Effect of Small Molecule Supplements During in Vitro Culture of Mouse Zygotes and Parthenogenetic Embryos on Hypoblast Formation and Stem Cell Derivation

Overview

Journal Stem Cell Rev Rep

Publisher Springer

Specialty Cell Biology

Date 2012 May 26

PMID 22628112

Authors

K Versieren

M Van der Jeught

T OLeary

G Duggal

J Gerris

S Chuva De Sousa Lopes

B Heindryckx

P De Sutter

Affiliations

Soon will be listed here.

Abstract

Background: Small molecule inhibitors are organic components that modulate signalling pathways and have the ability to change the differentiation state of cells. They have been used to increase the efficiency of induced pluripotent stem cell generation and to support stem cell derivation and culture. In this study, we aimed to evaluate the effects of small molecules on the development of mouse zygotes and parthenogenetic embryos.

Methods And Results: Three inhibitors (SC-1, PD0325901 and BIO) were added to the culture medium from the 2-cell stage onwards. We have observed that addition of an inhibitor of the fibroblast growth factor (FGF) pathway (SC-1 or PD0325901) compromises the segregation of hypoblast from the inner cell mass (ICM). Given no difference was observed in size of the ICM, but more epiblast cells were found in these embryos, we can conclude that this is caused by redirection of all ICM cells to the epiblast. We also determined the consequences of reduced hypoblast and increased epiblast formation on stem cell derivation efficiency. No significant difference was found between derivation rates from treated embryos as compared to controls. However, only under 2i + ROCKi conditions, stem cells could be derived with an efficiency of more than 90%. Addition of BIO, an activator of the WNT pathway, did not have any effects on hypoblast development or stem cell derivation.

Conclusion: We have demonstrated that FGF signalling is crucial for hypoblast generation and small molecules can be efficiently used to inhibit this process both in zygotes and parthenogenetic embryos.

References

Furue M, Okamoto T, Hayashi Y, Okochi H, Fujimoto M, Myoishi Y . Leukemia inhibitory factor as an anti-apoptotic mitogen for pluripotent mouse embryonic stem cells in a serum-free medium without feeder cells. In Vitro Cell Dev Biol Anim. 2005; 41(1-2):19-28. DOI: 10.1290/0502010.1. View

Marikawa Y, Alarcon V . Establishment of trophectoderm and inner cell mass lineages in the mouse embryo. Mol Reprod Dev. 2009; 76(11):1019-32. PMC: 2874917. DOI: 10.1002/mrd.21057. View

Zhen Y, Sorensen V, Jin Y, Suo Z, Wiedlocha A . Indirubin-3'-monoxime inhibits autophosphorylation of FGFR1 and stimulates ERK1/2 activity via p38 MAPK. Oncogene. 2007; 26(44):6372-85. DOI: 10.1038/sj.onc.1210473. View

Yamanaka Y, Lanner F, Rossant J . FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst. Development. 2010; 137(5):715-24. DOI: 10.1242/dev.043471. View

Chen L, Wang D, Wu Z, Ma L, Daley G . Molecular basis of the first cell fate determination in mouse embryogenesis. Cell Res. 2010; 20(9):982-93. DOI: 10.1038/cr.2010.106. View

Nichols J, Silva J, Roode M, Smith A . Suppression of Erk signalling promotes ground state pluripotency in the mouse embryo. Development. 2009; 136(19):3215-22. PMC: 2739140. DOI: 10.1242/dev.038893. View

Gauthaman K, Fong C, Bongso A . Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion. Stem Cell Rev Rep. 2009; 6(1):86-95. DOI: 10.1007/s12015-009-9107-8. View

Thouas G, Korfiatis N, French A, Jones G, Trounson A . Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts. Reprod Biomed Online. 2003; 3(1):25-29. DOI: 10.1016/s1472-6483(10)61960-8. View

Liu N, Enkemann S, Liang P, Hersmus R, Zanazzi C, Huang J . Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis. J Mol Cell Biol. 2010; 2(6):333-44. DOI: 10.1093/jmcb/mjq029. View

10.

Li X, Meng G, Krawetz R, Liu S, Rancourt D . The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells Dev. 2008; 17(6):1079-85. DOI: 10.1089/scd.2007.0247. View

11.

Lawitts J, Biggers J . Optimization of mouse embryo culture media using simplex methods. J Reprod Fertil. 1991; 91(2):543-56. DOI: 10.1530/jrf.0.0910543. View

12.

van Amerongen R, Nusse R . Towards an integrated view of Wnt signaling in development. Development. 2009; 136(19):3205-14. DOI: 10.1242/dev.033910. View

13.

Ishiwata I, Tokeida Y, Iguchi M, Ishiwata C, Kiguchi K, Yasumoto S . New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation. Hum Cell. 2002; 14(4):283-91. View

14.

Chen Z, Liu Z, Huang J, Amano T, Li C, Cao S . Birth of parthenote mice directly from parthenogenetic embryonic stem cells. Stem Cells. 2009; 27(9):2136-45. DOI: 10.1002/stem.158. View

15.

Li W, Ding S . Small molecules that modulate embryonic stem cell fate and somatic cell reprogramming. Trends Pharmacol Sci. 2009; 31(1):36-45. DOI: 10.1016/j.tips.2009.10.002. View

16.

Olson M . Applications for ROCK kinase inhibition. Curr Opin Cell Biol. 2008; 20(2):242-8. PMC: 2377343. DOI: 10.1016/j.ceb.2008.01.002. View

17.

Nichols J, Jones K, Phillips J, Newland S, Roode M, Mansfield W . Validated germline-competent embryonic stem cell lines from nonobese diabetic mice. Nat Med. 2009; 15(7):814-8. DOI: 10.1038/nm.1996. View

18.

Rossant J . Lineage development and polar asymmetries in the peri-implantation mouse blastocyst. Semin Cell Dev Biol. 2004; 15(5):573-81. DOI: 10.1016/j.semcdb.2004.04.003. View

19.

Kawase E, Suemori H, Takahashi N, Okazaki K, Hashimoto K, Nakatsuji N . Strain difference in establishment of mouse embryonic stem (ES) cell lines. Int J Dev Biol. 1994; 38(2):385-90. View

20.

Versieren K, Heindryckx B, Lierman S, Gerris J, De Sutter P . Developmental competence of parthenogenetic mouse and human embryos after chemical or electrical activation. Reprod Biomed Online. 2010; 21(6):769-75. DOI: 10.1016/j.rbmo.2010.07.001. View