Culture media was developed as a result of discovering that colony formation is not key to self renewal, according to paper in PLOS One.
A group of scientists have found that embryonic stem cell (ESC) pluripotency can be retained even when these cells do not have close cell-cell contact through colony formation. Based on this discovery, the team was able to create a culture method independent of animal-derived matrices.
Currently human ESCs are grown on Matrigel-coated culture plates, which use a gelatinous extract from mouse tumor cells that contain extracellular matrices (ECMs). hESCs first attach to these scaffolds and then grow in undifferentiated colonies before differentiating into specialized cells. This process of colony formation was assumed to be pivotal to maintaining pluripotency, according to Noboru Sato, M.D., Ph.D., an assistant professor of biochemistry at University of California, Riverside.
Dr. Sato and colleagues identified a signaling pathway called Rho-Rock, which the ESCs use during colony formation and which plays a role in physical interactions. When the investigators blocked the pathway in mice with a chemical called Y27632, normal colony formation of mouse ESCs was considerably impaired as expected. Surprisingly, however, the ESCs maintained their pluripotency. The researchers say that this pathway is essential for the regulation of basic cell-cell communications in both mouse and human ES cells.
The scientists then developed a culture medium replacing the Matrigel coating with poly-D-lysine, a chemically synthesized ECM, and combined it with the Rock inhibitor. This system fully warranted ESC self-renewal and was entirely independent of animal-derived matrices as well as tight cell contacts. “We found that the growth of the hESCs under this novel culture condition was almost identical to the growth of hESCs on Matrigel-coated culture plates with no compromise in pluripotency,” says Dr. Sato.
The research was a collaboration between UCR and the National Institute of Environmental Health Sciences. The results appear online in the August 20 issue of the Public Library of Science ONE.
The group is now focusing on applying the technique to induced pluripotent stem cells.