**4. Hair follicle reconstruction**

Regeneration ability of organs and even tissues is significantly limited in humans. At the same time, loss of teeth, hair, and even mammary glands due to different reasons is quite abundant. These organs are comparable by their ontogenetic epithelial-mesenchymal origin i.e., like almost all organs in the body, they arise from organ germs which undergo subsequent stages of reciprocal epithelial-mesenchymal crosstalk and close interactions. In adult human body, reproduction of these interactions in the right order and availability of germ-initiating cells to regenerate the entire architecture of multicomponent tissues and organs seems to be impossible or at least dramatically impeded. Development of novel technologies proposes a new hope for people. Tissue engineering is a promising approach to replace the lost tissues and organs. However, the problem how to obtain complex structures which can imitate an organ or develop the organ after grafting is far from solution. In many cases, practical technologies imply transplantation of specialized cells in suspension or sheets. More complicated structures are much more difficult to grow.

Reconstruction of folliculogenesis *in vitro* has been in the minds of scientists for a long period of time taking into account vast knowledge and impressive progress in skin biology beginning from the pioneering works on cultivation of epidermal cells [71] and the epidermis being one of the first tissues that has been reconstructed using tissue engineering [72]. Skin comprises epithelial and mesenchymal components which form body coverage and multiple skin appendages like HF and different types of glands. Since it became possible to reconstruct and reconstitute damaged skin, the problem of HF and gland reconstruction emerged for proper functioning of skin grafts as well as for fundamental studies. It has become especially attractive because a new direction of tissue engineering and regenerative medicine has been developed which utilized the ability of pluripotent cells to self-organize in culture into organ-like structures reproducing functional activity of the corresponding organ [73]. However postnatal cells represent quite a different story.

During recent decades, HF biology was profoundly studied. As mentioned above, HF in mammals is the only organ which in normal physiological condition undergoes degeneration (catagen) to small aggregates of resting cells (telogen) with subsequent full regeneration leading to complete restoration of multicellular organ generating corneal shaft (anagen). Multiple mechanisms regulating organogenesis and physiological regeneration of HFs are found, the cellular structure of HF is studied in detail, properties and functions of cell subpopulations are elucidated [74–78].

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

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 large-

Regeneration ability of organs and even tissues is significantly limited in humans. At the same time, loss of teeth, hair, and even mammary glands due to different reasons is quite abundant. These organs are comparable by their ontogenetic epithelial-mesenchymal origin i.e., like almost all organs in the body, they arise from organ germs which undergo subsequent stages of reciprocal epithelial-mesenchymal crosstalk and close interactions. In adult human body, reproduction of these interactions in the right order and availability of germ-initiating cells to regenerate the entire architecture of multicomponent tissues and organs seems to be impossible or at least dramatically impeded. Development of novel technologies proposes a new hope for people. Tissue engineering is a promising approach to replace the lost tissues and organs. However, the problem how to obtain complex structures which can imitate an organ or develop the organ after grafting is far from solution. In many cases, practical technologies imply transplantation of specialized cells in suspension or sheets. More complicated

Reconstruction of folliculogenesis *in vitro* has been in the minds of scientists for a long period of time taking into account vast knowledge and impressive progress in skin biology beginning from the pioneering works on cultivation of epidermal cells [71] and the epidermis being one of the first tissues that has been reconstructed using tissue engineering [72]. Skin comprises epithelial and mesenchymal components which form body coverage and multiple skin appendages like HF and different types of glands. Since it became possible to reconstruct and reconstitute damaged skin, the problem of HF and gland reconstruction emerged for proper functioning of skin grafts as well as for fundamental studies. It has become especially attractive because a new direction of tissue engineering and regenerative medicine has been developed which utilized the ability of pluripotent cells to self-organize in culture into organ-like structures reproducing functional activity of the corresponding organ [73]. However postna-

During recent decades, HF biology was profoundly studied. As mentioned above, HF in mammals is the only organ which in normal physiological condition undergoes degeneration (catagen) to small aggregates of resting cells (telogen) with subsequent full regeneration leading to complete restoration of multicellular organ generating corneal shaft (anagen). Multiple mechanisms regulating organogenesis and physiological regeneration of HFs are found, the

enhanced hair growth compared to minoxidil [70].

scale culture technologies.

52 Hair and Scalp Disorders

**4. Hair follicle reconstruction**

structures are much more difficult to grow.

tal cells represent quite a different story.

Numerous studies utilized the ability of epithelial and mesenchymal cells of different organs to produce mutual influence even in culture conditions in attempts to reproduce morphogenetic processes. After both compartments were determined in HF, this approach was applied to reconstruct folliculogenesis *in vitro*. It was proved that DP cells affect interfollicular keratinocytes in many aspects. Coculturing of keratinocytes and DP cells in Transwell system stimulated expression of follicular markers in keratinocytes [79]. Direct contact between cells of different types enhances this interaction [80].

Due to specific inducing properties of DP cells morphogenesis of HF may be reproduced in culture. While keratinocytes possess intrinsic ability to form structures resembling different stages of HF morphogenesis, HF mesenchyme regulates and intensifies this process. DP-conditioned medium stimulates morphogenesis in keratinocyte culture demonstrating high rate of tube formation in the collagen gel [81]. However, production of fully functional HFs from postnatal cells is still challenging.

Scientists tried to improve effectiveness of HF morphogenesis using more potent cells including keratinocytes from HFs or embryonic cells. As it can be predicted, embryonic tissues demonstrate higher potential to develop organs after inoculation into culture. Significant portion of studies were performed using embryonic tissues in order to avoid roadblocks of postnatal conditions. Mouse embryonic skin explants may successfully develop HFs in culture [82]. Dissociated cells from embryonic tissues aggregate and form the organ germs which can develop into mature primordium and then into a functioning organ. Such results were demonstrated for tooth and hair germs. Buds from dissociated skin of murine embryos developed into mature HFs in culture or after transplantation to immunocompromised animals [83–85]. However, it is quite difficult to reproduce this type of experiments using postnatal HF cells. Long-term cultivation of HFs from postnatal skin is usually completed with gradual degradation of the structure and degeneration of HF. Even in optimized conditions, HFs begin to degenerate after 20 days in culture, on average, while apoptotic cells appear approximately on day 5 [86]. HFs maintained *in vitro* are unable to keep cycling [87, 88]. In the study on rat HFs cultured on gelfoam supports, Philpott and Kealey were able to demonstrate signs of cycling but all follicles appeared to remain blocked in pro-anagen [89]. Non-follicular keratinocytes failed to reconstruct HFs in combination with DP cells but the latter improved significantly the quality of engineered skin grafts applied onto acute skin defects in nude mice [90]. Human DP cells used in skin equivalent together with epidermal cells enriched with HF keratinocytes could generate HF bud 14 days after transplantation into nude mice but further development was impeded [91]. Interestingly, the authors also noticed a quality improvement in composite skin substitutes containing DP cells as compared to dermal fibroblasts, these substitutes more accurately mimicked a well-ordered epithelium. More impressive results in terms of folliculogenesis were reported in xenogenic equivalents combining human foreskin keratinocytes and murine dermal cells [92, 93]. Six weeks after grafting, the authors recorded bulbous pegs and HFs, which however lacked sebaceous glands and were not able to erupt through the epidermal surface. These experiments declare the development of skin substitutes with a high degree of homology to native skin as a long-term objective. At the same time, such an approach may be a useful tool to elaborate an effective technique for HF production. To overcome problems with HF eruption through the epithelial sheet and prevent epithelial cyst formation, a nylon thread was used as a guide for the infundibulum direction via insertion into the bioengineered germ [94]. This method showed perfect results in terms of the shaft formation after transplantation onto nude mice. Both mouse and human HF germs were reconstructed in the study. Human bioengineered HF germ was composed of the bulge region-derived epithelial cells and scalp HF-derived intact DPs of an androgenetic alopecia patient. These germs developed pigmented hair shafts within 21 days after intracutaneous transplantation into the back skin of nude mice [94].

Inamatsu with colleagues compared the process of neofolliculogenesis after intracutaneous transplantation of postnatal DP cells and embryonic dermal condensate in mice [95]. They showed that the dermal condensate-triggered development of HFs is similar to that in embryogenesis. Postnatal DP induced formation of new follicles by a different way, it induces the onset of the anagen-like stage without embryonic-like development.

Nevertheless, a number of studies have demonstrated successful reconstruction of folliclelike structures from cells cultivated *in vitro*. They are focused on development of these structures into HF after transplantation as it was discussed above. This is achieved by two ways: skin equivalents with cells capable of hair follicle induction or aggregates made of keratinocytes and trichogenic mesenchyme with subsequent transplantation. Zheng and co-authors [96, 97] used DP and keratinocyte suspension to inject into mice. It was shown that keratinocytes aggregated first, DP cells stimulated their proliferation enlarging hair follicle primordium and then the shaft began to grow into the cavity which had been formed in the aggregate. It was found that the way of cell combination affects epithelio-mesenchymal interactions. In mixed culture, aggregates were smaller; keratinocytes proliferated better and escaped apoptosis [98]. In the study by Havlickova with co-authors, human keratinocytes of the outer root sheath and DP were placed into specially constructed pseudodermis comprising collagen matrix and dermal fibroblasts. The authors found that cells preserved viability, expressed specific markers, and supported proliferation. They were also able to produce specific reaction on substances stimulating or inhibiting hair growth [99]. However, the appropriate architecture of hair follicle bud was lost. Thus, authors supposed to use the model for drug testing. They think such model should (1) imitate at least one typical feature of the human HF; (2) manifest the predicted reaction to the known modulators of HF cycle and development; and (3) exhibit the reactions which are reproducible *in vivo* [99]. Scientists from Technical University in Berlin reported the production of microfollicles *in vitro* by mixing DP aggregates with the basement membrane components and the outer root sheath keratinocytes [100]. They found expression of HF markers such as vimentin, cytokeratins, trichohyalin, and chondroitin sulfate. Remarkably, they observed hair-like fibers sprouting from the nascent microfollicles. Different types of free aggregates present another modification of culture conditions. Cells may be placed on partially-adhesive substrates [101]. Being seeded on poly(ethylene-co-vinyl alcohol), DP cells first aggregate in this model and then are covered with keratinocytes. The authors found keratinocytes expressed keratin 6 and, thus, underwent differentiation.

Another way to closely reproduce HF morphogenesis *in vitro* may involve specialized HF cells derived from the pluripotent cells. For example, the derivation of functional DP-like cells from human embryonic stem cells was recently reported [102]. Derivation of multipotent progenitors of the epithelial lineage [103, 104] gives an opportunity to combine trichogenic cells at their early stages of commitment with hopefully better results.

Using the advantage of induced pluripotency Yang with colleagues [105] obtained folliculogenic CD200+/ITGA6+ epidermal stem cells from human fibroblast-derived induced pluripotent cells. Patch *in vivo* assay demonstrated the ability of these cells to generate all HF lineages including the hair shaft, and the inner and outer root sheaths. The regenerated HFs possessed a stem cell population and produced hair shafts expressing hair specific keratins. The ability of pluripotent cells to self-organize and spontaneously differentiate into all cell lineages was used by Takagi and colleagues [106] who carefully selected conditions that enabled direct derivation of HFs from induced pluripotent cells through embryoid bodies using the clustering-dependent embryoid body transplantation method.

Currently, there are a number of ongoing clinical trials using cultured cells to treat alopecia [107]. It is noteworthy that interfollicular unspecialized cells are used in many cases, put it simply, dermal fibroblasts and epidermal keratinocytes. Therapeutic mechanisms of such preparations are unclear and positive effect depends on patient's remaining HFs. However, the latter are absent in many cases. Therefore, modern approaches to HF restoration are badly needed for severe cases of hair loss or alopecia. Taking into account great progress in skin and HF biology, biotechnology, and tissue engineering we hope to meet highly effective methods of HF production in the nearest future.
