Cell dissociation, a technique for isolating cells in procedures such as sub-cloning, poses one of the greatest obstacles to effective human embryonic stem cell research due to its damaging effects on human ES cells. 99% of human ES cells cultured in this way are destroyed by an extensive apoptotic response that is curiously absent in mouse ES cells. Earlier research by the researchers said that inhibition of a protein known as the Rho-associated kinase (ROCK) reduced this rate of cell death by 30%, yet fundamental questions remained about the mechanisms involved.
The researchers applied live-cell imaging to the earliest phase of dissociation in human and mouse ES cells and the results showed something interesting; mouse ES cells hardly moved but the human ES cells skittered about in a so-called “death dance”, immediately sprouting finger-shaped bulges, known as blebs, which grew until the cells burst and died.
The researchers traced this early-onset blebbing, whose duration and severity exceeded anything ever before observed, to the hyperactivation of myosin, a type of protein responsible for cell motility.
Contrary to their expectations, the researchers went on to find that this myosin hyperactivation, mediated by activation of the ROCK kinase, which is the direct cause of apoptosis in dissociated human ES cells, and not the blebbing itself. Further implicated in this process is the inhibition of another protein known for its role in cell motility, Rac, which together with ROCK activation strongly promotes myosin hyperactivation leading to cell death.
They say the results provide a first ever comprehensive explanation of mechanisms underlying dissociation-induced apoptosis in human ES cells. While vastly improving the efficiency of colony formation in dissociation culture, the findings also promise safer and more effective cellular therapy treatments for a range of debilitating degenerative diseases.
Citation: Ohgushi et al., 'Molecular Pathway and Cell State Responsible for Dissociation-Induced Apoptosis in Human Pluripotent Stem Cells', Cell Stem Cell (2010), doi:10.1016/j.stem.2010.06.018
- Shinya Yamanaka Reprograms Human Adult Cells To Function Like Embryonic Stem Cells
- The Imperfect Replacement For Human Embryonic Stem Cells
- Can IPS Replace The Need For Embryonic Stem Cells?
- 5 Things You Should Know About Stem Cell Research
- Technique For Rapidly Reprogramming Adult Cells Into Stem Cells Published In PLoS Biology