Identification of ROBO2 as a Useful Cell Surface Marker for Live Human Dermal Papilla Cell Isolation Utilizing a Novel Culture Condition With WNT and FGF Signalling Activation

ABSTRACT The dermal papilla (DP) is essential to hair follicle development and regeneration. Isolation of human DPs still largely depends on manual microdissection and human DP cells (DPCs) lose their intrinsic properties in vitro. Establishing a culture condition that maintains the biological properties of DPCs allows for identification of cell surface markers that enable cell sorting of living human DPCs. A new DPC culture condition was developed using the combination of a WNT activator, CHIR99021, and recombinant FGF9 (CH + F9). Global gene expression profiling and bioinformatic analyses of DPCs grown under this condition were conducted to identify DPC surface markers enabling live DPC isolation. Compared to a conventional culture condition, CH + F9 increased the expression of the representative DPC biomarkers WNT5A , LEF1 and BMP4 by 3.4‐, 3.8‐ and 60.5‐fold ( p < 0.01) and better maintained their expression levels after long‐term serial passaging. Aggregated CH + F9‐treated DPCs unevenly upregulated DP biomarkers further, suggesting that highly potent DPCs can be enriched using a marker for such cells. Bioinformatics analysis of CH + F9‐treated and control DPCs identified cell marker candidates, including roundabout guidance receptor 2 (ROBO2). Importantly, ROBO2 + DPCs expressing the representative DP biomarkers WNT5A , VCAN , NOG were successfully sorted from mixed cell suspensions of keratinocytes, fibroblasts and DPCs mimicking enzymatically dissociated human skin. These findings suggest that the new culture condition and cell surface marker for human DPCs established in this study provide useful tools for drug discovery and regenerative medicine to address hair loss diseases.