An efficient method for the derivation of mouse embryonic stem cells

Vítzslav Bryja ¹, Sonia Bonilla ¹, Luká ajánek ¹, Clare L. Parish ¹, Catherine M. Schwartz ², Yongquan Luo ³, Mahendra S. Rao ⁴, Ernest Arenas ^1*

¹ Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
² Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden; Gerontology Research Center, Stem Cell Biology Unit, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
³ Gerontology Research Center, Stem Cell Biology Unit, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland
⁴ Gerontology Research Center, Stem Cell Biology Unit, Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland; Department of Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, Maryland

^* To whom correspondence should be addressed. E-mail: ernest.arenas{at}ki.se.

	Abstract

Mouse embryonic stem cells (mESC) represent a unique tool for many researchers, however, the process of ESC derivation is often very inefficient and requires high specialization, training and expertise. In order to circumvent these limitations we aimed to develop a simple and efficient protocol based on the use of commercially available products. Here we present an optimized protocol that we successfully applied to derive ESCs from several knockout mouse strains (Wnt-1, Wnt-5a, Lrp6 and parkin) with 50-75% efficiency. The methodology is based on the use of mouse embryonic fibroblast feeders, knockout serum replacement (SR) and minimal handling of the blastocyst. In this protocol, all centrifugation steps (as well as the use of trypsin inhibitor) were avoided and replaced by an ESC medium containing fetal calf serum (FCS) after the trypsinizations. We define the potential advantages and disadvantages of using SR and FCS in individual steps of the protocol. We also characterize the ESCs for the expression of ESC markers by immunohistochemistry, western blot and a stem cell focused microarray. In summary, we provide a simplified and improved protocol to derive mESCs that can be useful for laboratories aiming to isolate transgenic mESCs for the first time.