Acknowledgments We thank Elisa Ronconi for her assistance in
We thank Elisa Ronconi for her assistance in preparing Fig. 1.
We thank P Romagnani and G Remuzzi for their comment () on our paper (). The goal of our paper was to obtain a cell population enriched with adult renal stem ezh2 pathway exploiting the self-renewal characteristic, through the formation of clonal nephrospheres, adapting the sphere-forming assay to fresh human renal tissue. We avoided the use of markers because they are known to be promiscuous and expressed not only by stem cells but also by progenitors and differentiated cells (). With our approach, we obtained cells with properties of stem cells, as has been previously shown by others that have provided reliable evidence in obtaining stem cells from different human tissues (). After staining with fluorescent PKH26 dye (), the brightest renal cells (PKHhigh) in the nephrospheres were the only ones able to generate new filial nephrospheres. Thus our PKHhigh cells showed self-renewal capacity, with a calculated Sphere Forming Efficiency (SFE) of about 72%, and were also able to differentiate into epithelial, podocytic, and endothelial lineages in vitro. The SFE of fresh human renal tissue cells was 0.69% (see Table S2 in our paper). Therefore, our PKHhigh cells represent a cell population enriched with cells that show characteristics of stem cells. The previous use of CD133 and CD24 to indicate human renal progenitor cells () led us to evaluate their expression in PKHhigh cells, showing that the distribution of these markers was heterogeneous. Romagnani and Remuzzi say that “CD133+/CD24− cells do not exist in vivo,” and our results can be “related to technical differences and/or culture manipulation”. In recent years, with the availability of validated conjugated antibodies, direct staining is widely used, even when coexpression studies are needed (). We obtained the same results on nephrosphere cells using either FITC or PE conjugated CD24 antibodies. Moreover, in our PKHhigh cells the absence or the not detectable low expression of CD24 characterizes a cellular subset that is different from the one in which CD24 is well expressed. This difference is supported also by the different stem cell capacities. In fact, CD133+/CD24−/PKHhigh cells generated new filial nephrospheres and differentiated toward the epithelial, podocytic, and endothelial lineages; CD133+/CD24+/PKHhigh cells generated new filial nephrospheres as well, but differentiated toward epithelium and podocytes only, like the cells described by the Romagnani group (); the CD133−/CD24−/PKHhigh cells were not able to form nephrospheres. Otherwise, in (Fig. 1A), in (Fig. 2A,B), and in (Fig. 1A) the existence of CD133+/CD24− cells cannot be excluded. By FACS analysis, after indirect staining of freshly isolated adult renal cells, it is possible to notice that a low percentage of CD133+/CD24− events have been detected. In addition, (Fig. 3A) show a consistent amount of CD133+/CD24− events by FACS analysis on CD133+ cultured renal cells after direct staining. Of course, we agree that it is important to establish the existence of the putative stem cell population in fresh kidney tissue. However, we found that CD133+/CD24− and CD133+/CD24+ cells were also present in our cell populations that were unable to generate new filial nephrospheres and therefore not capable of self-renewal (PKHlow, PKHneg cells, and differentiated primary cell cultures). This means that the expression of these markers is not related “per se” to the self-renewal and multipotent capacities. Stem cell properties are dispersed among the cells also carrying these markers, but the specific stem cell phenotype is probably something hidden under the PKHhigh status and a transcriptomic analysis of the self-renewing PKHhigh cells may reveal it. We are convinced that the specific signature, identified on a homogeneous cell population with self-renewal and multipotent capacity, has to be obtained to precisely identify stem cells on fresh tissue, overcoming the problems of promiscuity shown by the markers currently available. We are currently trying to obtain this signature on our self-renewing PKHhigh cells and then we will go back to the tissue to identify and localize stem cells in the context of a possible renal stem cell niche on both physiological and pathological fresh renal tissues.