Investigators at the University of California at Riverside have demonstrated that the Rho-Rock-Myosin signaling pathway is essential for the regulation of basic cell-cell communications in both mouse and human embryonic stem cells, a finding that could have important implications for drug-discovery strategies, according to a UC-Riverside official.
Compounds that affect cell-cell or cell-matrix contact could potentially be used to control the undifferentiated cell growth of hESCs, said Noboru Sato, an assistant professor in the department of biochemistry at UC-Riverside. In addition, synthetic, or animal-free, matrix coatings could replace Matrigel coating in combination with chemical compounds that alter cell-cell and cell-matrix contact states, to achieve completely animal-free culture environments.
This changes the quality of the cells, so they are cleaner and easier to handle versus those grown on Matrigel, Sato told CBA News last week.
Although tight colony formation has been an important morphological criterion to determine the undifferentiated state of hESCs, the scientists’ results indicate that hESCs can maintain an undifferentiated state without forming tight colonies. This suggests that for the screening of compounds that support the undifferentiated growth of hES cells, formation of tight colonies may not be the necessary criterion for the screening.
The work was published online last month in PLoS ONE. .
The researchers showed that endogenous Rho signaling is required for the maintenance of cell-cell contacts in ESCs. They conducted siRNA-mediated loss-of-function experiments, and demonstrated that that Rock, the major effector kinase downstream of Rho, played a role in the formation of cell-cell junctional assemblies via the regulation of myosin II.
Through the use of a Rock-specific inhibitor, the investigators showed that cell-cell adhesion was reversibly controllable and dispensable for the self-renewal of murine ESCs. They confirmed this finding using a chimera assay.
“[Cell-cell interaction mechanisms]” are quite important to understand the basic function of stem cells, and also how to steer the growth phenotype in a cell culture dish.”
In addition, a culture system comprising a poly-D-lysine-coated plate, the defined medium mTeSR, and the Rock-specific inhibitor made hESC self-renewal completely independent of animal-derived matrices, tight cell contacts, or fibroblastic niche-forming cells. This finding was determined using a teratoma formation assay.
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“I have been working in this field for almost seven or eight years. I think many researchers have been working on identifying genes that are basic for ESCs. They want to determine how the genes interact with each other to regulate the stem cells’ basic biological functions,” Sato said. Scientists are also interested in signaling pathways that regulate ESC self-renewal.
Although cell-cell interactions are quite fundamental to cellular self renewal, very few studies address the subject of cell-cell interactions in ESCs, so “I started looking at how ESCs communicate with each other by focusing on basic signaling pathways,” said Sato.
He added that he and his team have now found that Rho and Rho-associated kinase signaling regulate cell-cell interactions in both mouse and human ESCs. “The surprising thing is that these cell-cell interactions may not directly affect the cellular self-renewal and function of ESCs.”
Sato and his team determined this not just by looking at gene expression or gene functions that are specific for stem cells, but fundamental cell-cell interaction mechanisms as well. As Sato put it, “Those are quite important to understand the basic function of stem cells, and also how to steer the growth phenotype in a cell culture dish.”
He went on to say that he and his colleagues are trying to apply these techniques to induced pluripotent stem cell culture, because the use of stem cells is rapidly shifting towards iPS-based techniques.
Growing iPS cells in animal-free conditions has significant implications “because in terms of hESCs, even if we discover how to grow hESCs in animal-free conditions, unless you derive them from the beginning from embryos, they are already contaminated with animals.”
This work could have an impact if the techniques described by Sato and his team could be used for culturing and deriving iPS cells, because the use of adult skin cell-derived iPS cells is more practical.