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generate one next step questions. ( scientific question ) from the Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells experment

Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells (embryonic stem cells/inner cell masses/differentiation in vsitro/embryonal carcinoma cells/growth factors) GAIL R. MARTIN Department of Anatomy, University of California, San Francisco, California 94143 Communicated byJ. Michael Bishop, September 14, 1981 ABSTRACT This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should make possible new approaches to the study of early mammalian development. NORMAL MOUSE development in utero BLASTOCYST transfer to extra-uterine site injection into blastocyst TERATOMA differentiation in situ TERATOCARCINOMA l1 subcutaneous injection culture in vitro Teratocarcinomas are tumors that arise with relatively high efficiency when normal 1- to 7.5-day-old inbred mouse embryos are transplanted to an extra-uterine site in a histocompatible host (1, 2). The stem cell lines that are isolated from them are known as embryonal carcinoma cell (ECC) lines. Such cells have morphological, biochemical, and immunological properties in common with pluripotent embryonic cells and therefore have been used extensively as an in vitro model system for the study of the developing embryo (3-5). In some cases these properties include the ability to differentiate in vitro in a manner that closely parallels the normal behavior of the isolated embryonic inner cell mass (ICM) (6, 7). The most compelling evidence for the close relationship between the tumor stem cells and normal embryonic cells is the fact that stem cells taken either from teratocarcinomas or from embryonal carcinoma cell cultures can participate in the development ofcompletely normal adult mice when combined with embryonic cells by the technique of blastocyst injection (8, 9). These findings, schematically represented in Fig. 1, raise questions about the origin of the teratocarcinoma stem cell. One hypothesis is that the tumor stem cells arise as a consequence of a stable, but reversible, epigenetic change in normal pluripotent embryonic cells. Such "transformed" cells presumably continue proliferation in the undifferentiated state because neoplastic conversion has reduced their efficiency of response to the normal signals for differentiation (11). An alternative idea is that embryonal carcinoma cells are not transformed but rather represent a selected population of completely normal embryonic cells that are programmed to divide until they receive the appropriate signals for differentiation; when the cells are in an TERATOCARCINOMA STEM CELL LINE differentiation in vitro DIFFERENTIATED CELL CULTURES FIG. 1. Relationship between normal embryos and teratocarcinoma stem cells. Adapted from Martin (10). extra-embryonic site the signals they receive are apparently not conducive to completely normal embryogenesis. It is difficult to study the process by which teratocarcinoma stem cells derive from normal embryonic cells as long as the transforming or selective events occur in vivo. As indicated by the dotted line in Fig. 1, it has long seemed logical to assume that pluripotent stem cells capable of forming teratocarcinomas might be isolated directly from embryos. Recently, Evans and Kaufman (12) carried out experiments in which pregnant strain 129 female mice were subjected to a treatment, involving ovariectomy and appropriate hormonal stimulation, that prevents normal implantation of blastocysts. Cells with the properties of teratocarcinoma stem cells were isolated from the embryos that had been maintained in such "delay" in the reproductive tract. Other attempts have been made to isolate cells with the properties of teratocarcinoma stem cells directly from normal early Abbreviations: ICM, inner cell mass; DME medium, Dulbecco's modified Eagle's medium; ESC, embryonic stem cell(s). The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 7634 Proc. Natl. Acad. Sci. USA 78 (1981) 7635 mouse embryos that have not been subjected to alteration in vivo. The approaches employed included embryo culture in a variety of media, and the use, as a starting material, of normal embryos at different stages of development and of giant embryonic cell masses created by embryo aggregation. None of these approaches has led to the establishment ofpluripotent cell cultures, although several differentiated cell lines have been isolated from early embryos (13). This report describes a method, involving culture in conditioned medium, for isolating and establishing pluripotent cell lines with the properties of teratocarcinoma stem cells directly from normal early mouse embryos in vitro. This method should be useful not only for further elucidating the relationship between teratocarcinoma stem cells and their normal embryonic progenitors but also for generating new, genetically marked pluripotent cell lines that -can be used fo