Abstract
The progression in the hair follicle cycle from the telogen to the anagen phase is the key to regulating hair regrowth. Dermal papilla (DP) cells support hair growth and regulate the hair cycle. However, they gradually lose key inductive properties upon culture. DP cells can partially restore their capacity to promote hair regrowth after being subjected to spheroid culture. In this study, results revealed that DP spheroids are effective at inducing the progression of the hair follicle cycle from telogen to anagen compared with just DP cell or minoxidil treatment. Because of the importance of paracrine signaling in this process, secretome and exosomes were isolated from DP cell culture, and their therapeutic efficacies were investigated. We demonstrated that miR-218-5p was notably up-regulated in DP spheroid–derived exosomes. Western blot and immunofluorescence imaging were used to demonstrate that DP spheroid–derived exosomes up-regulated β-catenin, promoting the development of hair follicles.
miR-218-5p plays a crucial role in exosome-mediated hair regrowth
One of the main challenges in miRNA treatment is delivery. The in vitro model does not accurately reflect whether miR-218-5p plays an important role in regulating hair follicle growth when considering the diverse types of cells in the follicles (46). Here, an mmu-miR-218-5p mimic was mixed with polyethylenimine (PEI) solution (in vivo-jetPEI; Polyplus Transfection, Illkirch, France) according to the manufacturer’s instructions. The PEI/negative control, PEI/mimics, or PEI/inhibitors polyplex solution (10 μl per site) was carefully administered, subcutaneously, into the dorsal skin of depilated mice (Fig. 6E). By targeting SFRP2, miR-218-5p mimics promoted hair follicle development, while miR-218-5p inhibitors inhibited the onset of the anagen phase in the hair follicle cycle. We can see notable hair regrowth effects with the treatment of miR-218-5p mimic, as compared with the control group and the miR-218-5p inhibitors. However, these effects (50 to 90% hair coverage) were less potent than those from exosome treatment (95 to 100% hair coverage) on day 20. We reason that this is because exosomes contain a variety of miRNAs and proteins, and miR-218-5p is not the only one (although a very important one) that was able to contribute to promoting hair growth.
We used a different strategy to promote hair growth. As illustrated in Fig. 6G, both miR-218-5p–loaded exosomes and miR-218-5p mimics encapsulated by in vivo PEI promoted the transcript of miR-218-5p. The genetic structure of miR-218-5p showed that it would target SFRP2 directly. To demonstrate the regulatory effect that miR-218-5p and WNT signaling have on the hair growth cycle, we examined transcriptional mediators, SFRP2, and β-catenin by Western blot. Skin samples (day 15) showed that miR-218-5p mimics robustly increased endogenous β-catenin expression, while treatment with miR-218-5p inhibitors showed decreased β-catenin expression (Fig. S10).
Together, these data support the theory that the main therapeutic pathway involves the stimulation of WNT signaling by miR-218-5p–overexpressed exosomes through a down-regulation of SFRP2, a WNT signaling inhibitor, and up-regulation of β-catenin.
Given the temporary efficacy of finasteride and minoxidil, and the limited number of treatments available, the need to discover new therapies for preventing hair loss and enhancing hair regrowth is urgent (47). Alternative solutions are challenging, especially bioproducts.
Replenishing DP cells with in vitro–cultured DP cells is a reasonable approach for driving the telogen-to-anagen transition in the hair follicle cycle. However, DP cells will lose their hair-inducing capacity over time when they are cultured on a flat, plastic surface. Horne and co-workers (48) observed that cultured DP cells (over six passages) could not induce any hair follicles to transition to anagen after implantation in situ. DP cells have to agglomerate in the hair follicle bulb to promote folliculogenesis (49, 50). Osada et al. have shown that DP cells regained their hair follicle inductive capacity after they were cultured in suspension as spheroids (14). In our study, we verified a higher expression of CD133 and β-catenin in DP spheroids compared with DP cells, which means that DP spheroids could exhibit better hair growth inductive capacity in vivo.
Secretome from DP spheroids is also different from that of dissociated cells. Researchers have shown that dermal spheres are morphologically akin to DP cells in the anagen phase with expression profiles different from 2D cells but greatly similar to intact DP cells (51). Evidence of how the transplanted hair follicles affect the environment of the bald area and paracrine effects involved were not sufficient. Our studies demonstrated that DP cells exerted their regulatory capacity on hair cycles mainly through a paracrine mechanism. The expression of β-catenin was up-regulated not only in the injection sites but also in nontreated sites. DP cells release various growth factors and vesicles to regulate follicular biology, and it was established that cultivation in spheroids most efficiently preserves the initial phenotype of these cells in vitro. Rather than introducing more DP cells to accumulate into hair follicles, exosomes were injected to regulate hair follicle cycles. Spheroid-derived exosomes, with a higher level of miR-218-5p compared with 2D DP-XOs, also enhanced the expression of β-catenin and down-regulated SFRP2, which positively regulated hair follicle growth and maintained the anagen phase of the hair cycle.
C57BL/6 mice are useful models for screening agents that promote hair growth because their skin produces pigment only during the anagen phase (52). Human hair loss has hormonal, environmental, and genetic causes. Because of its complexity, cell or secretome therapy may be more advantageous in treating hair loss compared with minoxidil, since the primary therapeutic mechanism of minoxidil is the increase in cutaneous blood flow to the treated site. Increased blood flow causes hyperpolarization of cell membranes, allowing more nutrients to reach the follicles and cells. However, if DP cells remain dormant, little hair regrowth is likely to occur. In this study, we demonstrated that spheroids drive the hair cycle from telogen to anagen in healthy mice. Disease-related models or hormone-related hair loss models are needed to fully demonstrate the advantages of exosome therapy in comparison to minoxidil. Another limitation of this study lies in the interaction of exosomes with follicle cells. We focused on animal studies rather than cell studies because we were not sure which types of follicle cells were being regulated. More cell studies are needed to explore mechanisms in the future.
Collectively, we demonstrated that miR-218-5p–overexpressed exosomes accelerated the onset of anagen, and spheroid culture provided a potential avenue for cell therapy. miR-218-5p regulated hair follicle development by down-regulating WNT signaling inhibitor SFRP2, thus promoting β-catenin, creating a positive feedback loop. Our study may offer the following advantages compared with current hair regeneration practice. For people not suitable for invasive surgical procedures, they can benefit from the administration of the factors and exosomes by a minimally invasive approach, e.g., needle-free injection (23), microneedle patches (53–55), and sprays (56, 57). Compared with the commercially available minoxidil, exosomes represent a natural product with fewer side effects. Overall, studies outlined here may represent new therapeutic strategies for hair treatment.
https://advances.sciencemag.org/content/6/30/eaba1685
The progression in the hair follicle cycle from the telogen to the anagen phase is the key to regulating hair regrowth. Dermal papilla (DP) cells support hair growth and regulate the hair cycle. However, they gradually lose key inductive properties upon culture. DP cells can partially restore their capacity to promote hair regrowth after being subjected to spheroid culture. In this study, results revealed that DP spheroids are effective at inducing the progression of the hair follicle cycle from telogen to anagen compared with just DP cell or minoxidil treatment. Because of the importance of paracrine signaling in this process, secretome and exosomes were isolated from DP cell culture, and their therapeutic efficacies were investigated. We demonstrated that miR-218-5p was notably up-regulated in DP spheroid–derived exosomes. Western blot and immunofluorescence imaging were used to demonstrate that DP spheroid–derived exosomes up-regulated β-catenin, promoting the development of hair follicles.
miR-218-5p plays a crucial role in exosome-mediated hair regrowth
One of the main challenges in miRNA treatment is delivery. The in vitro model does not accurately reflect whether miR-218-5p plays an important role in regulating hair follicle growth when considering the diverse types of cells in the follicles (46). Here, an mmu-miR-218-5p mimic was mixed with polyethylenimine (PEI) solution (in vivo-jetPEI; Polyplus Transfection, Illkirch, France) according to the manufacturer’s instructions. The PEI/negative control, PEI/mimics, or PEI/inhibitors polyplex solution (10 μl per site) was carefully administered, subcutaneously, into the dorsal skin of depilated mice (Fig. 6E). By targeting SFRP2, miR-218-5p mimics promoted hair follicle development, while miR-218-5p inhibitors inhibited the onset of the anagen phase in the hair follicle cycle. We can see notable hair regrowth effects with the treatment of miR-218-5p mimic, as compared with the control group and the miR-218-5p inhibitors. However, these effects (50 to 90% hair coverage) were less potent than those from exosome treatment (95 to 100% hair coverage) on day 20. We reason that this is because exosomes contain a variety of miRNAs and proteins, and miR-218-5p is not the only one (although a very important one) that was able to contribute to promoting hair growth.
We used a different strategy to promote hair growth. As illustrated in Fig. 6G, both miR-218-5p–loaded exosomes and miR-218-5p mimics encapsulated by in vivo PEI promoted the transcript of miR-218-5p. The genetic structure of miR-218-5p showed that it would target SFRP2 directly. To demonstrate the regulatory effect that miR-218-5p and WNT signaling have on the hair growth cycle, we examined transcriptional mediators, SFRP2, and β-catenin by Western blot. Skin samples (day 15) showed that miR-218-5p mimics robustly increased endogenous β-catenin expression, while treatment with miR-218-5p inhibitors showed decreased β-catenin expression (Fig. S10).
Together, these data support the theory that the main therapeutic pathway involves the stimulation of WNT signaling by miR-218-5p–overexpressed exosomes through a down-regulation of SFRP2, a WNT signaling inhibitor, and up-regulation of β-catenin.
Given the temporary efficacy of finasteride and minoxidil, and the limited number of treatments available, the need to discover new therapies for preventing hair loss and enhancing hair regrowth is urgent (47). Alternative solutions are challenging, especially bioproducts.
Replenishing DP cells with in vitro–cultured DP cells is a reasonable approach for driving the telogen-to-anagen transition in the hair follicle cycle. However, DP cells will lose their hair-inducing capacity over time when they are cultured on a flat, plastic surface. Horne and co-workers (48) observed that cultured DP cells (over six passages) could not induce any hair follicles to transition to anagen after implantation in situ. DP cells have to agglomerate in the hair follicle bulb to promote folliculogenesis (49, 50). Osada et al. have shown that DP cells regained their hair follicle inductive capacity after they were cultured in suspension as spheroids (14). In our study, we verified a higher expression of CD133 and β-catenin in DP spheroids compared with DP cells, which means that DP spheroids could exhibit better hair growth inductive capacity in vivo.
Secretome from DP spheroids is also different from that of dissociated cells. Researchers have shown that dermal spheres are morphologically akin to DP cells in the anagen phase with expression profiles different from 2D cells but greatly similar to intact DP cells (51). Evidence of how the transplanted hair follicles affect the environment of the bald area and paracrine effects involved were not sufficient. Our studies demonstrated that DP cells exerted their regulatory capacity on hair cycles mainly through a paracrine mechanism. The expression of β-catenin was up-regulated not only in the injection sites but also in nontreated sites. DP cells release various growth factors and vesicles to regulate follicular biology, and it was established that cultivation in spheroids most efficiently preserves the initial phenotype of these cells in vitro. Rather than introducing more DP cells to accumulate into hair follicles, exosomes were injected to regulate hair follicle cycles. Spheroid-derived exosomes, with a higher level of miR-218-5p compared with 2D DP-XOs, also enhanced the expression of β-catenin and down-regulated SFRP2, which positively regulated hair follicle growth and maintained the anagen phase of the hair cycle.
C57BL/6 mice are useful models for screening agents that promote hair growth because their skin produces pigment only during the anagen phase (52). Human hair loss has hormonal, environmental, and genetic causes. Because of its complexity, cell or secretome therapy may be more advantageous in treating hair loss compared with minoxidil, since the primary therapeutic mechanism of minoxidil is the increase in cutaneous blood flow to the treated site. Increased blood flow causes hyperpolarization of cell membranes, allowing more nutrients to reach the follicles and cells. However, if DP cells remain dormant, little hair regrowth is likely to occur. In this study, we demonstrated that spheroids drive the hair cycle from telogen to anagen in healthy mice. Disease-related models or hormone-related hair loss models are needed to fully demonstrate the advantages of exosome therapy in comparison to minoxidil. Another limitation of this study lies in the interaction of exosomes with follicle cells. We focused on animal studies rather than cell studies because we were not sure which types of follicle cells were being regulated. More cell studies are needed to explore mechanisms in the future.
Collectively, we demonstrated that miR-218-5p–overexpressed exosomes accelerated the onset of anagen, and spheroid culture provided a potential avenue for cell therapy. miR-218-5p regulated hair follicle development by down-regulating WNT signaling inhibitor SFRP2, thus promoting β-catenin, creating a positive feedback loop. Our study may offer the following advantages compared with current hair regeneration practice. For people not suitable for invasive surgical procedures, they can benefit from the administration of the factors and exosomes by a minimally invasive approach, e.g., needle-free injection (23), microneedle patches (53–55), and sprays (56, 57). Compared with the commercially available minoxidil, exosomes represent a natural product with fewer side effects. Overall, studies outlined here may represent new therapeutic strategies for hair treatment.
https://advances.sciencemag.org/content/6/30/eaba1685
