In vivo monitoring of survival and proliferation of hair stem cells in hair follicle regeneration animal model

Objectives The purpose of this study is to investigate in vivo trafficking of hair follicle stem cells (Newborn mouse Fibroblasts, NF cells) expressing enhanced firefly luciferase (effluc) using non-invasive bioluminescence imaging.

Methods NF cells expressing effluc (NF-effluc) were established by infected with a retrovirus expressing the retrovirus effluc reporter gene driven by CMV promoter and Thy1.1 linked with the IRES2 sequences. mRNA expression of effluc was evaluated by RT-PCR analysis. The function of effluc gene was verified by luciferase assay. We performed transplantation of the NF-effluc with primary keratinocytes to the flank of nude mice. Non-invasive bioluminescence imaging with D-luciferin was performed on day 1, 3, 7, 14 and 21 after the transplantation. In addition, hair follicle regeneration by the implanted cells was investigated using immunohistochemistry and microscopy after skin excision.

Results Expression of effluc was confirmed by RT-PCR with the NF-effluc. The luciferase activity of NF-effluc showed 41,175-fold higher compared to untransfected NF cells. Bioluminescence signal from the transplantation site decreased gradually over 2 weeks, after then, the signal plateaued (day 1: 1.1 x 107 ± 1.3 x 106 P/cm2/s/sr, day 3: 4.7 x 106 ± 7.3 x 105 P/cm2/s/sr, day 7: 2.7 x 106 ± 5.9 x 105 P/cm2/s/sr, day 14: 1.1 x 106 ± 4.1 x 105 P/cm2/s/sr, day 21: 6.6 x 105 ± 7.8 x 104 P/cm2/s/sr) in the hair follicle generation animal model. Hair follicle regeneration was confirmed at the implantation site of NF-effluc. The expression of Thy 1.1 in hair follicle-like structure was demonstrated by immunohistochemistry on day 14 after transplantation.

Conclusions We have established NF expressing bioluminescence reporter gene. Hair generation by the hair follicle formation can be feasibly and non-invasively monitored with the optical molecular imaging strategy in in vivo animal model