**Author details**

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 intracu-

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

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

taneous transplantation into the back skin of nude mice [94].

54 Hair and Scalp Disorders

the onset of the anagen-like stage without embryonic-like development.

Ekaterina P. Kalabusheva1,2,\*, Elina S. Chermnykh1,2 , Vasily V. Terskikh<sup>1</sup> and Ekaterina A. Vorotelyak1,2

\*Address all correspondence to: vorotelyak@yandex.ru

1 N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow, Russia

2 Pirogov Russian National Research Medical University, Moscow, Russia
