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

Corresponding author Jong-Hyuk Sung 1st author Mei Zheng

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.

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

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.

Methods (2) Proliferation assay ROS detection 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.

Methods (3) RT-qPCR assay Growth factor PCR array 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).

Methods (4) Anagen induction Organ culture 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.

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

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

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

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.

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.  

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.

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.

Conclusions What 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.

Call for correspondence Why not join the debate on this article through our correspondence section? Rapid responses should not exceed 350 words, four references and one figure Further details can be found here