1 STEMORE Co. Ltd., Incheon, South Korea

1. Hypoxia improves hair inductivity of dermal papilla cells via nuclear NAPDH oxidase 4-mediated reactive oxygen species generation 1STEMORE Co. Ltd., Incheon, South Korea 2College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, South Korea M. Zheng1, Y.-J. Jang1, N. Choi2, D.-Y. Kim2, T. W. Han2, J. H. Yeo2, J. Lee2, J.-H. Sung1, 2* British Journal of Dermatology. DOI: 10.111/bjd.17706

2. Corresponding author Jong-Hyuk Sung 1st author Mei Zheng

3. Introduction What’s already known? • Dermal papilla cells (DPCs) play a key role in hair regeneration and morphogenesis but they are difficult to isolate and expand for use in cell therapy. • Tremendous efforts have been made to increase proliferation of DPCs and promote its hair formation ability.

4. Objective • To investigate the mitogenic and hair inductive effects of hypoxia on DPCs. • To examine the underlying mechanism of hypoxia-induced stimulation of DPCs.

5. Methods (1) • Cell culture (#c-12071) • hDPCs (passage 5~9) were obtained from PromoCell (#c-12071) and cultured in Follicle Dermal Papilla Cell Growth Medium (PromoCell, Heidelberg, Germany) with 1% antibiotic-antimycotic (Gibco, CA, USA). • Cells were maintained in a humidified incubator at 37°C under 5% CO2 and balanced N2 (normoxia). • Hypoxic cells were grown in a humidified hypoxia workstation at 37 °C under 1%-20% O2 and 5% CO2 and balanced N2.

6. Methods (2) • Proliferation assay • A cell counting kit-8 or direct counting methods were used to detect cell proliferation. • ROS detection • The ROS-specific fluorescent probe, 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA, Molecular Probes), was used to measure total intracellular ROS levels. • ROS generation was analyzed by flow cytometry (FACS Caliber; Becton Dickinson, Heidelberg, Germany) or fluorescence microscopy to determine the mean fluorescence intensity.

7. Methods (3) • RT-qPCR assay • Total cellular RNA was extracted using a TRIzol reagent (Invitrogen, Carlsbad, NY, USA), followed by reverse transcription using a cDNA synthesis kit (Nanohelix, Madison, WI, USA). • Quantitative real-time PCR (q-RT-PCR) reactions were performed using an Exicycler 96 (Applied Biosystems, Foster City, CA). • Growth factor PCR array • The DPCsʼ growth factor expression profile was analyzed using a Human Transduction PathwayFinder RT2 Profiler PCR Array (PAHS-041ZA, Qiagen, Hilden, Germany).

8. Methods (4) • Anagen induction • Mice were maintained and anesthetized according to the method described by Kim24. • Organ culture • Hair growth activity during organ culture of mouse vibrissa HFs was measured according to the method described by Jindo and Tsuboi25. • Three dimensional (3D) hanging drop culture and Patch assay • An established patch assay was performed 26 was used to determine DPCsʼ hair inductivity under hypoxia.

9. Results (1) • Hypoxia upregulates DPCs᾽ proliferation and delay cell senescence. • Hypoxia enhances the hair inductivity of DPCs.

10. Results (2) • Hypoxia enhances the paracrine effects and alkaline phosphatase of DPCs.

11. Results (3) • Hypoxia induces ROS generation in DPCs and mediate the hypoxic stimulation of DPCs. • Hypoxia induces ROS generation by regulating NOX4.

12. Discussion (1) • Hair transplantation is a common way to treat severe hair loss • However, hair transplantation is based on redistribution of hair follicles without any new follicle formation. • Therefore, many researchers have focused on developing methods of regenerating new hair follicles.

13. Discussion (2) • For example, DPCs have been developed as new drug candidates in phase II clinical trials. • As only a small number of DPCs exist at the bottom of the hair follicle, it takes 6–8 weeks to obtain enough cells to use in therapy. • Moreover, cultured DPCs lose their ability to induce hair follicles after in vitro subculture.

14. Discussion (3) • This study aims at large-scale production of DPCs in vitro and maintaining their hair inductivity in vivo. • This study found that hypoxia significantly increases the proliferation and enhances hair inductivity of DPCs, and also delays cell senescence even at late passage. • Increased ROS which were produced by nuclear NOX4 contributes to DPC stimulation by hypoxia.

15. Discussion (4) • In summary, hypoxia increases DPC proliferation and migration, and growth factor secretion. • NOX4-mediated ROS generation in DPCs’ nuclear region plays a key role in its stimulation by hypoxia. • Therefore, preconditioning DPCs under hypoxia can improve its hair regenerative potential for cell therapy.

16. ConclusionsWhat does this study add? • Hypoxia (2% O2) culture of DPCs increases proliferation, delays senescence, and enhances hair inductivity of DPCs. • Reactive oxygen species (ROS) play a key role in hypoxia-induced stimulation of DPCs.

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