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The capacity to identify and isolate lineage‐specific progenitor cells from developing and mature tissues would enable the development of cell replacement therapies for disease treatment. The enteric nervous system (ENS) regulates important gut functions, including controlling peristaltic muscular contractions, and consists of interconnected ganglia containing neurons and glial cells. Hirschsprung's disease (HSCR), one of the most common and best understood diseases affecting the ENS, is characterized by absence of enteric ganglia from the distal gut due to defects in gut colonization by neural crest progenitor cells and is an excellent candidate for future cell replacement therapies. Our previous microarray experiments identified the neural progenitor and stem cell marker  SRY‐related homoebox transcription factor 2  ( Sox2 ) as expressed in the embryonic ENS. We now show that  Sox2  is expressed in the ENS from embryonic to adult stages and constitutes a novel marker of ENS progenitor cells and their glial cell derivatives. We also show that  Sox2  expression overlaps significantly with SOX10, a well‐established marker of ENS progenitors and enteric glial cells. We have developed a strategy to select cells expressing  Sox2 , by using G418 selection on cultured gut cells derived from  Sox2  β geo /+  mouse embryos, thus allowing substantial enrichment and expansion of neomycin‐resistant  Sox2 ‐expressing cells.  Sox2  β geo   cell cultures are enriched for ENS progenitors. Following transplantation into embryonic mouse gut,  Sox2  β geo   cells migrate, differentiate, and colocalize with the endogenous ENS plexus. Our studies will facilitate development of cell replacement strategies in animal models, critical to develop human cell replacement therapies for HSCR. S TEM  C ELLS  2011;29:128–140

Prospective Identification and Isolation of Enteric Nervous System Progenitors Using Sox2