A Rho Kinase (ROCK) Inhibitor, Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells

Kim, Ki-Chul; Min, Sol; Kim, Dae-Hwan; Kim, Hyewon; Roh, Sangho

doi:10.3390/molecules26092658

Cited by 9 publications

(8 citation statements)

References 47 publications

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“…The ROCK inhibitor, Y27632, has been used to promote cell viability and decrease cell stress following cell isolation [ 24 , 41 , 42 , 43 ]. However, it has also been reported that the ROCK inhibitor causes a loss of 3D architecture and cell spreading [ 17 , 44 ], which aligns with the aberrant cell growth observed here ( Supplementary Figure S1 ).…”

Section: Resultsmentioning

confidence: 99%

“…By day 14, relative mRNA expression of all markers had dropped in all media conditions, suggesting that further work could be done to improve longevity of the cultures. A fourth media condition was also investigated, consisting of a ROCK inhibitor for 24 h, followed by 0.5 µM EGFR inhibitor (R24M), to help reduce cell isolation-induced stress [ 24 ]. Little improvement was observed between R24M and M media for Mist1, Aqp5, and Tmem16a mRNA expression.…”

Section: Discussionmentioning

confidence: 99%

“…In particular, the aim was to test several factors that have been previously shown to impact secretory characteristics for their individual effects on Mist1, as well as to test the effects of combining factors. A DoE approach was designed in JMP Pro 15 (SAS) using seven factors (FGF10, neurturin, EGFR inhibitor, ROCK inhibitor, Apolipoprotein E, insulin, and TGFβR1 inhibitor) identified from the literature [ 12 , 16 , 17 , 18 , 24 , 25 , 26 , 27 , 28 , 29 ] and tested with a microbubble (MB)-hydrogel culture system [ 13 ].…”

Section: Methodsmentioning

confidence: 99%

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Optimizing Soluble Cues for Salivary Gland Tissue Mimetics Using a Design of Experiments (DoE) Approach

Piraino

Benoit

DeLouise

2022

Cells

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The development of therapies to prevent or treat salivary gland dysfunction has been limited by a lack of functional in vitro models. Specifically, critical markers of salivary gland secretory phenotype downregulate rapidly ex vivo. Here, we utilize a salivary gland tissue chip model to conduct a design of experiments (DoE) approach to test combinations of seven soluble cues that were previously shown to maintain or improve salivary gland cell function. This approach uses statistical techniques to improve efficiency and accuracy of combinations of factors. The DoE-designed culture conditions improve markers of salivary gland function. Data show that the EGFR inhibitor, EKI-785, maintains relative mRNA expression of Mist1, a key acinar cell transcription factor, while FGF10 and neurturin promote mRNA expression of Aqp5 and Tmem16a, channel proteins involved in secretion. Mist1 mRNA expression correlates with increased secretory function, including calcium signaling and mucin (PAS-AB) staining. Overall, this study demonstrates that media conditions can be efficiently optimized to support secretory function in vitro using a DoE approach.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Optimizing Soluble Cues for Salivary Gland Tissue Mimetics Using a Design of Experiments (DoE) Approach

Piraino

Benoit

DeLouise

2022

Cells

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“…Therefore, we speculate the likelihood of other mechanisms that are associated with miRNA export upstream to RhoA/ROCK signaling. Another mechanism is the antiapoptotic action of RI can antagonize the apoptotic action of miR-155 on embryonic cells [18,[66][67][68][69]. Additionally, ROCK pathway inhibition enhances trophoblast adhesion and viability in humans.…”

Section: Discussionmentioning

confidence: 99%

ROCK Inhibitor (Y-27632) Abolishes the Negative Impacts of miR-155 in the Endometrium-Derived Extracellular Vesicles and Supports Embryo Attachment

Saadeldin

Tanga

Bang

et al. 2022

Cells

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Extracellular vesicles (EVs) are nanosized vesicles that act as snapshots of cellular components and mediate cellular communications, but they may contain cargo contents with undesired effects. We developed a model to improve the effects of endometrium-derived EVs (Endo-EVs) on the porcine embryo attachment in feeder-free culture conditions. Endo-EVs cargo contents were analyzed using conventional and real-time PCR for micro-RNAs, messenger RNAs, and proteomics. Porcine embryos were generated by parthenogenetic electric activation in feeder-free culture conditions supplemented with or without Endo-EVs. The cellular uptake of Endo-EVs was confirmed using the lipophilic dye PKH26. Endo-EVs cargo contained miR-100, miR-132, and miR-155, together with the mRNAs of porcine endogenous retrovirus (PERV) and β-catenin. Targeting PERV with CRISPR/Cas9 resulted in reduced expression of PERV mRNA transcripts and increased miR-155 in the Endo-EVs, and supplementing these in embryos reduced embryo attachment. Supplementing the medium containing Endo-EVs with miR-155 inhibitor significantly improved the embryo attachment with a few outgrowths, while supplementing with Rho-kinase inhibitor (RI, Y-27632) dramatically improved both embryo attachment and outgrowths. Moreover, the expression of miR-100, miR-132, and the mRNA transcripts of BCL2, zinc finger E-box-binding homeobox 1, β-catenin, interferon-γ, protein tyrosine phosphatase non-receptor type 1, PERV, and cyclin-dependent kinase 2 were all increased in embryos supplemented with Endo-EVs + RI compared to those in the control group. Endo-EVs + RI reduced apoptosis and increased the expression of OCT4 and CDX2 and the cell number of embryonic outgrowths. We examined the individual and combined effects of RI compared to those of the miR-155 mimic and found that RI can alleviate the negative effects of the miR-155 mimic on embryo attachment and outgrowths. EVs can improve embryo attachment and the unwanted effects of the de trop cargo contents (miR-155) can be alleviated through anti-apoptotic molecules such as the ROCK inhibitor.

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“…Excessive cellular contraction can induce cell death as observed in human embryonic stem cells, in which actin-myosin contractions are a critical effector of cell death in response to cell dissociation 10 . Previously, we have reported that Y-27632 inhibits the dissociation-induced cell death of salivary gland stem cells 11 . TGF-β is a major pro brotic mediator and triggers broblast activation and differentiation into myo broblasts resulting in increased ECM production and cell contraction 12 .…”

Section: Introductionmentioning

confidence: 92%

Y-27632-mediated long-term expansion of salivary gland epithelial cells

Kim¹,

Kim

et al. 2022

Preprint

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In salivary glands, epithelial cells perform functional activities such as the production and secretion of saliva. Therefore, the use of salivary gland epithelial cells to study general biology or drug screening may contribute to the development of therapeutic strategies for salivary gland diseases. However, the underlying mechanism of long-term in vitro expansion methods for salivary gland epithelial cells (SG-Epis) are unknown. Here, we present the roles of the rho-associated kinase (ROCK) inhibitor Y-27632 in maintaining SG-Epis and its underlying mechanisms. The treatment with Y-27632 significantly increased the proliferative potential and maintained the expression of Krt8 and Krt14 for 17 passages. Conversely, in the absence of Y-27632, SG-Epis lost their epithelial morphology. The treatment with Y-27632, however, maintained the epithelial morphology and downregulated the mRNA levels of Tgf-β1, Ctgf, and Rock2. Treatment with TGF-β1 reveals that TGF-β/CTGF/p38 signaling is responsible for the maintenance of SG-Epis. RNA interference study shows that ROCK2/JNK signaling is also required for proliferation and maintenance of SG-Epis. The culture method using Y-27632 provides a solution for efficient in vitro expansion of SG-Epis.

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A Rho Kinase (ROCK) Inhibitor, Y-27632, Inhibits the Dissociation-Induced Cell Death of Salivary Gland Stem Cells

Cited by 9 publications

References 47 publications

Optimizing Soluble Cues for Salivary Gland Tissue Mimetics Using a Design of Experiments (DoE) Approach

Optimizing Soluble Cues for Salivary Gland Tissue Mimetics Using a Design of Experiments (DoE) Approach

ROCK Inhibitor (Y-27632) Abolishes the Negative Impacts of miR-155 in the Endometrium-Derived Extracellular Vesicles and Supports Embryo Attachment

Y-27632-mediated long-term expansion of salivary gland epithelial cells

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