Hair follicles harbor a heterogeneous regenerative cell pool and represent a putative low-to-non-invasively available source of stem cells. We previously reported a technology for culturing human melanocytes from the hair follicle outer root sheath (ORS) for autologous pigmentation of tissue engineered skin equivalents. This study translated the ORS technology to horses. We de-veloped a culture of equine melanocytes from the ORS (eMORS) from equine forelock hair follicles cultured by means of an analogue human hair follicle-based in vitro methodology. The procedure was adjusted to equine physiology by addition of equine serum to the culture medium. The hair follicles were isolated by macerating forelock skin rests, enzymatically digested and subjected to air-medium-interface cultivation method. The procedure resulted in differentiated equine melanocytes, which exhibited typical morphology, presence of melanosomes, expression of cytoskeleton proteins vimentin, α-SMA, Sox2, S100ß and tyrosinase as well as tyrosinase activity followed by production of melanin. According to all assessed parameters, eMORS could be ranked as partially melanotic melanocytes. The results of the study offer an experimental base for further insight into hair follicle biology in equine and for comparative studies of hair follicles across different species.

Um eine verbesserte Therapieoption mit weniger Schmerzen und Nebenwirkun-gen für Patienten mit Depigmentierungsstörungen wie Vitiligo zu entwickeln, wurde eine Methode zur nichtinvasiven Gewinnung von autologen Melanozyten aus der Haarwurzel genutzt. Die Haarwurzel als einfach zugängliches Stammzell-reservoir bietet die Möglichkeit, Vorläufermelanozyten aus der äußeren Haar-wurzelscheide zu isolieren, differenzieren und zu proliferieren. Für zukünftige autologe Transplantationsversuche wurden in dieser Arbeit die kultivierten hu-manen Melanozyten aus der äußeren Haarwurzelscheide (Human Melanocytes from the Outer Root Sheath, HUMORS) auf drei unterschiedlichen Scaffolds getes-tet. Hinsichtlich mitochondrialer Aktivität (Marker für Zellproliferation), mela-nozytenspezifischer Markerexpression und ihrer Funktionalität (Tyrosinase-Enzymaktivität und Melaningehalt) wurden die Zellen auf Collagen Cell Carrier® (CCC), Poly-ε-Caprolacton-Scaffolds (PCL) und kollagen basierten Hydrogelen (cGEL) kultiviert und charakterisiert. Alle Scaffolds waren biokompatibel, immu-nologisch nur gering aktiv und wiesen eine dreidimensionale Struktur auf, die der extrazellulären Matrix nachempfunden war. Einen positiven Effekt auf die Prolife-ration wiesen die HUMORS auf den Collagen Cell Carrier® auf. Bei Untersuchun-gen der melanotischen Aktivität überzeugten die HUMORS auf dem cGEL Typ4 durch einen signifikant höheren Melaningehalt. Da Melanin das entscheidende Produkt der Repigmentierung bei Vitiligoläsionen ist, stellte sich damit das cGEL Typ4 als vielversprechender Zellträger für die Kultivierung und vorgesehene Transplantation der Melanozyten heraus.

Yes / Background and Objective: Though devices for hair growth based on low levels of light have shown encouraging results, further improvements of their efficacy is impeded by a lack of knowledge on the exact molecular targets that mediate physiological response in skin and hair follicle. The aim of this study was to investigate the expression of selected light-sensitive receptors in the human hair follicle and to study the impact of UV-free blue light on hair growth ex vivo. Material and Methods: The expression of Opsin receptors in human skin and hair follicles has been characterised using RT-qPCR and immunofluorescence approaches. The functional significance of Opsin 3 was assessed by silencing its expression in the hair follicle cells followed by a transcriptomic profiling. Proprietary LED-based devices emitting two discrete visible wavelengths were used to access the effects of selected optical parameters on hair growth ex vivo and outer root sheath cells in vitro. Results: The expression of OPN2 (Rhodopsin) and OPN3 (Panopsin, Encephalopsin) was detected in the distinct compartments of skin and anagen hair follicle. Treatment with 3.2 J/cm2 of blue light with 453 nm central wavelength significantly prolonged anagen phase in hair follicles ex vivo that was correlated with sustained proliferation in the light-treated samples. In contrast, hair follicle treatment with 3.2 J/cm2 of 689 nm light (red light) did not significantly affect hair growth ex vivo. Silencing of OPN3 in the hair follicle outer root sheath cells resulted in the altered expression of genes involved in the control of proliferation and apoptosis, and abrogated stimulatory effects of blue light (3.2 J/cm2; 453 nm) on proliferation in the outer root sheath cells. Conclusions: We provide the first evidence that 1) OPN2 and OPN3 are expressed in human hair follicle, and 2) 453 nm blue light at low radiant exposure exerts a positive effect on hair growth ex vivo, potentially via interaction with OPN3. / This study was supported by the European Marie-Curie Actions Programme, Grant agreement no.: 607886

Um eine verbesserte Therapieoption mit weniger Schmerzen und Nebenwirkun-gen für Patienten mit Depigmentierungsstörungen wie Vitiligo zu entwickeln, wurde eine Methode zur nichtinvasiven Gewinnung von autologen Melanozyten aus der Haarwurzel genutzt. Die Haarwurzel als einfach zugängliches Stammzell-reservoir bietet die Möglichkeit, Vorläufermelanozyten aus der äußeren Haar-wurzelscheide zu isolieren, differenzieren und zu proliferieren. Für zukünftige autologe Transplantationsversuche wurden in dieser Arbeit die kultivierten hu-manen Melanozyten aus der äußeren Haarwurzelscheide (Human Melanocytes from the Outer Root Sheath, HUMORS) auf drei unterschiedlichen Scaffolds getes-tet. Hinsichtlich mitochondrialer Aktivität (Marker für Zellproliferation), mela-nozytenspezifischer Markerexpression und ihrer Funktionalität (Tyrosinase-Enzymaktivität und Melaningehalt) wurden die Zellen auf Collagen Cell Carrier® (CCC), Poly-ε-Caprolacton-Scaffolds (PCL) und kollagen basierten Hydrogelen (cGEL) kultiviert und charakterisiert. Alle Scaffolds waren biokompatibel, immu-nologisch nur gering aktiv und wiesen eine dreidimensionale Struktur auf, die der extrazellulären Matrix nachempfunden war. Einen positiven Effekt auf die Prolife-ration wiesen die HUMORS auf den Collagen Cell Carrier® auf. Bei Untersuchun-gen der melanotischen Aktivität überzeugten die HUMORS auf dem cGEL Typ4 durch einen signifikant höheren Melaningehalt. Da Melanin das entscheidende Produkt der Repigmentierung bei Vitiligoläsionen ist, stellte sich damit das cGEL Typ4 als vielversprechender Zellträger für die Kultivierung und vorgesehene Transplantation der Melanozyten heraus.

Bone transplantation is regarded as the preferred therapy to treat a variety of bone defects. Autologous bone tissue is often lacking at the source, and the mesenchymal stem cells (MSCs) responsible for bone repair mechanisms are extracted by invasive procedures. This study explores the potential of autologous mesenchymal stem cells derived from the hair follicle outer root sheath (MSCORS). We demonstrated that MSCORS have a remarkable capacity to differentiate in vitro towards the osteogenic lineage. Indeed, when combined with a novel gelatin-based hydrogel called Osteogel, they provided additional osteoinductive cues in vitro that may pave the way for future application in bone regeneration. MSCORS were also compared to MSCs from adipose tissue (ADMSC) and bone marrow (BMMSC) in a 3D Osteogel model. We analyzed gel plasticity, cell phenotype, cell viability, and differentiation capacity towards the osteogenic lineage by measuring alkaline phosphatase (ALP) activity, calcium deposition, and specific gene expression. The novel injectable hydrogel filled an irregularly shaped lesion in a porcine wound model displaying high plasticity. MSCORS in Osteogel showed a higher osteo-commitment in terms of calcium deposition and expression dynamics of OCN, BMP2, and PPARG when compared to ADMSC and BMMSC, whilst displaying comparable cell viability and ALP activity. In conclusion, autologous MSCORS combined with our novel gelatin-based hydrogel displayed a high capacity for differentiation towards the osteogenic lineage and are acquired by non-invasive procedures, therefore qualifying as a suitable and expandable novel approach in the field of bone regeneration therapy

Hair follicle outer root sheath (ORS) is a putative source of stem cells with therapeutic capacity. ORS contains several multipotent stem cell populations, primarily in the distal compartment of the bulge region. However, the bulge is routinely obtained using invasive isolation methods, which require human scalp tissue ex vivo. Non-invasive sampling has been standardized by means of the plucking procedure, enabling to reproducibly obtain the mid-ORS part. The mid-ORS shows potential for giving rise to multiple stem cell populations in vitro. To demonstrate the phenotypic features of distal, middle, and proximal ORS parts, gene and protein expression profiles were studied in physically separated portions. The mid-part of the ORS showed a comparable or higher NGFR, nestin/NES, CD34, CD73, CD44, CD133, CK5, PAX3, MITF, and PMEL expression on both protein and gene levels, when compared to the distal ORS part. Distinct subpopulations of cells exhibiting small and round morphology were characterized with flow cytometry as simultaneously expressing CD73/CD271, CD49f/CD105, nestin, and not CK10. Potentially, these distinct subpopulations can give rise to cultured neuroectodermal and mesenchymal stem cell populations in vitro. In conclusion, the mid part of the ORS holds the potential for yielding multiple stem cells, in particular mesenchymal stem cells.