In vitro induction of hair follicle signatures using human dermal papilla cells encapsulated in fibrin microgels

Abstract

Cellular spheroids have been described as an appropriate culture system to restore human follicle dermal papilla cells (hFDPc) intrinsic properties; however, they show a low and variable efficiency to promote complete hair follicle formation in in vivo experiments. In this work, a conscientious analysis revealed a 25% cell viability in the surface of the dermal papilla spheroid (DPS) for all culture conditions, questioning whether it is an appropriate culture system for hFDPc. To overcome this problem, we propose the use of human blood plasma for the generation of fibrin microgels (FM) with encapsulated hFDPc to restore its inductive signature, either in the presence or in the absence of blood platelets. FM showed a morphology and extracellular matrix composition similar to the native dermal papilla, including Versican and Collagen IV and increasing cell viability up to 85%. While both systems induce epidermal invaginations expressing hair-specific keratins K14, K15, K71, and K75 in in vitro skin cultures, the number of generated structures increases from 17% to 49% when DPS and FM were used, respectively. These data show the potential of our experimental setting for in vitro hair follicle neogenesis with wild adult hFDPc using FM, being a crucial step in the pursuit of human hair follicle regeneration therapies.

FIGURE 1

Morphometric, viability, and proliferation characterization of dermal papilla spheroids (DPS) and fibrin microgels (FM). (A) Visual inspection under the phase‐contrast microscope of: (i) human follicle dermal papilla cells (hFDPc) suspension, (ii) DPS, (iii) FM just after generation, and (iv) FM 48 h after generation. Scale bar = 200 μm. In (B) morphometric analysis of both DPS and FM for different cell numbers. (C) Size evolution of FM during 9 days after formation. In the x‐axis: culture time, (D₋₂); formation day, 1 day in culture (D₋₁); 2 days in culture (D₀); 5 days in culture (D₃); 7 days in culture (D₅); and 10 days in culture (D₇). Ten samples were analysed to extract morphology values. (D) Cell morphology using CellTracker™ Orange Dye (red fluorescence) inside the FM of different cell numbers after 48 h in culture: (i) 6000 cells; (ii) 3000 cells; (iii) 1500 cells; and (iv) 750 cells. Scale bar = 50 μm. (E) Viability of hFDPc in DPS (i–iv) and FM (v–vii) using Live/Dead® assay at D₀ 6000 cells (i)18.3%‐ (v) 40.2% viability; 3000 cells (ii) 25.1%‐ (vi) 44.5% viability; 1500 cells (iii) 25.4%‐ (vii) 50% viability and 750 cells (iv) 23%‐ (viii) 85% viability. Proliferation, measured as BrdU incorporation, of hFDPc containing 3000 cells at D₀: (ix) 2D culture (12% proliferation); (x) DPS (5% proliferation); (xi) PPP‐FM (16.7% proliferation); and (xii) PRP‐FM (16.7% proliferation). Viability (F) in terms of ATP at D₀ of hFDPc for DPS and FM. (G) Size evolution of DPS during 5 days in culture after formation culture. Proliferation (H) in terms of ATP for FM and DPS containing 3000 cells. ATP concentration was normalized in terms of cell number. D₀ was established 48 h after FM generation. In blue, cell nuclei stained with DAPI, in pink, BrdU‐positive cells. Three samples were analysed to extract cell viability and proliferation. Scale bar = 150 μm. Statistical significance: *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 2

Protein expression characterization in dermal papilla spheroids (DPS) and fibrin microgels (FM). In red, immunofluorescence of human follicle dermal papilla cells (hFDPc) at D₀ in 2D (A–C); DPS (D–H); and FM (I–L) containing 3000 cells for ALP (A, E, I); Versican (B, F, J); αSMA (C, G, K), and Col IV (D, H, L). In blue, cell nuclei stained with DAPI. D₀ was established 48 h after FM generation. White arrows point to cell protein expression in a magnified view. Immunofluorescence of hFDPc inside FM containing 3000 cells at day D₁₅ for (M) Versican and (N) Col IV. Merge images for (O) PPP‐FM and (P) PRP‐FM: in red, Versican, in green, Col IV. In blue, cell nuclei stained with DAPI. D₁₅ was established 17 days after FM generation. Scale bar = 100 μm. Gene expression analysis of genes associated with hair follicle induction using RT‐qPCR (Q). Data were normalized to levels of the reference gene YWHAZ. Error bars represent the standard deviation calculated from two independent experiments for each condition.

FIGURE 3

Epidermal invaginations expressing hair follicle signatures. Structures induced in organotypic skin cultures by dermal papilla spheroids after 3 weeks in culture (A–I): epidermal invagination structure (A, D, E,) expressing hair follicle specific keratins K15/K75 (B) and K14/K71 (C, G, H); induction of keratinocyte intussusception (F) expressing (I) K14/K71 hair follicle specific keratins. Structures induced in organotypic skin cultures by fibrin microgels (FM) after 3 weeks in culture (J–O): tubular structures (J, M) expressing hair follicle specific keratins K14/K71 (K, L, N, O). White and black arrows point to the FM in the dermal compartment of the construct. In blue, nuclei are stained with DAPI. Scale bar = 200 μm.