The role of RhoA/ROCK singaling pathway in organoid research

Ethics statement: This study constituted a comprehensive analysis of previously released studies and thus, was not subject to the approval of the institutional review board.

The Rho/ROCK signaling axis coordinates multiple downstream cascades critical to organoid development by regulating cytoskeletal dynamics, mechanical tension, and cell–matrix interactions. At the core of this pathway is Ras homolog family member A (RhoA), a small GTPase activated in response to mechanical stimuli or growth factor signaling. In its active, GTP-bound state, RhoA binds to and activates its primary effector, ROCK, which exists in two isoforms: ROCK1 and ROCK2 [14].

Upon activation, ROCK mediates several key signaling pathways. One of the best characterized is the ROCK–myosin light chain (MLC) pathway, in which ROCK directly phosphorylates MLC while concurrently inhibiting myosin light chain phosphatase. This dual action enhances actomyosin contractility, thereby reinforcing cytoskeletal tension and enabling morphogenetic movements in developing organoids [15–18].

In parallel, ROCK activates LIM domain kinase (LIMK), which phosphorylates and inactivates cofilin, a key actin-severing protein. The resulting ROCK–LIMK–cofilin axis stabilizes filamentous actin (F-actin) and supports cytoskeletal remodeling. These structural changes are vital for cell migration, neurite outgrowth, and the maintenance of epithelial polarity in three-dimensional cultures [19,20].

Additionally, ROCK regulates focal adhesion kinase (FAK) activity, thereby facilitating integrin-dependent adhesion and dynamic cell motility. This ROCK–FAK pathway is particularly important during organoid expansion and matrix remodeling, as cells continually re-establish contacts with their surrounding extracellular environment [21].

Furthermore, ROCK modulates the phosphatase and tensin homolog (PTEN)–AKT pathway, a key regulator of cell survival and proliferation. By influencing PTEN, ROCK indirectly controls AKT (also known as protein kinase B) activity. This mechanism is crucial for balancing survival and apoptotic signals, particularly during the early stages of organoid formation when dissociated stem cells are highly vulnerable to cell death [22].

Beyond these specific cascades, Rho/ROCK signaling is an integral component of broader mechanotransduction networks. Notably, it interfaces with the Hippo signaling pathway, impacting yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ) activity. This cross-regulation plays an important role in stem cell fate decisions, proliferation, and spatial tissue patterning [23,24].

In summary, these interlinked mechanisms establish Rho/ROCK signaling as a master regulator of the physical and biochemical processes underlying organoid morphogenesis. Therefore, fine-tuning ROCK activity is essential for supporting cell survival, differentiation, and proper tissue organization. Disruption of this balance—whether by excessive inhibition or constitutive activation—can compromise the structural integrity and biological fidelity of organoids (Fig. 1) [25,26].

Fig. 1.

Overview of Rho/Rho-associated coiled-coil containing protein kinase (ROCK) pathway in organoids. This figure illustrates the key molecular interactions in the Rho/ROCK pathway, including downstream effectors such as myosin light chain (MLC), LIM domain kinase (LIMK), and focal adhesion kinase (FAK), and highlights its role in cytoskeletal dynamics and stem cell behavior. F-actin, filamentous actin; PTEN, phosphatase and tensin homolog.

Crosstalk with Ras and other pathways

Rho/ROCK signaling does not operate in isolation; it interacts with other key signaling cascades to fine-tune cellular responses during organoid development. Among these, the Ras–mitogen-activated protein kinase (MAPK) pathway stands out as a significant counterpart. While RhoA predominantly regulates cytoskeletal organization and contractility, Ras promotes cell proliferation and survival via the RAF–MEK–ERK signaling cascade [27]. The interplay between Rho and Ras signaling is complex and context-dependent, displaying both cooperative and antagonistic interactions. For example, active Ras signaling can suppress RhoA activity through the activation of p190RhoGAP (GTPase-activating protein), reducing cellular contractility and fostering a more migratory or proliferative phenotype [28,29]. This antagonism is especially apparent during epithelial-to-mesenchymal transition, where suppression of Rho activity enhances cell mobility. Conversely, cytoskeletal changes mediated by RhoA can influence the spatial distribution and activation dynamics of Ras effectors. Tension-induced alterations in actin architecture affect the localization of scaffold proteins such as kinase suppressor of Ras, modulating the intensity and duration of ERK activation [30]. These feedback loops underscore the importance of cytoskeletal context in shaping mitogenic signals (Fig. 2).

Fig. 2.

The complexity of Rho/Rho-associated coiled-coil containing protein kinase (ROCK) signaling. This diagram depicts the crosstalk between Rho/ROCK and diverse cellular pathways. Same-colored circles share similar cellular functions. RhoA, Ras homolog family member A; FAK, focal adhesion kinase; PTEN, phosphatase and tensin homolog; MLC, myosin light chain; YAP/TAZ, yes-associated protein/transcriptional coactivator with PDZ-binding motif; TGF, transforming growth factor; Wnt, wingless/integrated; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; LIMK, LIM domain kinase; Ras, Rat sarcoma viral oncogene homolog; MEK, MAP/ERK kinase; ERK, extracellular signal-regulated kinase; EMT, epithelial-mesenchymal transition.

Beyond Ras, Rho/ROCK signaling also interacts with the Hippo pathway, particularly through the mechanical regulation of YAP and TAZ. ROCK-induced cytoskeletal tension promotes the nuclear localization of YAP/TAZ, thereby enhancing the transcription of genes associated with proliferation and differentiation [31]. This intersection indicates the presence of a mechanochemical feedback network, where matrix stiffness, RhoA activation, and Ras signaling converge to direct organoid morphogenesis.

In organoid cultures, a balanced interplay between Rho/ROCK and Ras signaling is crucial. Excessive Ras activity without corresponding mechanical cues can lead to uncontrolled proliferation, while hyperactive RhoA/ROCK signaling may inhibit expansion or result in abnormal morphology [32]. Thus, fine-tuned crosstalk between these pathways is vital for maintaining the equilibrium between stem cell renewal and differentiation, ultimately preserving tissue architecture and function.

Mechanobiology and extracellular matrix interactions

Organoid development is governed not only by biochemical signals but also by mechanical cues derived from the extracellular matrix (ECM). The mechanobiology of stem cells—how they sense and respond to physical forces—plays a critical role in determining organoid structure, polarity, and lineage specification [33–36].

The Rho/ROCK pathway functions as a critical mechanotransducer, converting physical inputs from the ECM into intracellular signals. ECM stiffness is detected via integrins, which connect the external matrix to the actin cytoskeleton through focal adhesions. Mechanical loading or substrate tension activates integrins, which in turn stimulate FAK and subsequently trigger RhoA signaling [37,38]. Activated RhoA then activates ROCK, promoting actomyosin contractility via MLC phosphorylation and thereby reinforcing cytoskeletal tension [39]. This dynamic feedback loop enables cells to adjust their shape, polarity, and adhesion in response to the matrix environment. In softer ECM conditions, reduced ROCK activity leads to lower tension, preserving stemness, whereas stiffer matrices increase contractility and drive stem cell differentiation toward epithelial or mesenchymal lineages [40].

Furthermore, ECM stiffness modulates Hippo signaling by affecting the nuclear localization of YAP and TAZ. In rigid matrices, increased cytoskeletal stress due to ROCK activation favors YAP/TAZ translocation into the nucleus, thereby promoting gene expression associated with proliferation and tissue growth [23]. In organoid systems, controlling matrix mechanics is crucial for directing morphogenesis. Hydrogels with adjustable stiffness have been utilized to fine-tune Rho/ROCK activity and replicate tissue-specific microenvironments. For instance, intestinal and hepatic organoids exhibit different responses to ECM density, underscoring the need for precise biomechanical design in organoid engineering [41].

In summary, Rho/ROCK-mediated mechanosensing serves as a central integrator of ECM stiffness, cytoskeletal dynamics, and transcriptional responses, making it indispensable for designing and optimizing physiologically relevant organoid models.

Applications and limitations of ROCK inhibition

The use of ROCK inhibitors, particularly Y-27632 and fasudil, has become a standard component in many organoid protocols because of their ability to enhance stem cell survival, reduce dissociation-induced apoptosis, and improve clonogenicity during the early stages of organoid formation. These inhibitors suppress RhoA–ROCK–MLC-mediated actomyosin contractility, thereby reducing cellular tension and enabling epithelial cells to aggregate and survive after single-cell dissociation (Table 1) [16,42]. This effect is especially critical during initial plating and passaging, when cells experience mechanical stress or suboptimal adhesion conditions.

Table 1.

Key ROCK inhibitors and their applications

ROCK inhibition has been widely employed across various organoid systems to enhance initial cell survival and support early morphogenesis. In intestinal organoids, Y-27632 increases the viability of Lgr5⁺ stem cells and promotes crypt-like budding. In liver and pancreas models, it supports the expansion of EpCAM⁺ hepatic progenitors and facilitates organoid establishment from limited patient-derived samples. In lung and airway organoids, ROCK inhibition prevents anoikis in basal stem cells, while in neural systems, it reduces apoptosis and improves the uniformity of cerebral organoid spheres during early culture (Table 2) [43–47].

Table 2.

Organ-specific applications of ROCK inhibition in organoid culture

Although transient use of ROCK inhibitors is crucial for early organoid establishment, prolonged or unregulated exposure can cause undesirable effects that compromise tissue development and fidelity. One significant limitation is the potential disruption of epithelial morphogenesis. Sustained inhibition of the RhoA–ROCK–MLC pathway reduces actomyosin contractility, which is essential for epithelial folding, lumen formation, and overall structural integrity during organoid maturation [48].

Another concern is the delayed or incomplete differentiation of stem cells. ROCK inhibition can interfere with the cytoskeletal remodeling necessary for lineage specification, leading to the persistence of progenitor-like states or skewed differentiation trajectories [49]. This effect is particularly problematic in long-term cultures, where precise temporal transitions between cell states are required to mimic in vivo development. In some systems, excessive ROCK suppression results in aberrant tissue architecture, including flattened, cystic, or multilayered structures that deviate from the physiological morphology of the target organ [41]. These anomalies are attributed to the loss of mechanical tension, which normally guides coordinated growth and polarity cues within the organoid.

Therefore, a strategic approach is needed when incorporating ROCK inhibitors into organoid protocols. Most studies recommend limiting their use to the first 24 to 48 hours after seeding or passaging, followed by withdrawal to permit natural morphogenic processes to resume. Additionally, combining ROCK inhibition with biomimetic ECM environments or temporal gene control systems may mitigate these limitations and improve organoid reproducibility [50].

Perspectives

Future research should integrate single-cell omics, live imaging, and biomechanical assays to map the spatiotemporal activity of Rho. Genetic manipulations (e.g., CRISPR targeting of ROCK1/2), in combination with tunable ECM systems, may provide a precise approach to enhance reproducibility and complexity in organoid cultures. This strategy will facilitate translational applications ranging from personalized disease modeling to therapeutic tissue engineering (Fig. 3). Rho/ROCK signaling is indispensable for the successful generation and maturation of organoids. Its diverse functions in regulating cytoskeletal dynamics, adhesion, and mechanosensation position it as a central hub in stem cell and organoid biology. By harnessing its modulatory potential, researchers can refine organoid systems to more closely mimic physiological conditions, thereby advancing both basic science and clinical applications.

Fig. 3.

Perspectives on the role of the Rho/Rho-associated coiled-coil containing protein kinase (ROCK) signaling in future organoid research. This diagram explains how Rho/ROCK signaling will be handled for various applications. ECM, extracellular matrix.