Metformin Promotes the Hair-Inductive Activity of Three-Dimensional Aggregates of Epidermal and Dermal Cells Self-Assembled In Vitro

Introduction: Although it has been reported that the anti-diabetic drug metformin has multiple extra-hypoglycemic activities, such as anti-oxidation, anti-aging and even anti-tumor, topical metformin also can induce hair regeneration, but the precise mechanism involved in that process is still unclear. Objectives: To assess the effect of metformin on hair growth in a mouse hair follicle reconstitution model generated by in vitro self-assembled three-dimensional aggregates of epidermal and dermal cells (3D aggregates). Methods: Epidermal cells and dermal cells were isolated and cultured from the mouse skin of fifty C57BL/6 mouse pups (1-day-old). For tracing the distribution of dermal cells during the self-assembly process of 3D aggregates, the dermal cells were labeled with Vybrant Dil cell-labelling solution and mixed with epidermal cells at 1:1 ratio. Formed 3D aggregates were treated with 10 mM metformin and then were grafted into recipient BALB/c nude mice. The biomarkers (HGF, CD133, ALP, β-catenin and SOX2) associated with the hair-inductive activity of dermal cells were detected in the grafted skin tissues and in cultured 3D aggregates treated with metformin using immunofluorescent staining, quantitative real-time RT-PCR (qRT-PCR), and western blotting. Furthermore, the expression levels of CD133 were also examined in dermal cells with different passage numbers using qRT-PCR and western blotting. Results: Metformin directly stimulates the activity of alkaline phosphatase (ALP) of cultured 3D aggregates, upregulates both the protein and mRNA expression levels of molecular markers (HGF, CD133, ALP, β-catenin and SOX2) and improves the survival rate of reconstituted hair follicles. Moreover, we also found that metformin increases the expression of CD133 in dermal cells thus maintaining their trichogenic capacity that would normally be lost by serial subculture. Conclusions: These results suggest that metformin can promote hair follicle regeneration in vitro through up-regulation of the hair inductive capability of dermal cells, warranting further evaluation in the clinical treatment of male or female pattern hair loss.

Induction of Hair Keratins Expression by an Annurca Apple-Based Nutraceutical Formulation in Human Follicular Cells

Hair disorders may considerably impact the social and psychological well-being of an individual. Recent advances in the understanding the biology of hair have encouraged the research and development of novel and safer natural hair growth agents. In this context, we have previously demonstrated—at both preclinical and clinical level—that an Annurca apple-based dietary supplement (AMS), acting as a nutraceutical, is endowed with an intense hair-inductive activity (trichogenicity), at once increasing hair tropism and keratin content. Herein, in the framework of preclinical investigations, new experiments in primary human models of follicular keratinocytes and dermal papilla cells have been performed to give an insight around AMS biological effects on specific hair keratins expression. As well as confirming the biocompatibility and the antioxidant proprieties of our nutraceutical formulation, we have proven an engagement of trichokeratins production underlying its biological effects on human follicular cells. Annurca apples are particularly rich in oligomeric procyanidins, natural polyphenols belonging to the broader class of bioflavonoids believed to exert many beneficial health effects. To our knowledge, none of the current available remedies for hair loss has hitherto shown to stimulate the production of hair keratins so clearly.

Lmx1b influences correct post-mitotic coding of mesodiencephalic dopaminergic neurons

The Lim Homeobox transcription factor 1 beta (LMX1b) has been identified as one of the transcription factors important for the development of mesodiencephalic dopaminergic (mdDA) neurons. During early development, Lmx1b is essential for induction and maintenance of the Isthmic Organizer (IsO), and genetic ablation results in the disruption of inductive activity from the IsO and loss of properly differentiated mdDA neurons.To study the downstream targets of Lmx1b without affecting the IsO, we generated a conditional model in which Lmx1b was selectively deleted in Pitx3 expressing cells from embryonic day (E)13 onward. Supporting previous data, no significant changes could be observed in general dopamine (DA) marks, like Th, Pitx3 and Vmat2 at E14.5. However, in depth analysis by means of RNA-sequencing revealed that Lmx1b is important for the expression level of survival factors En1 and En2 and for the repression of mdDA subset mark Ahd2 during (late) development. Interestingly, the regulation of Ahd2 by Lmx1b was found to be Pitx3 independent, since Pitx3 levels were not altered in Lmx1b conditional knock-outs (cKO) and Ahd2 expression was also up-regulated in Lmx1b/Pitx3 double mutants compared to Pitx3 mutants. Further analysis of Lmx1b cKOs showed that post-mitotic deletion of Lmx1b additional leads to a loss of TH+ cells at 3 months age both in the VTA and SNc. Remarkably, different cell types were affected in the SNc and the VTA. While TH+AHD2+ cells were lost the SNc, TH+AHD2- neurons were affected in the VTA, reflected by a loss of Cck expression, indicating that Lmx1b is important for the survival of a sub-group of mdDA neurons.