Vascularized Dermopapilla 3D scaffold free Spheroids: an advanced micro-physiological system for hair growth research
Podium 50
Presented by: Francesca Rescigno
INTRODUCTION
The hair follicle (HF) is a self-renewing “mini-organ” acting as a biological clock which undergoes to continuous cycles of growth (anagen) regression (catagen) and quiescence state (telogen): these phases have a defined, perpetual unidirectional and highly regulated precise scheme in which a complex and fine-tuned interaction of signals induces deep metabolic and morphological changes.
AIM OF THE WORK
Based on previously developed system (DermoPapilla model) a specialized 3D scaffold free spheroid Vascularized DermoPapilla system was developed by co-colturing primary human hair dermopapilla cells (HHDPC) and microvascular endothelial cells (HMVEC) and then characterized compared to the simplified DermoPapilla model.
METHODS
The DP spheroid were produced with 5000 cells/well and cultivate for a period of 7 and 14 days in a 96 wells plate specific for hanging drop technology according to internal optimized procedure.
Spheroids viability was monitored by ATP realese quantification and the establishment of the co-culture and microvessels formation inside dermal ECM was monitored by immunofluorescence (IF) in Whole mount samples after clarification by Thunder imager for CD31 and/or FN1.
NanoString® analysis was applied to investigate the hair follicle-cycle gene signature and its dynamic progression during the culture time. The high through-put approach was applied using Nanostring nCounter® WNT-Codeset containing 182 genes.
RESULTS
After 5 days of culture, vessels-like structures expressing CD31 were visible as a well-organized network inside the stromal compartment, positive for fibronectin.
Nanostring preliminary results suggest an anagen-like phase between 3 and 5 days of culture, as confirmed by the up-regulation of anagen key promoter genes, such as CCND3, WNT-1, WNT-4, WNT-5A and WNT-10A and the downregulation of DKK-1 and BMP-4, a growth cellular inhibitor and an early anagen activator, respectively. Endothelial cells increased VEGF expression, a key modulator of angiogenesis and vasculogenesis and an anagen promoter: the interaction between stromal dermal papilla compartment and the vascular network increased the biological relevance mirroring the native features of hair follicle tissue. The full analysis of Nanostring nCounter® WNT-Codeset results is still in progress.
CONCLUSION
As advanced in vitro approach to preclinical testing of new ingredients for hair growth the Vascularized DermoPapilla spheroids system seems to have many advantages compared to ex vivo human hair follicle ( difficult availability, poor through-put analysis, high inter - individual variability) or to bi-dimensional monolayer (intrinsic limitation for primary cells due to limited number of passage in culture, cells deeply modify their genotypic profile when cultured in monolayer, simplified structure and poor cell-matrix interactions, poor predictive power) in particular for the presence of: micro vessels, cells-matrix interactions, possibility to follow transcriptional changes.
With respect to other test systems applicable to hair growth R&D studies, thanks to the presence of 3D physiological microenvironment and microvessels, the spheroids organization mimics a miniaturized functional organ providing the guidance for cells orientation, polarization, and multi-compartmentalization thanks to the crosstalk between cells, the 3D microphysiological environment and the presence of microvessels.
The hair follicle (HF) is a self-renewing “mini-organ” acting as a biological clock which undergoes to continuous cycles of growth (anagen) regression (catagen) and quiescence state (telogen): these phases have a defined, perpetual unidirectional and highly regulated precise scheme in which a complex and fine-tuned interaction of signals induces deep metabolic and morphological changes.
AIM OF THE WORK
Based on previously developed system (DermoPapilla model) a specialized 3D scaffold free spheroid Vascularized DermoPapilla system was developed by co-colturing primary human hair dermopapilla cells (HHDPC) and microvascular endothelial cells (HMVEC) and then characterized compared to the simplified DermoPapilla model.
METHODS
The DP spheroid were produced with 5000 cells/well and cultivate for a period of 7 and 14 days in a 96 wells plate specific for hanging drop technology according to internal optimized procedure.
Spheroids viability was monitored by ATP realese quantification and the establishment of the co-culture and microvessels formation inside dermal ECM was monitored by immunofluorescence (IF) in Whole mount samples after clarification by Thunder imager for CD31 and/or FN1.
NanoString® analysis was applied to investigate the hair follicle-cycle gene signature and its dynamic progression during the culture time. The high through-put approach was applied using Nanostring nCounter® WNT-Codeset containing 182 genes.
RESULTS
After 5 days of culture, vessels-like structures expressing CD31 were visible as a well-organized network inside the stromal compartment, positive for fibronectin.
Nanostring preliminary results suggest an anagen-like phase between 3 and 5 days of culture, as confirmed by the up-regulation of anagen key promoter genes, such as CCND3, WNT-1, WNT-4, WNT-5A and WNT-10A and the downregulation of DKK-1 and BMP-4, a growth cellular inhibitor and an early anagen activator, respectively. Endothelial cells increased VEGF expression, a key modulator of angiogenesis and vasculogenesis and an anagen promoter: the interaction between stromal dermal papilla compartment and the vascular network increased the biological relevance mirroring the native features of hair follicle tissue. The full analysis of Nanostring nCounter® WNT-Codeset results is still in progress.
CONCLUSION
As advanced in vitro approach to preclinical testing of new ingredients for hair growth the Vascularized DermoPapilla spheroids system seems to have many advantages compared to ex vivo human hair follicle ( difficult availability, poor through-put analysis, high inter - individual variability) or to bi-dimensional monolayer (intrinsic limitation for primary cells due to limited number of passage in culture, cells deeply modify their genotypic profile when cultured in monolayer, simplified structure and poor cell-matrix interactions, poor predictive power) in particular for the presence of: micro vessels, cells-matrix interactions, possibility to follow transcriptional changes.
With respect to other test systems applicable to hair growth R&D studies, thanks to the presence of 3D physiological microenvironment and microvessels, the spheroids organization mimics a miniaturized functional organ providing the guidance for cells orientation, polarization, and multi-compartmentalization thanks to the crosstalk between cells, the 3D microphysiological environment and the presence of microvessels.