The impact of culture dimensionality on behavioral epigenetic memory contributing to pluripotent state of iPS cells

Abstract

Three‐dimensional (3D) culture platforms have been explored to establish physiologically relevant cell culture environment and permit expansion scalability; however, little is known about the mechanisms underlying the regulation of pluripotency of human induced pluripotent stem cells (hiPSCs). This study elucidated epigenetic modifications contributing to pluripotency of hiPSCs in response to 3D culture. Unlike two‐dimensional (2D) monolayer cultures, 3D cultured cells aggregated with each other to form ball‐like aggregates. 2D cultured cells expressed elevated levels of Rac1 and RhoA; however, Rac1 level was significantly lower while RhoA level was persisted in 3D aggregates. Compared with 2D monolayers, the 3D aggregates also exhibited significantly lower myosin phosphorylation. Histone methylation analysis revealed remarkable H3K4me3 upregulation and H3K27me3 maintenance throughout the duration of 3D culture; in addition, we observed the existence of naïve pluripotency signatures in cells grown in 3D culture. These results demonstrated that hiPSCs adapted to 3D culture through alteration of the Rho‐Rho kinase‐phospho‐myosin pathway, influencing the epigenetic modifications and transcriptional expression of pluripotency‐associated factors. These results may help design culture environments for stable and high‐quality hiPSCs.

Mechanobiological conceptual framework for assessing stem cell bioprocess effectiveness

“…This confirms that the cells adapt to culture environment through the alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and the transcription factors that contribute to their inherent states (Thanuthanakhun et al, 2021). Studies using ESCs and iPSC cultures revealed that the time-dependent regulation of two key bivalent epigenetic marks, histone H3 trimethylation at lysine 4 (H3K4me3) and histone H3 trimethylation at lysine 27 (H3K27me3), could contribute to the maintenance of the stem cell state and differentiation potential (Harikumar and Meshorer, 2015;Li et al, 2018;Thanuthanakhun et al, 2021). This regulation can take place through epigenetic changes and may be manifested as a "memorizer."…”

“…In addition, the actin-myosin cytoskeleton is a major "integrator" and "organizer" of mechanical and biochemical signaling inputs that continuously senses, organizes, and integrates these signals using its control "effector" protein belonging to the Rho family of GTPases (Arnold et al, 2017;Kimura et al, 1996). Specially, the antagonistic activities of RhoA and Rac1 GTPases play a role in the dynamics of myosin-mediated contraction and relaxation during cell migration in several cellular settings (Thanuthanakhun et al, 2021). This confirms that the cells adapt to culture environment through the alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and the transcription factors that contribute to their inherent states (Thanuthanakhun et al, 2021).…”

“…Specially, the antagonistic activities of RhoA and Rac1 GTPases play a role in the dynamics of myosin-mediated contraction and relaxation during cell migration in several cellular settings (Thanuthanakhun et al, 2021). This confirms that the cells adapt to culture environment through the alteration of the Rho-Rho kinase-phospho-myosin pathway, influencing the epigenetic modifications and the transcription factors that contribute to their inherent states (Thanuthanakhun et al, 2021). Studies using ESCs and iPSC cultures revealed that the time-dependent regulation of two key bivalent epigenetic marks, histone H3 trimethylation at lysine 4 (H3K4me3) and histone H3 trimethylation at lysine 27 (H3K27me3), could contribute to the maintenance of the stem cell state and differentiation potential (Harikumar and Meshorer, 2015;Li et al, 2018;Thanuthanakhun et al, 2021).…”

“…In particular, cells remember their behavioral changes in past environments. For instance, it has been shown that mechanotransduction through cytoskeletal contractility, Rho family GTPase signaling, and subsequent changes in epigenetic marks, such as DNA methylation, histone modifications, and chromatin state, play major roles in cellular homeostasis (Matthews et al, 2006;Rosowski et al, 2015;Thanuthanakhun et al, 2021). The ability of cells to maintain a constant level of cytoskeletal tension in response to external and internal disturbances is defined by tensional homeostasis (Ambriz et al, 2018;Lenormand et al, 2007;Mizuno et al, 2007;Walker et al, 2020a;Webster et al, 2014).…”

Mechanobiological conceptual framework for assessing stem cell bioprocess effectiveness

“…As shown in Figure 4, we developed a conceptual framework based on the criteria and indicators of intrinsic disorder within a bioprocess. When abnormal physical force is exerted on cells, cytoskeletal rearrangements are expected to counterbalance the extracellular mechanical forces, trigger signaling cascades, and eventually cause epigenetic modifications (Matthews et al, 2006;Rosowski et al, 2015;Thanuthanakhun et al, 2021). The exact role of this phenomenon and whether it induces the epigenetic modification is still under investigation, but accumulating evidence suggests that mechanical force-driven epigenetic memory formation are important for PSC bioprocess.…”