Surviving separation

Japanese researchers may have found a simple solution to the problem of keeping human embryonic stem (hES) cells alive after dissociation of the embryo into individual cells.

ROCK-blocked human embryonic stem cells survive dissociation to grow in culture

Japanese researchers may have found a simple solution to the problem of keeping human embryonic stem (hES) cells alive after dissociation of the embryo into individual cells. Inhibition of the enzyme rho-associated coiled-coil kinase (ROCK), which is believed to play a role in the initiation of apoptosis or programmed cell death, results in a marked reduction in dissociation-induced apoptosis, and an increase in cloning efficiency.

Human embryonic stem (hES) cells hold much potential for the treatment of many diseases, but the cells have proven far more difficult to grow in culture than murine embryonic stem (mES) cells. The poor survival of dissociated cells in culture has hindered efforts to isolate or subclone specific populations of cells after the induction of differentiation or gene transfer.

The researchers, at the RIKEN Center for Developmental Biology, Kobe, Japan’s National Center for Geriatrics and Gerontology and Kyoto University, have recently published their findings in Nature Biotechnology1. They showed that the cloning efficiency of cells treated with the compound Y-27632, which selectively inhibits ROCK, increased from around 1% in untreated cells to 27% in the treated cells (Fig. 1 - Click on link below).

The cells treated with Y-27632 could also be grown in a serum-free suspension culture and induced to differentiate into precursor cells that resemble the cells of the embryonic brain and neural system.

Team leader Yoshiki Sasai says while it isn’t well understood why hES cells are so prone to apoptosis after dissociation, a better understanding of the role of ROCK in triggering apoptotic cell death may provide some answers.

“This is one of the most interesting questions for future study: why hES cells, but not mouse ones, are so prone to go apoptotic upon dissociation,” he says. “We have no answer for it, and are currently comparing these two systems both upstream and downstream of ROCK.”

In the meantime, the use of Y-27632 to improve cell survival and cloning efficiency will enable researchers to manipulate hES cells with greater ease, allowing large-scale dissociation culture, and easier isolation of genetically modified cells after gene transfer as well as the ability to establish cell lines of hES cells from a single cell.

Ultimately, Sasai says, the use of hES cells may lead to new therapies for degenerative diseases and intractable conditions including Lou Gehrig’s disease (amyotrophic lateral sclerosis (ALS)) and other neurological diseases.
Reference

1. Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Nishikawa, S., Muguruma, K. & Sasai, Y. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology 25, 681–686 (2007).

Published: 01 Sep 2007

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http://www.rikenresearch.riken.jp/research/287/image_1119.html Figure 1: Increased survival of hES cells in dissociation culture (red) after treatment with ROCK inhibitor Y-27632 (right); compared with untreated hES cells (left).

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Medicine