**3. Dermal papilla cells and their inductive properties**

have a deep insight into cellular and molecular mechanisms of HF development and regeneration. The research on HF is a rapidly developing area of skin biology. This mini-organ can be successfully used for a wide range of studies into the mechanisms of morphogenesis, stem cell behavior, cell differentiation, and apoptosis [2–4]. Moreover, as mentioned above, HF investigations can provide invaluable insights into the possible causes of human hair disor-

HF is a skin appendage with very complex structure undergoing lifelong periods of morphogenesis. The major part of HF is produced by the epithelium. Highly proliferative matrix cells in HF bulb gradually move upwards in the course of differentiation. They are progenitors for the inner and outer root sheaths and hair shaft later on. Mesenchymal portion of HF includes dermal papilla (DP) and HF connective tissue sheath. DP is located inside the bulb and is separated from the matrix cells by a basement membrane. In a lower part of the bulb, DP contacts with the dermal sheath that surrounds the entire follicle. This structure is maintained throughout the hair-growth phase – anagen. With time, when HF transits into the destructive phase–catagen–the lower two-thirds of the HF epithelial strand degenerate and the hair shaft production stops. DP also becomes smaller but its losses are much less significant and are mainly caused by reduced content of an extracellular matrix [5] and migration of cells between the DP and the adjacent dermal sheath [6]. During catagen, HF is reduced to a tiny epidermal strand surrounded by a basement membrane. As it retracts, the DP is pulled

upward to the upper permanent portion of the HF containing bulge stem cells [7].

Then HF enters the resting phase – telogen. The transition from telogen to anagen is the beginning of a new HF cycle, a process that continues throughout life. In mice, the lengths of anagen and catagen phases are similar from one cycle to the next, while each telogen becomes longer than the previous one. This results in progressive asynchrony in HF cycling with age [8].

Now it is quite clear that the regeneration cycle is maintained by activity of HF stem cells. HF is considered to be a valuable source of adult stem cells (SCs) with morphogenetic potential. SCs may be isolated from epidermal and mesenchymal compartments of HF. Additionally neural crest-derived cells are found in HF, at least in the facial skin [9, 10]. Matrix cells had been thought to be stem cells for a long time as they proliferate very intensively at the beginning of the growth phase. However in experiments with murine HFs, Cotsarelis and co-authors discovered special population of cells at the bottom of a permanent portion of the HF known as the bulge [11]. These cells retained thymidine DNA label after 4 weeks of chase period unlike matrix cells which lost the label as early as 1 week after cessation of labeling. From these pioneering experiments, the bulge has been proved in many studies to be the main reservoir of SCs in HF. It contains morphologically undifferentiated and slow-cycling under the normal conditions of cells. The bulge is a swelling and contiguous part of outer root sheath. As many HFs lack anatomically well-defined bulge region, the term 'bulge' is often referred loosely to the permanent region of the follicle below the sebaceous gland [12, 13]. The bulge region is also the point of attachment of the arrector pilorum muscles [11]. SCs of melanocyte lineage

ders and provide conditions for development of HF restoration technologies.

**2. Hair follicle: structure, cycling and stem cells**

48 Hair and Scalp Disorders

Dermal papilla cells represent mesenchymal cell subpopulation with stem properties. According to their specific features, they may be attributed to classical fibroblast-like cells with special functions. At the same time, DP cells meet stem cell criteria. In a number of works, including those coming from our lab, it was demonstrated that DP cells, according to their characteristics, may be attributed to mesenchymal stem cells along with those derived from bone marrow and adipose tissue [23–26]. These characteristics are widely accepted criteria for mesenchymal stem cells including fibroblastic morphology, ability to adhere and to differentiate into osteogenic, chondrogenic, and adipogenic lineages [24, 27, 28]. It should be mentioned that DP differentiation abilities are often pronounced not to the same extent as those of classical mesenchymal stem cells [23]. As other lineages of differentiation were demonstrated some authors believe DP cells to be multipotent [28]. In laboratory animals, it was demonstrated that this type of cells is able to incorporate into skin structures by grafting [29] and stimulate hair growth and angiogenesis. As it was found in detail during the last 20 years, the ability to induce and regulate HF morphogenesis has been considered to be the main characteristic and core biological function of DP cells. They are indispensable component for embryonic development of HF and postnatal cycling [30]. They serve as the niche for providing signals to matrix progenitors in specifying the size, shape, and pigmentation of hair fiber [5, 31]. Multiple pathways and molecules regulating epithelio-mesenchymal interactions were discovered [32]. Using double reporter Lef1-RFP/K14-H2BGFP mice, Rendl with co-authors discovered detailed genetic signature of isolated mouse DP cells [33]. In ontogenesis, DP first appears as cell condensates on the dermis. As HF develops, epidermal cells proliferate actively and envelope the dermal condensates [34]. Exposed to these new niche conditions, DP cells acquire the expression of BMP-4, its inhibitor noggin, and the surface markers N-CAM and p-75. Additionally, they secret specific extracellular matrix protein versican and show a high level of alkaline phosphatase activity.

Inducing capacity of DP cells, i.e., their ability to induce HF development in embryogenesis and regulate postnatal HF cycling, is the most interesting and intriguing trait of these cells. It is noteworthy that not only DP but also skin dermis on the whole has an ability to regulate differentiation of integumental epithelia. As an example, keratinocytes of the palmoplantar thick skin exclusively express keratin 9 [35]. Recombination of the epidermis from different body sites with palmoplantar dermis caused onset of keratin 9 expression and differentiation of keratinocytes into thick skin [36]. Later it was shown that paleness and thickness of the palmoplantar skin is determined by Wnt signaling. Fibroblasts present in thick skin secrete Wnt inhibitor Dikkopf1, which causes thickening of the epidermis and decreases pigmentation both *in vivo* and *in vitro* [37]. HFs and, thus, DPs are distributed over the entire surface of the mammal body. In spite of different embryological origin of DPs from different sites of the body, their functional properties are quite similar. Analysis of global gene expression using microarrays demonstrated a very high degree of similarity between facial and trunk dermal hair-associated cells indicating phenotypic convergence within HF niche [38].

 DP cells have been quite profoundly investigated but their isolation and long-term cultivation is not a trivial task yet. They quickly lose intrinsic biological characteristics, especially hair inductive capacity, with passaging [39–41]. This process correlates with decrease in expression of DP markers including alkaline phosphatase, versican, Wnt5a, and some others [42–45].

A number of studies reported various approaches to maintain innate properties in cultured DP cells. Earlier approach implied cocultivation with keratinocytes or addition of keratinocyte-derived factors [46, 47] based on close interaction of DP cells with keratinocytes in their natural niche. Another group of studies considers specific signaling pathways participating in hair growth activation and epithelial-mesenchymal interactions. Wnt and BMP signaling were shown to play a key role in HF morphogenesis [18, 44, 48, 49]. Wnt proteins demonstrated high effectiveness in respect of DP maintenance [50, 51]. Shimizu and Morgan found that Wnt 3a can maintain the hair-inductive properties of DP cells when they are cultured *in vitro* [49]. The medium containing recombinant Wnt-10b protein promoted the proliferation of DP cells, which successfully maintained their ability to induce HFs up to at least 10 serial passages [52]. Noteworthy, Wnt10b has been shown to be expressed in developing HFs, with the earliest and most marked localization in placodes [53] while Wnt10b-producing cells promoted hair folliculogenesis [54]. Canonical Wnt pathway in cultured DP cells was shown to be modulated by several compounds: ciprofloxacin [55], valproic acid [56, 57], and glycogen synthase kinase (GSK)-3 –inhibitors [43, 58]. Addition of vitamin D3 to culture medium upregulated expression of Wnt10b and TGF-β2 in murine DP cell providing significantly enhanced hair growth in hemivascularized sandwich assay [59].

Members of TGFbeta signaling pathway, Bmp 4 and Bmp 6 were used successfully to maintain specific characteristics of DP cells in culture [44, 48]. EGF and VEGF demonstrated stimulation of DP proliferation [60, 61]. However, these factors failed to maintain specific DP markers (unpublished data). It is not surprising as cell proliferation may necessarily correlate with hair inductive activity. On the other hand, combination of FGF and PDGF-AA promoted DP growth in culture and increased *de novo* HF induction in chamber assay using treated DP cells [62].

In ontogenesis, DP first appears as cell condensates on the dermis. As HF develops, epidermal cells proliferate actively and envelope the dermal condensates [34]. Exposed to these new niche conditions, DP cells acquire the expression of BMP-4, its inhibitor noggin, and the surface markers N-CAM and p-75. Additionally, they secret specific extracellular matrix protein

Inducing capacity of DP cells, i.e., their ability to induce HF development in embryogenesis and regulate postnatal HF cycling, is the most interesting and intriguing trait of these cells. It is noteworthy that not only DP but also skin dermis on the whole has an ability to regulate differentiation of integumental epithelia. As an example, keratinocytes of the palmoplantar thick skin exclusively express keratin 9 [35]. Recombination of the epidermis from different body sites with palmoplantar dermis caused onset of keratin 9 expression and differentiation of keratinocytes into thick skin [36]. Later it was shown that paleness and thickness of the palmoplantar skin is determined by Wnt signaling. Fibroblasts present in thick skin secrete Wnt inhibitor Dikkopf1, which causes thickening of the epidermis and decreases pigmentation both *in vivo* and *in vitro* [37]. HFs and, thus, DPs are distributed over the entire surface of the mammal body. In spite of different embryological origin of DPs from different sites of the body, their functional properties are quite similar. Analysis of global gene expression using microarrays demonstrated a very high degree of similarity between facial and trunk dermal

versican and show a high level of alkaline phosphatase activity.

50 Hair and Scalp Disorders

hair-associated cells indicating phenotypic convergence within HF niche [38].

significantly enhanced hair growth in hemivascularized sandwich assay [59].

Members of TGFbeta signaling pathway, Bmp 4 and Bmp 6 were used successfully to maintain specific characteristics of DP cells in culture [44, 48]. EGF and VEGF demonstrated stim-

 DP cells have been quite profoundly investigated but their isolation and long-term cultivation is not a trivial task yet. They quickly lose intrinsic biological characteristics, especially hair inductive capacity, with passaging [39–41]. This process correlates with decrease in expression of DP markers including alkaline phosphatase, versican, Wnt5a, and some others [42–45]. A number of studies reported various approaches to maintain innate properties in cultured DP cells. Earlier approach implied cocultivation with keratinocytes or addition of keratinocyte-derived factors [46, 47] based on close interaction of DP cells with keratinocytes in their natural niche. Another group of studies considers specific signaling pathways participating in hair growth activation and epithelial-mesenchymal interactions. Wnt and BMP signaling were shown to play a key role in HF morphogenesis [18, 44, 48, 49]. Wnt proteins demonstrated high effectiveness in respect of DP maintenance [50, 51]. Shimizu and Morgan found that Wnt 3a can maintain the hair-inductive properties of DP cells when they are cultured *in vitro* [49]. The medium containing recombinant Wnt-10b protein promoted the proliferation of DP cells, which successfully maintained their ability to induce HFs up to at least 10 serial passages [52]. Noteworthy, Wnt10b has been shown to be expressed in developing HFs, with the earliest and most marked localization in placodes [53] while Wnt10b-producing cells promoted hair folliculogenesis [54]. Canonical Wnt pathway in cultured DP cells was shown to be modulated by several compounds: ciprofloxacin [55], valproic acid [56, 57], and glycogen synthase kinase (GSK)-3 –inhibitors [43, 58]. Addition of vitamin D3 to culture medium upregulated expression of Wnt10b and TGF-β2 in murine DP cell providing  Ohyama and co-authors studied molecular signature of freshly dissected human DP cells and found gene expression profiles that distinguish intact human DP from conventionally cultured human DP cells and fibroblasts. Because the bioinformatics analysis performed by the authors implied the involvement of Wnt, BMP, and FGF signaling pathways in the maintenance of specific DP properties they used the mixture of recombinant proteins and small molecules for stimulation of BMP, FGF, and Wnt pathways, respectively. This approach allowed them to maintain or even restore innate DP gene expression profile and trichogenic properties demonstrated in an *in vivo* hair induction assay [43].

Recently it was shown that systemically administered pharmacological inhibitors of Janus kinase (JAK) family of protein tyrosine kinases, as downstream effectors of the IFN-γ and γc cytokine receptors, prevented the development of alopecia areata in a mouse model reducing the accumulation of effector T cells in the skin [63]. During the course of the study, the authors noticed unexpected regrowth of HFs after topical treatment with JAK-STAT inhibitors. They checked it in a separate study and were able to demonstrate direct stimulation of HFs growth both in mice, and the human xenografts and HF organ culture model [64]. Moreover, treatment of human DP spheres with the inhibitor of JAK1/3 signaling tofacitinib enhanced inductivity of human DP cells grown in spheres significantly which resulted in larger and significantly greater numbers of HFs obtained in the patch assay.

Another way to get closer to native DPs is to cultivate DP cells in spheres. It has been noticed long ago that DP cells demonstrate aggregative behavior in culture [65]. This may be readily used for creation of three-dimensional (3D) environment by hanging drop or non-adherent biomaterial culture systems. This approach can partially recover expression of core markers in human DP cultures [66]. Cells in spheres stop to proliferate and establish multiple cellcell contacts that may enhance Wnt signaling. They returned to a more native state judged by alpha-smooth muscle actin expression. It is noteworthy that not all strains demonstrated this behavior indicating large differences between cultures derived from different donors. Nevertheless, many studies with DP cells have been conducted recently using 3D cultivation [31, 64, 67, 68]. They showed prolongation of specific markers expression and enhancement of hair inductive capacity after preliminary DP cell aggregation [42, 43, 66].

Taking into account complex natural HF niche it seems quite reasonable to introduce elements of this niche into DP culture systems. Huang with co-authors [68] combined DP with SCs derived from the adipose tissue (ASC) which normally surrounds HF and is shown to influence HF cycling presumably via PDGF signaling [69]. It was found that core-shell patterning of combined spheres with DP cells inside and ASC outside had a beneficial effect on DP markers expression (Hey 1 and Versican) and the rate of hair formation in *in vivo* patch assay. Mature adipocytes incorporated into the same type of spheres had no impact on these processes. A simple mixture of cells within spheres without the formation of core-shell structures yielded much worse results [68]. The authors assume that the mixture of ASCs and DPs in simple mixed spheres interrupted the direct cell-cell interactions and association in DP cells or diluted the signals from ASCs to the DP sphere. It was reported that extracts and conditioned medium from neural stem cells were able to stimulate keratinocyte growth and enhanced hair growth compared to minoxidil [70].

Further search for suitable factors and conditions for effective cultivation and propagation of DP cells will allow one to elucidate mechanism of their self-maintenance and develop largescale culture technologies.
