December 2, 2009 – The invention of techniques for reprogramming differentiated cells to a pluripotent state has changed our understanding of the process of cellular differentiation and opened up exciting new prospects for the emerging field of regenerative medicine. Before applications can be made, however, a number of potential safety issues with the original method for creating such induced pluripotent stem (iPS) cells will need to be resolved. Advances have been made toward eliminating problematic transgenes from the original iPS recipe, and toward finding new delivery systems to replace the original retroviral vector, which had been a subject of concern due to the scattershot manner in which it introduces its payload. The culture system used for nurturing iPS cells in vitro itself represented a third hurdle, for past efforts have tended to use heterologous (or non-self) fibroblast cells (primarily from embryonic mice) as feeders, which raises possible issues of viral or other contamination.

Now, in a report published in the online journal PLoS One, Kazutoshi Takahashi and colleagues at the Kyoto University Center for iPS Cell Research and Application (CiRA) have demonstrated the feasibility of growing iPS cells on beds of fibroblast feeder cells derived from the same human donor. These autologous feeder cells significantly mitigate the risk of the transfer of pathogens to the iPS cells which they sustain.

The team began by confirming that both human neonatal and adult fibroblasts are able to serve as feeders for human iPS cell growth. Using existing fibroblast lines, they transferred an iPS cell line that had been cultured for 20 passages on one fibroblast feeder line to four new human fibroblast feeder systems (three derived from adults, the fourth from a neonate), and observed the newly cultured iPS cells' proliferation for at least 19 additional passages. They found no significant differences in plating efficiency, reactivation of transgenes, or typical pluripotency gene expression.

They next tried generating new iPS cell lines from the fibroblasts themselves. Using the conventional technique of introducing the transgenes Oct3/4, Sox2, Klf4, and c-Myc via a viral vector, they were able to reprogram all four human cell lines into a state of pluripotency, and to grow each of them for at least 18 passages in culture, using the same fibroblasts they had been derived from as feeders. Not only did these iPS cells show the same potential for proliferation and self-renewal as other lines cultured using the conventional feeder system, they showed no defects in stability or gene expression pattern, and were capable of teratoma formation, demonstrating their capacity for differentiation in vivo.

This is the first time that human iPS cells have been derived from adult fibroblasts and cultured on autologous feeder cells. However, as the culture medium used still includes xenogeneic components from animal sources, such as albumin, insulin, and the digestive enzyme trypsin, more work will be needed to refine iPS cell culture before these cells can be used safely in clinical applications.

"Human induced pluripotent stem cells on autologous feeders"
Kazutoshi Takahashi, Megumi Narita, Midori Yokura, Tomoko Ichisaka and Shinya Yamanaka

• PLoS ONE website