Overexpression of Insulin-like Growth Factor-II in Mouse Embryonic Stem Cells Promotes Myogenic Differentiation Katja Prelle,* Anna M. Wobus,† Ottheinz Krebs,* Werner F. Blum,‡ and Eckhard Wolf* ,1 *Department of Molecular Animal Breeding and Genetics, Ludwig-Maximilian University, Feodor-Lynen-Straße 25, 81377 Munich, Germany; †Institute of Plant Genetics and Crop Plant Research, 06466 Gatersleben, Germany; and ‡Lilly Germany GmbH, 61350 Bad Homburg, Germany Received September 29, 2000 Embryonic stem (ES) cells derived from androge- netic or parthenogenetic mouse embryos are impor- tant tools for studying the roles of imprinted genes in early development. Androgenetic ES cells have been shown to preferentially differentiate into the myo- genic lineage both in vitro and after formation of ter- atocarcinomas in vivo. To clarify if the maternally im- printed Igf2 gene which is expected to be overexpressed in androgenetic ES cells is sufficient to induce myogenic differentiation, R1 ES cells were transfected with human IGF-II expression vectors. Stable ES cell clones exhibiting human IGF-II mRNA and protein expression were studied vs ES cell clones without IGF-II overexpression in a standard in vitro differentiation system involving culture in “hanging drops” and observation of differentiation of the recov- ered embryoid bodies (EBs). EBs derived from IGF-II overexpressing ES cells showed stimulated myogenic differentiation evident by the appearance of myo- blasts already 3 days after plating and by higher levels of skeletal muscle-specific transcripts (myf5, myoD, myogenin) at earlier stages. Our study demonstrates for the first time that overexpression of IGF-II en- hances and accelerates myogenic differentiation of ES cells, which has implications for ES cell-derived tissue engineering. © 2000 Academic Press Key Words: IGF-II; ES cells; in vitro differentiation; myogenesis. Embryonic stem (ES) cells are pluripotent cell lines capable of differentiating into a variety of cell types in vitro and into somatic and germ-line cells of chimeric animals in vivo (for review, see [1]). ES cells provide a unique system for introducing targeted mutations into the mouse genome [2]. By in vitro differentiation via embryoid bodies into endodermal, ectodermal, and me- sodermal cells, ES cells offer a suitable model to study terminal differentiation into specialized cells of the cardiogenic, neurogenic and myogenic lineages [3]. Embryonic stem cells are usually derived from the inner cell mass of normal blastocysts, but may also be obtained from androgenetic [4] or parthenogenetic em- bryos [5]. The latter types of ES cells are interesting models for studying the roles of imprinted genes which are ex- pressed monoallelicly due to silencing of either the ma- ternal or the paternal allele (for review, see [6, 7]). An- drogenetic ES cells exhibit an overexpression of maternally imprinted genes while expression of pater- nally imprinted genes is expected to be eliminated. In vitro differentiation studies [8] and analysis of tumors produced by subcutaneous injection of androgenetic ES cells into syngeneic mice [9] demonstrated increased myogenic differentiation. A potential candidate responsi- ble for this altered differentiation pattern is insulin-like growth factor-II (IGF-II). The Igf2 gene is expressed from the paternal allele and plays an important role in embry- onic growth and differentiation. Knockout mice lacking functional Igf2 expression are 40% smaller at birth than normal littermates [10, 11]. Conversely, fetal overexpo- sure to IGF-II, either due to functional inactivation of the IGF-II/cation-independent mannose 6-phosphate recep- tor [12–14] or to expression of IGF-II transgenes [15], results in fetal overgrowth and perinatal death. Mouse chimeras made with androgenic stem cells possessing two active paternal copies of the imprinted Igf2 gene exhibit a range of defects [16] related to the elevated level of IGF-II. A role of IGF-II in myogenic differentiation was dem- onstrated by identifying this peptide as an autocrine differentiation factor in myoblasts [17] as well as a survival factor during the transition from proliferating to differentiating myoblasts [18]. IGF-II overexpress- ing myoblasts undergo enhanced differentiation char- acterized by accelerated expression of myogenin mRNA and extensive myotube formation [18]. Muscle 1 To whom correspondence should be addressed. Fax: 49-89-2180- 6849. E-mail: ewolf@lmb.uni-muenchen.de. Biochemical and Biophysical Research Communications 277, 631– 638 (2000) doi:10.1006/bbrc.2000.3737, available online at http://www.idealibrary.com on 631 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.