Thiazovivin, a Rho kinase inhibitor, improves stemness maintenance of embryo-derived stem-like cells under chemically defined culture conditions in cattle

Park, Sangkyu; Kim, Dae-Hwan; Jung, Yeon-Gil; Roh, Sangho

doi:10.1016/j.anireprosci.2015.08.003

Cited by 23 publications

(13 citation statements)

References 31 publications

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“…ROCK1 regulates actin-myosin contraction (McBeath et al, 2004), facilitates expansion of PSCs (Ohgushi et al, 2015;Park et al, 2015), and acute inhibition by small molecules leads to a "relaxed" cell phenotype with decreased stiffness (Kinney et al, 2014;Lee et al, 2006). However, we found that prolonged silencing of ROCK1 in hiPSCs (6 days) resulted in cells that were actually two-fold stiffer than either the CDH1 KD cells or the control CRISPRi cells.…”

Section: Discussionmentioning

confidence: 66%

“…Accutase (STEMCELL Technologies) was used to dissociate hiPSCs to single cells during passaging. Cells were passaged at a seeding density of 12,000 cells per cm 2 and the small molecule Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-276932 (10µM; Selleckchem) was added to the media upon passaging to promote survival (Park et al, 2015;Watanabe et al, 2007).…”

Section: Human Ipsc Line Generation and Culturementioning

confidence: 99%

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Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

Libby

Joy

et al. 2018

Preprint

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Morphogenesis results from the interactions of asymmetric cell populations to form complex multicellular patterns and structures comprised of distinct cell types. However, current methods to model morphogenic events offer little control over parallel cell type co-emergence and do not offer the capability to selectively perturb gene expression in specific subpopulations of cells. We have developed an in vitro system that can spatiotemporally interrogate cell-cell interactions and multicellular organization within human induced pluripotent stem cell (hiPSC) colonies. We examined the effects of independently knocking down molecular regulators of cortical tension and cell-cell adhesion using inducible CRISPRi: Rho-associated kinase-1 (ROCK1) and E-cadherin (CDH1), respectively. Induced mosaic knockdown of ROCK1 or CDH1 in hiPSC populations resulted in differential patterning events within hiPSC colonies indicative of cell-driven population organization. Patterned colonies retained an epithelial phenotype and nuclear expression of pluripotency markers. Gene expression within each of the mixed populations displayed a transient wave of differential expression with induction of knockdown that stabilized in coordination with intrinsic pattern formation. Mosaic patterning of hiPSCs enables the genetic interrogation of emergent multicellular properties of pluripotent cells, leading to a greater mechanistic understanding of the specific molecular pathways regulating the dynamics of symmetry breaking events that transpire during developmental morphogenesis.SIGNIFICANCEHuman embryonic development entails a series of multicellular morphogenic events that lead to primitive tissue formation. Attempts to study human morphogenic processes experimentally have been limited due to divergence from model organisms and the inability of current human in vitro models to accurately control the coincident emergence of heterogeneous cell populations in the spatially controlled manner necessary for proper tissue structure. We developed a human induced pluripotent stem cell (iPSC) in vitro model that enables temporal control over the emergence of heterotypic subpopulations of cells. We examined mosaic knockdown of two target molecules to create predictable and robust cell-patterning events within hiPSC colonies. This method allows for dynamic interrogation of intrinsic cell mechanisms that initiate symmetry breaking events and provides direct insight(s) into tissue developmental principles.

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Section: Discussionmentioning

confidence: 66%

Section: Human Ipsc Line Generation and Culturementioning

confidence: 99%

Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

Libby

Joy

et al. 2018

Preprint

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“…IVP of bovine fertilized embryos was conducted as previously described [15]. The thawed HanWoo semen (purchased from HanWoo improvement center, Seosan, Korea) was deposited on the top of a discontinuous Percoll gradient prepared by depositing 2 ml of 90% Percoll under 2 ml of 45% Percoll in a 15 ml centrifuge tube, and the sample was then centrifuged for 20 min at 252 x g .…”

Section: Methodsmentioning

confidence: 99%

“…There have been several attempts to generate authentic bESCs using small molecules. Recently, embryo-derived stem-like cells (eSLCs) from bovine embryos were successfully generated using three inhibitors (3i): PD18435 (MEK inhibitor), CHIR99021, and SU5402, a fibroblast growth factor receptor (FGFR) inhibitor [14, 15]. The eSLCs in 3i were able to proliferate and be maintained in culture for over 50 passages with the normal karyotype.…”

Section: Introductionmentioning

confidence: 99%

Microarray analysis of embryo-derived bovine pluripotent cells: The vulnerable state of bovine embryonic stem cells

2017

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Although there are many studies about pluripotent stem cells, little is known about pluripotent pathways and the difficulties of maintaining the pluripotency of bovine cells in vitro. Here, we investigated differently expressed genes (DEG) in bovine embryo-derived stem-like cells (eSLCs) from various origins to validate their distinct characteristics of pluripotency and differentiation. We identified core pluripotency markers and additional markers which were not determined as pluripotency markers yet in bovine eSLCs. Using the KEGG database, TGFβ, WNT, and LIF signaling were related to the maintenance of pluripotency. In contrast, some DEGs related to the LIF pathway were down-regulated, suggesting that reactivation of the pathway may be required for the establishment of true bovine embryonic stem cells (ESCs). Interestingly, oncogenes were co-down-regulated, while tumor suppressor genes were co-up-regulated in eSLCs, implying that this pattern may induce abnormal teratomas. These data analyses of signaling pathways provide essential information on authentic ESCs in addition to providing evidence for pluripotency in bovine eSLCs.

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“…Recent studies have reported that ROCK inhibitors, specifically Thiazovivin and Y-27632, can effectively maintain the pluripotency and increase the viability of stem cells after freezing and thawing 9 , 10 . In cows, several studies have shown that these small molecules are also able to establish embryo-derived stem cells 11 – 13 . However, little is known about the relationship between small molecules and the resistance to heat stress during short-term embryo holding at a wide temperature range.…”

Section: Introductionmentioning

confidence: 99%

Holding of bovine blastocysts at suprazero temperatures using small molecules

Kim

Sul

Jung

et al. 2017

Sci Rep

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Although assisted reproductive technology (ART) currently exists, the only embryo preservation technology that is available is cryopreservation. In the present study, small molecules were used to hold embryos at room temperature. The basic medium for embryo holding for a short period of time at 4 °C, 10 °C and 20 °C consisted of 1% BSA non-cryopreservation medium (BNC) instead of fetal bovine serum. To maintain survival and prevent damage during embryo incubation, three candidate small molecules were selected-CHIR99021, Y-27632 and Thiazovivin-and their concentrations were optimized. The viability and hatching rate of embryos incubated at 10 °C were greater for Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. However, the rate was lower for Thiazovivin-BNC compared to BNC. Although there were no surviving embryos after incubation at 20 °C, the viability and hatching rate of embryos significantly increased in Y-27632-BNC and CHIR99021+Y-27632-BNC compared to BNC. The pregnancy rate of embryos incubated at 20 °C was also greater in the CHIR99021+Y-27632-BNC group compared to that in the frozen group. The mechanism by which small molecules enhance survival of embryos during incubation was investigated, and expression of heat shock protein 70 was observed to increase. The findings of this work may be useful in improving ART in the agricultural field.Cattle are one of the most important livestock in the world. Their reproduction constitutes a large part of the agricultural economy in many countries including milk and meat. Considering this point, it is essential to use and develop assisted reproductive technology (ART) such as artificial insemination, embryo transfer (ET) and embryo production by in vitro fertilization (IVF) in this species 1 . However, most ART has focused on improving embryo production, genetic selection and pregnancy rates, while embryo storage has not received as much attention. In numerous instances during ART, embryos require storage for short or long periods of time. Cryopreservation with liquid nitrogen (LN 2 ) is generally used for long-as well as short-term embryo storage. However, physical damage is caused during the freeze-thaw process and the damage can limit embryo survival 2 . Thus, only high degree (high quality) embryos that can endure physical damage are normally used, while the rest, even though they still have the potential for full development, are mostly discarded. Moreover, freezing-induced damage reduces pregnancy rates either directly or indirectly 3 . Therefore, it is necessary to improve storage systems to improve ART efficiency.It has been reported that many cells, including embryos, can be stored at hypothermic temperatures 4, 5 . Recently, it was also documented that bovine embryos could be incubated for 7 days using a simple medium including FBS and that embryos incubated in the medium were able to develop into normal calves 6 . However, the low-temperature embryo holding protocol still had limitations including temperature tolerance, preservative quality, mobilit...

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Thiazovivin, a Rho kinase inhibitor, improves stemness maintenance of embryo-derived stem-like cells under chemically defined culture conditions in cattle

Cited by 23 publications

References 31 publications

Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

Spatiotemporal mosaic patterning of pluripotent stem cells using CRISPR interference

Microarray analysis of embryo-derived bovine pluripotent cells: The vulnerable state of bovine embryonic stem cells

Holding of bovine blastocysts at suprazero temperatures using small molecules

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