ROCK inhibitor Y-27632

The ROCK inhibitor Y-27632 is a selective and cell permeable inhibitor of the Rho-associated protein kinase (ROCK) (K_i values are 0.14, 26 ,25 and >250 µM at p160ROCK, PKC, PKA and MLCK respectively).

Y-27632 has many biological actions and is an important small molecule modulator of stem cells. It diminishes hESC and hiPSC dissociation-induced apoptosis, enhances survival and colony formation of dissociated hESC without affecting pluripotency or self-renewal. Y-27632 also improves hESC and iPSCs postthaw viability and survival rate during cryopreservation. It is frequently used as a 3D growth matrix component and for production of organoids (e.g. brain organoids). Additionally, Y-27632 inhibits smooth muscle contractility and shows antihypertensive effect.

Background

Rho kinase (Rho-associated coiled coil protein kinase or ROCK) is a major downstream effector for the small GTP-binding protein Rho ^[1]. Rho kinase is involved in various biological processes including cell mitosis adhesion, cytoskeletal adjustments, muscle cell contraction, tumor cell invasion and a series of cell biological phenomena ^[2]. Through a series of cascading effects, Rho kinase influences actin and microtubule regulation of apoptosis and thus plays a key role in early embryonic development ^[3].The Rho kinase signalling pathway is highly activated in many pathological conditions ^[4].

Rock inhibitor Y-27632 is a Rho kinase inhibitor which selectively inhibits p160ROCK (ROCK1) with a K_i value of 140 nM and binds to the Rho kinase ATP binding pocket in an ATP-competitive manner ^[5].

Uses

Y-27632 has been shown to have a diverse range of actions and has been used in a variety of biological systems. It is the most commonly used of the ROCK inhibitors in stem cell research and is used as a multifunctional reagent in many stem cell related processes ^[6]. Y-27632 demonstrates many positive effects across various cell types, in particular human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) ^[7] and has greatly improved a number of practical stem cell procedures ^[8]. For example it has been shown that Y-27632 increases cell adhesion and proliferation and increases post-thaw cell survival and post-passaging cell viability ^[9,10].

Cellular dissociation

Cell dissociation is one of the most common manipulations in stem cell research. During cellular dissociation both hESC and hIPSCs are vulnerable to dissociation-induced apoptosis or anoikis (a form of apoptosis induced by inappropriate cell-cell or cell-ECM interactions) ^[3,11]. Y-27632 is of particular interest as it allows hPSCs to escape this dissociation-induced apoptosis. Y-27632 prevents apoptosis of dissociated hPSCs and increases their survival rate and plating efficiency ^[11].

Cryopreservation

Cryopreservation is also an important part of stem cell research. Y-27632 improves the recovery of cryopreserved hPSCs and has greatly helped simplify the cryopreservation procedure. The slow-freezing and rapid-thawing procedure which uses DMSO as a cryoprotectant is commonly used in stem cell cryopreservation, however this method is not suitable for hPSCs, therefore vitrification (fast freezing, slow thawing) is used ^[12]. Vitrified hPSCs do however still suffer from high levels of cell death and do not passage well as single cells ^[11]. The addition of Y-27632 to freezing and post-thawing medium enhances colony formation efficiency and treatment with Y-27632 during cryopreservation also increases survival rate and cell adhesion of freeze-thawed dissociated hES ^[12].

#Protocol 1: ROCK Inhibition assay using Y-27632

• ROCK-I and ROCK-II were assayed against Long S6 substrate peptide in a buffer containing 0.1mM ³³P-g-ATP.
• Enzymes were incubated with substrate for 30 minutes at room temperature before the reaction was stopped with 0.5M orthophosphoric acid.
• Samples were transferred to a P81 unifilter plate before being washed and then remaining ³³P labelled substrate being detected with a scintillation counter.

• If using the compound as a cell culture supplement, you should dilute the stock solution into culture medium immediately before use.
• Cell culture media can be prewarmed before adding the compound to avoid potential precipitation of the compound.
• Y-2762 is often removed from medium within 20 hours
• The compound has been shown to be effective at a final concentration of 10 µM (Ungrin et al.; Watanabe et al.)
• Mix and filter supplemented media using a 0.2 µm low‑protein binding filter.
• Please avoid a final DMSO concentration above 0.1% due to potential cell toxicity