Dept of Health Sciences, University of Milano-Bicocca, ItalyAvailable online 8 January 2014We thank P Romagnani and G Remuzzi for their comment(Romagnani and Remuzzi, 2014)onourpaper(Bombelli et al.,2013). The goal of our paper was to obtain a cell populationenriched with adult renal stem cells exploiting theself-renewal characteristic, through the formation of clonalnephrospheres, adapting the sphere-forming assay to freshhuman renal tissue. We avoided the use of markers becausethey are known to be promiscuous and expressed not only bystem cells but also by progenitors and differentiated cells(Sagrinati et al., 2006; Pece et al., 2010). With our approach,we obtained cells with properties of stem cells, as has beenpreviously shown by others that have provided reliableevidence in obtaining stem cells from different human tissues(Reynolds and Weiss, 1992; Dontu et al., 2003 ).After staining with fluorescent PKH26 dye (Pece et al.,2010),thebrightestrenalcells(PKHhigh)inthenephrosphereswerethe only onesabletogeneratenew filial nephrospheres.Thus our PKHhigh cells showed self-renewal capacity, with acalculated Sphere Forming Efficiency (SFE) of about 72%, andwere also able to differentiate into epithelial, podocytic, andendothelial lineages in vitro. The SFE of fresh human renaltissue cells was 0.69% (see Table S2 in our paper). Therefore,our PKHhigh cells represent a cell population enriched withcells that show characteristics of stem cells. The previous useof CD133 and CD24 to indicate human renal progenitor cells(Sagrinati et al., 2006; Bussolati et al., 2005; Lazzeri et al.,2007; Ronconi et al., 2009; Sallustio et al., 2010; Lindgrenet al., 2011; Angelotti et al., 2012; Bussolati et al., 2012)ledus to evaluate their expression in PKHhigh cells, showing thatthe distribution of these markers was heterogeneous.Romagnani and Remuzzi say that “CD133+/CD24− cells donot exist in vivo,” and ourresultscan be “relatedtotechnicaldifferences and/or culture manipulation”.In recent years, withthe availability of validated conjugat-ed antibodies, direct staining is widely used, even whencoexpressionstudiesareneeded(Sallustioetal.,2010;Buzhoret al., 2011; Bussolati et al., 2012; Tanqri et al., 2013). Weobtained the same results on nephrosphere cells usingeitherFITCorPEconjugatedCD24antibodies.Moreover,inour PKHhigh cells the absence or the not detectable lowexpression of CD24 characterizes a cellular subset that isdifferentfromtheoneinwhichCD24iswellexpressed.Thisdifference is supported also by the different stem cellcapacities. In fact, CD133+/CD24−/PKHhigh cells generat-ed new filial nephrospheres and differentiated toward theepithelial, podocytic, and endothelial lineages; CD133+/CD24+/PKHhigh cells generated new filial nephrospheres aswell,butdifferentiatedtowardepitheliumandpodocytesonly,like the cells described by the Romagnani group ( Sagrinatiet al., 2006; Ronconi et al., 2009); the CD133−/CD24−/PKHhighcellswerenotabletoformnephrospheres.Otherwise,in Lazzeri et al. (2007) (Fig.1A),inRonconi et al. (2009)(Fig. 2A,B), and in Angelotti et al. (2012) (Fig. 1A) theexistence of CD133+/CD24− cells cannot be excluded. ByFACS analysis, after indirect staining of freshly isolated adultrenal cells, it is possible to notice that a low percentage ofCD133+/CD24− events have been detected. In addition,Bussolati et al. (2012) (Fig. 3A) show a consistent amount ofCD133+/CD24− events by FACS analysis on CD133+ culturedrenal cells after direct staining.