific damage to DNA in either hPSCs or somatic cells is processed differently and could lead to deleterious mutations.
One major goal of the CIRM SEED grant mission is to bring new researchers into the hPSC field. The results we obtained during the funding period indicate that we have succeeded in that objective, since initially our laboratory had little experience with hESC culture. However, through courses and establishing critical collaborations with other hESC laboratories, we developed expertise in hPSC culture techniques. Most conditions for hPSCs growth require cells (feeder cells) that serve as a matrix and provide some factors needed for the pluripotent cells to divide. In accomplishing this aim, we perfected a method to generate reproducible feeder cells that significantly reduces the time and cost of feeder cell maintenance, and also developed a non-enzymatic and non-mechanical way to expand hPSCs. We now have experience with at least 5 hPSC lines and have methods to introduce foreign DNAs into hESCs and iPSCs to monitor DNA repair in hPSCs.
In Aim II of our grant, we used our accumulated knowledge of hPSCs and DNA repair to investigate 4 DNA repair mechanisms in hPSCs and in somatic cells. Depending on the DNA damage, there is often a preferred DNA repair pathway that cells use to alleviate potential harm. We initiated our investigation by treating hPSCs using different DNA damaging agents, including ultraviolet light and gamma radiation. However, we found that hPSCs exposed to these agents rapidly died compared to treatments that allowed somatic cells to continue growing. Therefore, we developed methods to study DNA repair in hPSCs without directly treating the cells with external agents. We treated closed, circular DNA (plasmids) with damaging agents separately, outside the hPSCs and then introduced them into the hPSCs. The plasmid DNA has a sequence that codes for a protein that is produced only when the damage is repaired. The length of time for repair both in hPSCs and in somatic cells was followed by determining the protein production. We have shown superior DNA repair ability and elevated protection against DNA damage in hPSCs compared to somatic cells for ultraviolet light and oxidative damage, two common sources of damage in cells. A major pathway for joining double-strand DNA breaks in mammalian cells, non-homologous end-joining (NHEJ) repair (error prone), is greater in H9 cells than in iPSCs. Another way to repair double-strand DNA breaks that uses similar (i.e., homologous) sequences is lower in iPSCs compared to hESCs and somatic cells. Further study of these repair pathways is warranted, since several methods can be used to form iPSCs. Therefore, the genomic stability for iPSCs could depend on the method used for their generation.
DNA repair analysis is critical to understanding how hPSCs protect against damage, but if left unrepaired, cells can turn damage into mutations when the damage is copied by enzymes (DNA polymerases) before repair occurs. Therefore, to monitor the mutations that ultimately lead to cancer or alter hPSC biology, we are using a plasmid that is damaged outside the cells and will make copies in hPSCs and somatic cells. That plasmid is introduced into cells and then the copies are recovered. The number of mutations found in the plasmid DNA indicates the likelihood of observing mutations in hPSCs compared to mutations in somatic cells. Together, these results will yield data on the stability of hPSCs and also a basis to monitor cells for stability which could serve as an indicator of safety for clinical use.