Stem Cell Reports Repor t CRIPTO/GRP78 Signaling Maintains Fetal and Adult Mammary Stem Cells Ex Vivo Benjamin T. Spike, 1, * Jonathan A. Kelber, 3,4 Evan Booker, 2 Madhuri Kalathur, 2 Rose Rodewald, 1 Julia Lipianskaya, 1 Justin La, 1 Marielle He, 2 Tracy Wright, 3 Richard Klemke, 3 Geoffrey M. Wahl, 1 and Peter C. Gray 2, * 1 Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA 2 Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA 3 Department of Pathology, University of California, San Diego, La Jolla, CA 92037, USA 4 Present address: Department of Biology, California State University, Northridge, CA 91330, USA *Correspondence: bspike@salk.edu (B.T.S.), gray@salk.edu (P.C.G.) http://dx.doi.org/10.1016/j.stemcr.2014.02.010 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). SUMMARY Little is known about the extracellular signaling factors that govern mammary stem cell behavior. Here, we identify CRIPTO and its cell- surface receptor GRP78 as regulators of stem cell behavior in isolated fetal and adult mammary epithelial cells. We develop a CRIPTO antagonist that promotes differentiation and reduces self-renewal of mammary stem cell-enriched populations cultured ex vivo. By contrast, CRIPTO treatment maintains the stem cell phenotype in these cultures and yields colonies with enhanced mammary gland reconstitution capacity. Surface expression of GRP78 marks CRIPTO-responsive, stem cell-enriched fetal and adult mammary epithelial cells, and deletion of GRP78 from adult mammary epithelial cells blocks their mammary gland reconstitution potential. Together, these findings identify the CRIPTO/GRP78 pathway as a developmentally conserved regulator of fetal and adult mammary stem cell behavior ex vivo, with implications for the stem-like cells found in many cancers. INTRODUCTION Somatic stem cells govern the development, mainte- nance, and regeneration of tissues, and their dysregu- lation is associated with diverse pathologies, including cancer. Given the significance of these cells both biologi- cally and therapeutically, it is critical to define factors and signaling mechanisms that dictate their behavior, including those that define niches capable of promoting the stem cell phenotype in normal and disease settings. However, few such factors have been elucidated, and progress toward this goal has been impeded by the fact that most somatic stem cells, including those of the mam- mary gland, are rare and difficult to isolate and propagate ex vivo. The mammary epithelium consists principally of line- age-restricted basal keratin-14-positive (KRT14 + ) myo- epithelial cells and keratin-8-positive (KRT8 + ) luminal epithelial cells (Mikaelian et al., 2006). Although recent reports indicate extensive self-renewal within each of these lineage-committed populations (Van Keymeulen et al., 2011), classic single-cell transplant experiments indicate the presence of rare transplantable bipotent mammary stem cells (MaSCs) in the mature mammary gland (Shackleton et al., 2006). These cells can be signifi- cantly enriched through the use of cell-surface marker combinations such as CD24 and CD49f (Stingl et al., 2006). However, the functional significance of such markers to stem cell biology is often unclear, and the re- sulting enrichment generally remains too low to discern core molecular determinants of the stem cell state from the population at large. In an effort to circumvent these challenges, we recently characterized a highly enriched population of stem cells from murine embryonic mammary rudiments (Spike et al., 2012). The greater purity of these fetal mammary stem cells (fMaSC) relative to their adult counterparts makes them particularly useful in the study of MaSC biology. Interestingly, we found that fMaSCs share gene expression features with certain aggressive human breast cancers that are not shared between enriched populations of adult MaSCs and the same breast cancers. This distinc- tion may reflect intrinsic differences between the fetal and adult MaSCs or differential heterogeneity in the stem cell-enriched populations used for profiling. Alternatively, this observation may be due to critical differences in the tissue contexts from which these cells are derived, a possibility consistent with prior reports indicating an important role for microenvironmental factors in estab- lishing and maintaining the stem cell competence of both fetal and adult mammary cells (Makarem et al., 2013; Spike et al., 2012; Vaillant et al., 2011). However, the ability of specific factors to promote the MaSC pheno- type has rarely been directly demonstrated. CRIPTO (CR-1, TDGF1) is an oncofetal, GPI-anchored/ secreted signaling protein that plays key roles as a stem cell regulator (Adewumi et al., 2007; Bianco et al., 2010; Hough et al., 2009; Miharada et al., 2011). CRIPTO is Stem Cell Reports j Vol. 2 j 427–439 j April 8, 2014 j ª2014 The Authors 427