Observations From the Field: Efficient hiPS Cell Culturing Without ROCK Inhibitors

^{This article is posted on our Science Snippets Blog} 

Stem cell research undoubtedly has the potential to benefit many people with unmet medical needs. However, to fully reach this potential, improvements need to be made in several areas. One such area is cloning efficiency and cell survival after thawing and passaging human pluripotent stem cells, or more generally, improving the ability to generate and subsequently propagate and passage hES and hiPS cells. To address this, RIKEN scientists screened various chemical compounds added to cell culture media to identify pro-growth and survival factors. The result was the identification of the small molecule Y-27632, a selective inhibitor of Rho-associated coiled-coil kinase (ROCK), which increased hES cell cloning efficiency from ~1% to ~27%¹.

Although the jump in cloning efficiency was an important finding, the increased wide-spread usage of Y-27632 in stem cell cultures is concerning to some researchers. In fact, some cell cultures have become reliant on this inhibitor for survival, perhaps indicating that the culture system itself is lacking critical components for cell health and survival. Additionally, while several studies have found that Y-27632 does not affect human stem cell pluripotency or karyotype, other reports have demonstrated that continued exposure to Y-27632 can lead to various developmental abnormalities, including prevention of porcine oocyte maturation², impaired heart muscle formation³, and cytoskeletal defects in the chick embryo⁴.

With these concerns in mind, a human stem cell culture system that is not reliant on a ROCK inhibitor or similar compounds would be ideal. NutriStem^® hPSC XF Medium, a serum-free, xeno-free culture medium for hES and hiPS cells cultured on a range of substrates (including human recombinant vitronectin and laminin proteins), does not require the use of Y-27632 or any other additives for efficient thawing and routine cell passaging. Additionally, NutriStem^® hPSC XF Medium alone (without Y-27632 supplementation) is sufficient for hiPS cell expansion, even when passaging as single cells on a human recombinant laminin-521 substrate⁵.

Recently, we visited with the laboratory of Dr. Brian Cummings at the University of California, Irvine, to discuss their experiences using NutriStem^® hPSC XF Medium (without Y-27632) for routine hiPS cell culture.

Previously, the lab was using medium to culture hiPS cells on vitronectin prior to differentiating into neural cells. However, they were concerned that extensive ROCK inhibition using Y-27632, along with the high levels of bFGF in TeSR-E8 media (100 ng/mL), were affecting the quality of their hiPS cell cultures and their ability to differentiate into specific lineages. Knowing that NutriStem^® hPSC XF Medium does not require a ROCK inhibitor for either cell thawing or passaging, and that it also contains a low basal level of bFGF, the lab was open to switching media and quickly recognized the opportunity to improve the quality of their cultures.

The lab transferred their hiPS cells straight from TeSR-E8 to NutriStem^® hPSC XF Medium on Vitronectin XF without any intermediate steps, and evaluated the cells with and without adding ROCK inhibitor. According to one researcher in the lab, “The cells plated without ROCK inhibitor during passaging seem to do much better, have good survival, and a much cleaner morphology in comparison to those plated with ROCK inhibitor. On the other hand, omitting ROCK inhibitor while passaging with TeSR-E8 led to very low cell survival and required a much higher plating density to avoid a bottlenecking effect.” (Click the following link to view the full Case Study using NutriStem^® hPSC XF Medium in combination with Vitronectin XF without Y-27632)

The quest to find the perfect human pluripotent stem cell culture system is ongoing. However, in this quest, we must be careful not to potentially undermine the quality and safety of the cells themselves. Until we have a better understanding of the long-term effects of ROCK inhibitors, such as Y-27632, on stem cells in culture, using a medium like NutriStem^® hPSC XF Medium that does not require its addition for routine culture and maintenance can alleviate the concerns associated with extended ROCK inhibitor use, as well as provide many additional downstream benefits beyond efficient cell thawing and passaging.

References:

_{¹ Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, et al. (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25: 681–686.}

_{² Zhang Y, Duan X, Xiong B, Xiang-Shun C, Nam-Hyung K, et al. (2014) ROCK inhibitor Y-27632 prevents porcine oocyte maturation. Theriogenology 82: 49–56.}

_{³ Sakata H, Sakabe M, Matsui H, Kawada N, Nakatani K, et al. (2007) Rho kinase inhibitor Y27632 affects initial heart myofibrillogenesis in cultured chick blastoderm. Dev Dyn 236(2): 461–472.}

_{⁴ Duess JW, Puri P, Thompson J. (2016) Impaired cytoskeletal arrangements and failure of ventral body wall closure in chick embryos treated with rock inhibitor (Y-27632). Pediatr Surg Int 32: 45–58.}

_{⁵ Rodin S, Antonsson L, Hovatta O, & Tryggvason K. (2014). Monolayer culturing and cloning of human pluripotent stem cells on laminin-521–based matrices under xeno-free and chemically defined conditions. Nature Protocols, 9, 2354–2368.}