SKPs, corresponding to an increase in cumulative population doublings and a reduction in doubling time (Figure 1D; Figure S1F). G-banding analysis (Methods S1) demonstrated normal karyotype with no cytogenetic abnormalities in both SKPs and KPCs (Figure S1G), suggesting the maintenance of chromosomal stability in these derived stem cells. These results suggest that keloid-derived stem cells maintain a relatively higher proliferative rate than their normal skin counterparts, and this inherent rapid growth appears unrelated to mutational changes observed in malignant tumor. Up to date, efforts have been made to develop a cell-surface antigen profile for better purification and identification of mesenchymal stem cells (MSCs) from different tissue sources [10]. There is a consensus that human MSCs rarely express markers of hematopoietic cells such as CD45 and CD34 [10,64,66]. Several positive markers such as Stro-1, CD73, and CD106 were considered the most useful and relatively specific markers for MSCs [10], but others including CD90/Thy-1 were relatively non-specific, although frequently expressed on MSCs from different tissues [10,64,66]. To characterize stem cell phenotypic markers of KPCs using flow cytometry, we observed that a high percentage of both KPCs and SKPs expressed CD90 at passage 2–6 (Figure 1E). However, only 11% and 46.4% of KPCs expressed CD29 and CD73, respectively, as compared to 77.6% for CD29 and 77.1% for CD73 in SKPs (Figure 1E). Interestingly, the percentage of KPCs expressing Stro-1 (19.1%) and CD105 (4.5%) was consistently higher than that of SKPs (Figure 1E). Similar expression profiles of cell surface molecules were observed in SKPs and KPCs up to 10 passages (data not shown). The differential expression of mesenchymal stem cell marker profiles suggests KPCs are uniquely distinct from SKPs. We next examined several specific stem cell genes expression using quantitative real-time PCR (qPCR). KPCs expressed relatively higher levels of Oct-4, Rex-1, Nanog, and human telomerase reverse transcriptase (hTERT) mRNAs than SKPs (Figure 1F). In addition, comparative analysis of telomerase activity using Telo TAGGG telomerase PCR ELISA showed that KPCs displayed an inherently higher telomerase activity (5–6 folds) as compared to normal skin stem cells (Figure 1G). Unlike cancer, both KPCs and SKPs showed significantly lower level of telomerase activities than cancer cells (Figure 1G), and gradually decreased with increasing cell passages (Figure 1H) suggesting a tighter growth regulation. Taken together, these results suggest that KPCs differ from SKPs in terms of distinct surface marker expression profile, elevated expression of several specific stem cell genes, and increased capability of colony formation and proliferation, thus resembling benign tumor-like properties. Keloid-derived precursor cells are capable of multiple differentiation The multi-differentiation potential of KPCs was determin- ed and compared to that of SKPs. Under adipogenic and osteogenic induction conditions, both SKPs and KPCs could differentiate into adipocytes and osteoblasts as determined by Oil Red O staining (Figure S2A) and by Alizarin Red S staining (Figure S2D), respectively. Adipogenic differentiation was further confirmed by the increased expression of specific adipogenic markers including peroxisome proliferators-activat- ed receptor c2 (PPARc2), aP2 and lipoprotein lipase (LPL) as determined by RT-PCR (Figure S2C). Similarly, the osteogenic induction of KPCs and SKPs was further supported by the increased expression of osteocalcin, an osteogenic marker (Figure S2F). In addition, quantification of the Oil Red O and Alizarin Red S (Figures S2B and S2E) staining using 5 random Niche-Driven Overgrowth of KPC fields as well as RT-PCR results (Figures S2C and S2F) showed a moderate increase in the expression of adipogenic and osteogenic markers in differentiated KPCs compared to differentiated SKPs. However, further evidence is needed to determine whether KPCs have a relatively stronger capability to differentiate toward adipogenesis and osteogenesis as compared to SKPs. Meanwhile, we demonstrated that keloid- derived sphere colonies (Figure S3) were capable of multipotent differentiation into distinct lineages including mesoderm- derived adipocytes, osteoblasts and smooth muscle cell-like cells, and ectoderm-derived different types of neural cells (Figure S3D). These findings consistent with previous stem cell properties described in rodent and mammalian skin-derived precursors or SKPs [54,55,56] indicate that keloid derived stem cells may represent an active form of SKPs. KPCs are capable of regenerating connective tissues after in vivo transplantation To explore the in vivo regenerative capability, the expanded KPCs and SKPs (26106) were subcutaneously transplanted with Gelfoam as a carrier in immunocompromised mice. Similar transplants were carried out using human bone marrow mesenchymal stem cells (hBMMSCs) as another source of stem cells. As expected, hBMMSCs were not capable of regenerating tissue up to 8 weeks (data not shown). However, KPCs and SKPs from different donors consistently regenerated connective tissue-like transplants (5 out of 5 mice), with a more rapidly growing tumor-like mass using KPCs as compared to SKPs (Figure 2A). The human origin of cellular components of the transplants was confirmed by immunostaining with specific antibodies for human mitochondria (Figure 2B). Histological features of the transplants resembled early connective tissue phenotype including presence of type I collagen fibrils, however, the characteristic keloid–like thick collagen bundles were not observed (Figures 2B and 2C). Next, to assess the in vivo bone regenerative capability, KPCs were implanted subcuta- neously with hydroxyapatite/tricalcium phosphate (HA/TCP) as a carrier in immunocompromised mice for 8 weeks. Histological analyses revealed calcified nodules in both KPCs and SKPs generated transplants (Figure 2D). To further confirm that the isolated KPCs and SKPs are truly stem cells, we carried out a series of transplantation using limiting cell density dilution. As shown in Table S4, KPCs exhibited stronger capability for tumor xenograft formation at a minimal density of 16105cells after transplantation for 8 weeks as compared to their counterparts, SKPs. We next performed serial transplantation with hydrogel as a carrier using 26106 KPCs subcutaneously transplanted into immunocompromised mice. At 4 weeks post-primary transplantation, the transplants were harvested and digested single cells were re-transplanted into immunocom- promised mice for another 4 weeks. Our results indicated that KPCs recovered from secondary transplants maintained the in vivo ability to form connective-like tissues (Figure 2E). These results were unique and particularly striking, as KPCs transplanted to a heterologous environment were capable of proliferating and differentiating into scar-like connective tissues expressing type I collagen. However, we were not successful in replicating the human keloid tumor using the current transplant model, suggesting that KPCs are not the sole contributor to the exuberant scar growth. Therefore, we hypothesize that, aside from KPCs, the environmental niche where KPCs reside, is capable to drive stem cell proliferation and differentiation, and therefore regulates the tumor-like keloid growth. PLoS ONE | www.plosone.org 6 November 2009 | Volume 4 | Issue 11 | e7798