Genetic selection of sox1GFP-expressing neural precursors removes residual tumorigenic pluripotent stem cells and attenuates tumor formation after transplantation

Embryonic stem (ES) cells are derived from the inner cell mass of pre-implantation mouse embryos (Evans and Kaufman 1981; Martin 1981) and represent pluripotent cells that can give rise to most cell types (Nagy et al. 1990, 1993). ES cells can be permanently maintained in vitro as pluripotent cells and, upon exposure to appropriate differentiation signals, can differentiate into a vast range of cell lineages (Smith 1991; Desbaillets et al. 2000). These unique properties make ES cells a useful tool for analyzing critical steps of cell development using both animal models and in vitro differentiation culture systems (Hooper et al. 1987; Thomas and Capecchi 1987; Nagy et al. 1990, 1993; Dinsmore et al. 1996; Wutz and Jaenisch 2000). In addition, the capacity of ES cells to generate terminally-differentiated cell types provides a potentially unlimited resource for cell replacement therapy (Dinsmore et al. 1996; Brustle et al. 1999; Lumelsky et al. 2001; Bjorklund et al. 2002). We have previously shown that in vivo grafting of mouse ES (mES) cells could ameliorate behavioral deficits in rodent models of Parkinson’s disease (PD) by generating mature dopaminergic (DA) neurons in the brain (Bjorklund et al. 2002). In addition, we and others have shown that mature DA neurons can be efficiently generated in vitro by genetic modification of mES cells with the transcription factor, Nurr1 (Chung et al. 2002; Kim et al. 2002). However, we and others observed that grafting in vitro-differentiated ES cells does not eliminate abnormal and disruptive growth post-transplantation (data not shown; Arnhold et al. 2004). Thus, for clinical application of ES-derived cells for transplantation therapy, it is essential to safeguard from any potential tumor formation derived from the grafted cells. It is therefore important to establish a procedure that can purify only the desired cell type and thus avoid post-grafting tumor formation. Sox1 is a member of a family of transcription factors containing the HMG-box DNA binding domain (Kamachi et al. 2000). Three members of the Sox family (Sox1, Sox2 and Sox3) are expressed in the neuroectoderm. While expression of Sox2 and Sox3 starts at the pre-implantation and the epiblast stage, respectively (Pevny et al. 1998), the onset of Sox1 expression correlates with the formation of neural plate, and its expression is down-regulated as neural cells exit the cell cycle and differentiate (Uwanogho et al. 1995; Pevny et al. 1998; Wood and Episkopou 1999). These observations mean that Sox1 is an ideal marker for neural precursor (NP) cells. In addition, Sox1 has been shown to be critical in maintaining NPs at the undifferentiated state by counteracting the activity of proneural proteins and inhibiting neurogenesis (Bylund et al. 2003). In Sox1-green fluorescent protein (GFP) knock-in ES cells, GFP is specifically expressed by NP cells both in vivo and in vitro (Ying et al. 2003), thus making fluorescence-activated cell sorting (FACS) purification of NPs possible. To test whether purification of NP cells removes tumor-forming cells, we purified ES cell-derived NPs using sox1-GFP knock-in ES cells by FACS, and characterized GFP+ versus GFP− cells both in vitro and in vivo. Here, we show that sorting GFP+ cells greatly enriched the NP population and efficiently removed stage specific embryonic antigen-1 (SSEA1+) pluripotent stem cells from the NP population. Furthermore, transplantation of sorted cells did not generate tumors, strongly suggesting that this genetic procedure could play an important role in future cell therapy by efficiently removing tumor-forming cells.