Partial Maintenance and Long-Term Expansion of Murine Skin Epithelial Stem Cells by Wnt-3a In Vitro  Yukiteru Ouji, Shigeaki Ishizaka, Fukumi Nakamura-Uchiyama, Daisuke Okuzaki, Masahide Yoshikawa  Journal of Investigative Dermatology  Volume 135, Issue 6, Pages 1598-1608 (June 2015) DOI: 10.1038/jid.2014.510 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Characterization of CD49f+CD34+ cells isolated from mouse-derived primary skin epithelial cells (MPSECs). (a) The CD49f+CD34+ population was isolated from MPSECs as 0p-EpSCs (blue square). (b, c) 0p-EpSCs were cuboid in shape after attachment and immunopositive for Lhx2. Bar=20 μm. (d, e) 0p-EpSCs began proliferation at 24 hours after plating and formed small colonies composed of tightly packed cuboid cells by day 5. Cells in the center of the colony were strongly positive, whereas those located at the margin were weakly positive or nearly negative for CD34. Bar=20 μm. (f) Reverse transcriptase–PCR analysis of 0p-EpSCs showed expression of undifferentiated epithelial cell markers. Uncropped full-length gel images are presented in Supplementary Figure S2 online. (g) Most of the 0p-EpSCs expressed Lgr4, Lgr5, and Lgr6. (h) 0p-EpSCs induced hair reconstitution following co-transplantation with dermal fibroblasts. Hair pigmentation was likely caused by contamination of melanocytes in the cell fraction of dermal fibroblasts. DAPI, 4,6-diamidino-2-phenylindole. Journal of Investigative Dermatology 2015 135, 1598-1608DOI: (10.1038/jid.2014.510) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Proliferation and maintenance of CD49f+CD34+ cells by Wnt-3a. (a, b) In the presence of Wnt-3a, single 0p-EpSCs proliferated and formed colonies. Asterisks indicate identical areas. (c) Prominent colony formation was observed in the culture with Wnt-3a on day 10. *P<0.05. (d) Cell proliferation was assessed by determining cell number on day 10. *P<0.05. (e–g) FACS (e), reverse transcriptase–PCR (RT-PCR) (f), and immunocytochemistry (g) analyses of cells at the end of the first culture. Uncropped full-length gel images used for RT-PCR analysis are presented in Supplementary Figure S5 online. (h) Maintenance of the CD49f+CD34+ population was dependent on Wnt-3a. (i) Most of the 1p-EpSCs expressed lgr4, lgr5, and lgr6. (j) Canonical Wnt-responsiveness was maintained in 1p-EpSCs. ND, not determined. Journal of Investigative Dermatology 2015 135, 1598-1608DOI: (10.1038/jid.2014.510) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Long-term maintenance of CD49f+CD34+ population by Wnt-3a in sequential cultures. (a) Repeated cultures of CD49f+CD34+ cells. We used the number of passages to express CD49f+CD34+ cells from sequential cultures. (b) CD49f+CD34+ cells comprised approximately 8–10% of all cells. (c) Maintenance of the CD49f+CD34+ population was dependent on Wnt-3a. (d, e) 2p-EpSCs and 15p-EpSCs were cultured in the presence or absence of Wnt-3a for 10 days. Microscopic images obtained on day 10 (d) and the numbers of cells during the cultures (e) are shown. (f) 2p-EpSCs and 15p-EpSCs retained canonical Wnt-responsiveness. *P<0.05. (g) Reverse transcriptase–PCR analysis. Uncropped full-length gel images are presented in Supplementary Figure S7 online. (h) Cytochemistry confirmed the expression of Lhx2 and Keratin15 in cells cultured with Wnt-3a, whereas AE15 and AS were detected in cells cultured without Wnt-3a. MPSEC, mouse-derived primary skin epithelial cell. Journal of Investigative Dermatology 2015 135, 1598-1608DOI: (10.1038/jid.2014.510) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CD49f+CD34+ cells maintained in serial cultures showed sustained hair reconstitution ability. (a) The CD49f+CD34+ population in sequential cultures with Wnt-3a was able to reconstitute the skin. 0p-EpSCs, 1p-EpSCs, 2p-EpSCs, and 15p-EpSCs were transplanted into the skin of BALB/c nude mice with dermal papilla cells, which promoted hair follicle development and hair growth. Representative results from 2p-EpSCs and 15p-EpSCs are shown. Hair pigmentation was likely caused by contamination of melanocytes in the cell fraction of dermal fibroblasts. Bar=100 μm. (b, c) Reconstituted hair follicles were derived from transplanted C3H mouse-derived cells (15p-EpSCs). Images obtained before merging are presented in Supplementary Figure S9 online. Loricrin-, AE13-, and AE15-immunopositive cells were observed in the reconstituted skin. Bar=100 μm. DAPI, 4,6-diamidino-2-phenylindole; H&E, hematoxylin and eosin; MPSEC, mouse-derived primary skin epithelial cell. Journal of Investigative Dermatology 2015 135, 1598-1608DOI: (10.1038/jid.2014.510) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Continuous requirement of Wnt-3a for maintenance of CD49f+CD34+ population. (a, b) Changes in CD34+ population during culture (a) and mRNA expression of Lhz2, K15, and Sox9 on day 5 (b). *P<0.05. (c) Isolation of EpSCs from a Wnt-3a-rich environment resulted in disappearance of CD34+ cells on day 10. *P<0.05. (d) CD34+ cells (15p-EpSCs) and CD34− cells were separately isolated at the end of the 15th culture, and then mRNA expression and protein secretion of Wnt-3a, Dkk-1, and Wif-1 were examined. Uncropped full-length gel images are presented in Supplementary Figure S12 online. *P<0.05. (e) Effects of supernatant samples (conditioned medium, CM) on Wnt-responsiveness of 0p-EpSCs. *P<0.05. (f) Immunocytochemistry at the end of the 15th passage of cells cultured with Wnt-3a. Bar=20 μm. Journal of Investigative Dermatology 2015 135, 1598-1608DOI: (10.1038/jid.2014.510) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Possible mechanisms for retaining epithelial stem cell (EpSC) characteristics in repeated cultures. (a) Wnt-3a suppressed mRNA expressions of Wif-1 and Dkk-1 in cultures of EpSCs. *P<0.05. (b) Attenuation of Dkk-1 activity by anti-Dkk antibody led to a partial restoration of TOPFLASH activity. *P<0.05. (c) Schema of EpSC maintenance in culture in the presence of Wnt-3a. CM, conditioned medium. Journal of Investigative Dermatology 2015 135, 1598-1608DOI: (10.1038/jid.2014.510) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions