Therapeutic Potential of Stem Cells in Follicle Regeneration.
Hair loss is caused by a variety of factors: hereditary (trichodystrophy, androgenic alopecia), concomitant medical conditions, hormonal disorders (thyroid gland disorders, insulin resistance), autoimmune (patchy alopecia, systemic lupus erythematosus), nutritional disorders, environmental factors (medicines, UV radiation), psychological factors (stress, trichotillomania), and ageing. The damaging factors affect the hair cycle and decrease stem cell activity and hair follicle regeneration capability.
Alopecia is commonly regarded as a defect with apparently no significant health consequences. However, hair loss affects self-acceptance, which may result in depression and anxiety [1, 2]. It is not only an aesthetic issue. An early onset of androgenic alopecia is associated with an increased incidence of the metabolic syndrome and an increased risk of the cardiac ischaemic disease . The ubiquity of alopecia provides an encouragement to seek new, more effective therapies aimed at hair follicle regeneration and neoregeneration.
1.1. Stem Cells in the Hair Follicle. Hair follicles have a niche for mature stem cells--hair follicular stem cells (HFSCs)--a so-called "bulge" in the attachment region of arrector pili muscles, which contain epithelial and melanocyte stem cells. Moreover, HFSCs are also situated within the outer root sheath (ORS), within the region of the proximal end of the isthmus--this area is also known as the "bulge" . HFSCs take part in the regeneration of epidermal cells and the structure of hair follicles and sebaceous glands  (Figure 1).
Stem cells of the "bulge" can remain in their niche where they self-regenerate, but they can also move down to the hair matrix region, where they become progenitor cells which then form an internal hair follicle and the hair stem . The "bulge" region is not uniform, with two compartments distinguishable in it: the lower part, close to the hair matrix, which generates the internal hair follicle cell line, and the upper part, which self-regenerates, but which does not directly participate in the regeneration of the hair follicle [7, 8]. Since the heterogeneity of the "bulge" also depends on its relationship with the basal membrane, two populations of CD34+ cells are distinguished. One of them, the so-called suprabasal SCs, contains lower levels of a6-intergin and has a lower proliferative potential [6, 9]. The "isthmus" region is, apart from the "bulge," another one which also contains stem cells participating in the formation of interfollicular epidermis and sebaceous glands [7, 10].
Another type of stem cells within the hair follicle is dermal papilla cells (DPCs), probably originating from dermal condensation, which is the initial stage of the hair follicle development [11, 12]. DPCs play an important role in induction and regulation of hair growth and the formation of new hair follicles [11,13,14]. Signals from DPCs activate stem cells in the "bulge" and germinal matrix cells in the late telogen/ early anagen phase [11, 15] by activating the Wnt/[beta]-catenin pathway [11,16]. Moreover, DPCs have potential for differentiation into lines of adipocytes and osteocytes [11, 17], and they can be transformed into pluripotential cells [11, 18].
Alopecia involves changes in two types of hair stem cells, both human hair follicle stem cells (HFSCs) and dermal papilla cells (DPCs) [19, 20]. They ensure conditions for proper hair regeneration . In scarring alopecia (lupus erythematosus, lichen planus), inflammatory cell infiltration around the bulge results in an irreversible loss of HFSCs. Although the progenitor cells are damaged, HFSCs are preserved in patchy and androgenic alopecia. This is why this type of alopecia can be reversible .
Stem cells of the "bulge" are increasingly well characterised, especially in murine hair follicle, which facilitates their identification, although no universal marker has been found for them. One of them is cytokeratin 15 (CK15), which is why CK15+/integrin [alpha]6+ or CD34+/integrin [alpha]6+ cells have been identified as "bulge" cells . Studies on murine hair follicles have also revealed expression of, inter alia, CK19 [8, 22] and numerous transcription factors, that is, Sox9, Lgr5, Gli1, Hopx, LHX2, Nfatc1, and Tcf3 [8, 25]. However, expression of certain markers depends on the hair cycle phase and on the precise location of the cells within the bulge [7, 8]. Lgr5, a receptor involved in the Wnt signaling pathway, has been identified as an actual marker of the hair follicle stem cells . Stem cells of the upper and lower parts of the bulge in the telogen hair follicle affect the expression of CD34 and only of the lower part of Lgr5. Cells participating in the formation of a new anagen hair express Lgr5, but not CD34 . Cells of the upper part of the "bulge" present a higher expression Nfatc1, which is associated with a state of rest . Expression of Lgr6 [7, 10] and Lrig1 [7, 27] has been observed within the "isthmus." Meanwhile, progenitor cells of the germinal matrix are derived from stem cells of the bulge but, unlike them, exhibit a high level of P-cadherin [8, 28].
Human hair follicle stem cells are less known than murine HFSCs. It seems that certain markers are common to both human and mouse HFSCs, that is, CD34 [4, 23], K15 [4,19], K19 [4,29], and CD200 [4, 19,23]. The presence of other markers, i.e., Sox9 and LHX2, requires further studies . Markers found only in human stem cell follicles belong to PHLDA1 [4, 24] and EpCAM/Ber-EP4, which is a useful marker of the telogen secondary hair germ [4, 28].
Dermal papilla cells present different markers, including those from hair follicle cells and dermal fibroblasts . Alkaline phosphatase (ALP) is the most important for both human and murine HFs and is the most specific of the markers [11, 29, 30]; its high activity is a marker of DP cell differentiation [11, 31]. Moreover, expression of [alpha]-SMA [11, 17], laminin, and fibronectin  as well as CD133 [11, 32] has been observed in DPCs.
Marker expression changes in pathological states. Immunoreactivity of CK15 is decreased in people with patchy alopecia, and it is present in androgenic alopecia . Hair follicles in the frontal parts of the scalp exhibit a deficit of CD34 in androgenic alopecia, and its expression is preserved in hair follicles of the occipital region . Another marker CD200 of matrix cells is poorly expressed in patchy alopecia, which may be a sign of the disappearance of the immune privilege and can contribute to pathogenesis (reaction of autoreactive lymphocytes) [21, 33].
Stem cells in the bulge remain in the resting phase for most of their lives, but they can be activated depending on the hair cycle phase. Most of the concepts regarding the course and regulation of the hair cycle have been created during research on mouse models. During the hair cycle in mice, in the anagen phase, stem cells in the bulge are divided three times on average and stay within the niche, whereas cells of the germinal matrix divide intensely and differentiate, forming the growing hair stem. During the catagen phase, cells of the germinal matrix undergo apoptosis; stem cells of the bulge migrate out of it to the external hair follicle, and subsequently, at the end of the catagen phase, they form a new bulge around the hair stem and a new germinal matrix under the bulge. Stem cells in the bulge remain in the state of rest during the telogen phase, and between the telogen and anagen phases, they self-regenerate or migrate, creating a pool of germinal matrix cells which subsequently proliferate to form the hair matrix . The precedence has been shown for the derivative cells in the bulge, the so-called "SC" progenitor cells of the germinal matrix, in the expression of genes that affect stem cell activation, and precedence in proliferation during the regeneration cycle, even before the cells of the bulge [8, 15, 34]. The translation of the mouse hair cycle into the human hair cycle has some limitations due to the different lengths of anagen [35, 36], asynchrony of the human cycle [35, 37], or a different reaction to the influence of hormonal factors [35, 38]. Currently, studies are conducted on human scalp skin xenografted into immunocompromised mice to establish the course of the hair cycle in vivo in humans .
The activity of stem cells in the bulge is controlled by the microenvironment that surrounds it, a so-called "niche." This includes daughter cells of stem cells of the bulge, which activate their self-regeneration during early and late anagen phases .
Stem cells are significantly affected by mesenchymal cells of the dermal papilla, which are in close contact with cells of the germinal matrix, separated only by the basal membrane . They seem to be of key importance in the induction of hair growth and in signal transmission during its regeneration [8, 34]. Experiments have shown that hair regeneration is not possible after laser ablation because the hair follicle cycle stops at the telogen phase without progressing to the anagen phase [6, 7, 34, 40]. Injections of exosomes derived from DPCs to HFs have been found to accelerate the entry of anagen and catagen delay via the [beta]-catenin and Shh pathways . HFSCs are also affected by fibroblasts in the reticular and papillary layers of the dermis as well as of the subcutaneous tissue .
Within the niche there are also melanocyte stem cells, which are responsible for the formation of mature melanocytes that impart the colour to a growing hair. The survival and growth of MSCs depend on signals transmitted by hair follicle epithelial cells, for example, the TGF-[beta] or the Wnt pathway [7, 39]. The extracellular matrix is another component of the microenvironment. It directly affects stem cells by the formation of the basal membrane, with which stem cells are in contact modulated, for example, by integrins [6, 8].
Stem cells of hair follicles are also affected by the macro-environment surrounding hair follicles, for example, adipose tissue. It seems to undergo similar changes to those of the hair follicle: the thickness of the adipose tissue increases during the anagen phase, and adipocytes proliferate intensively [8, 42]. Adipocytes secrete BMP2 during the late catagen phase and early telogen phase, which favours the resting states in the niche, whereas secretion of BMP2 is reduced at the end of the telogen phase, which supports the activation of HFSCs [8,42,43]. Communication between adipose tissue and the epithelium runs in both directions. Mutations blocking the hair cycle have been found to inhibit adipogenesis, which suggests that epithelium cells send signals activating the proliferation of adipocytes [6, 42].
Nerve ends affect stem cells situated at the upper part of the Gli1+ bulge by a signal of the Shh pathway [6, 44]. Therefore, denervation can reduce the effect of SCs in the "isthmus" on wound healing [6, 44]. However, it seems that nerves are not indispensable components of the niche, because denervation does not impair hair follicle regeneration, which may suggest that Gli+ cells receive Shh signal from other sources .
The hair follicle absorbs nutrients from the surrounding microvascular network, which is transformed during the hair cycle--angiogenesis is increased during the anagen phase [6, 45]. Cells of the bulge and of the matrix can probably stimulate angiogenesis . Delayed induction of angiogenesis, which accompanies impaired angiogenesis, has been observed in mice [7,45]. It has been suggested that stem cells in general prefer a low-oxygen environment, where they secrete marker of hypoxia [6, 46]. The vascular network, especially that surrounding the "isthmus," containing venous vessels, can participate in maintaining the low-oxygen environment in the surrounding of the stem cell environment .
However, although the effect of the immune response has not been sufficiently elucidated, it is important that the role of maintaining the immune privilege of hair follicles, associated with decreased expression of MHC I molecules and with increased secretion of local immunosuppressors, should be maintained during the anagen phase [6, 47]. The loss of this privilege and an immune attack on cells of the matrix and the bulge are associated with alopecia [6, 48]. Dermal cells ydT are known to modulate posttraumatic regeneration of hair follicles by secreting FGF9 [7, 49]. Macrophages, in turn, increase the level of Wnt7b and Wnt10a ligands during the telogen phase after undergoing apoptosis, whereby activating HFSCs [6, 7, 50]. Macrophages play an important role in posttraumatic activation of HFSCs--arresting their recruitment into the wound delays hair growth, whereas transplantation of active macrophages is sufficient for induction of hair growth [6, 51]. Also important is the role of Treg, which presents a high level of Jag 1 from the Notch family, which affects the effective regeneration of HF  (Figure 2 and Table 1).
1.2. Stem Cell Use in Hair Follicle Regeneration. Hair follicles are immunologically privileged places, like the brain, eyes, and testicles, and they are under the influence of the neuroendocrine-immune network . In physiological conditions, this is affected by
(1) low expression or absence of the main MHC I antigens,
(2) the presence of malfunctional Langerhans cells,
(3) local expression of immunosuppressive substances (TGF-[beta]1 and [alpha]-melanocytes MSH) [32, 48]. Owing to this, they can be easily used in transplantation.
Multipotent stem cells can regenerate hair follicles with sebaceous glands in the skin. In the current state of knowledge, stem cells can be used to regenerate hair in several therapeutic strategies:
(1) Reversing the pathological mechanisms which contribute to hair loss (especially in androgenic alopecia)
(2) Regeneration of complete hair follicles from their parts (cells in the bulge can regenerate a whole hair)
(3) Neogenesis of hair follicles from a stem cell culture with isolated cells or tissue engineering [5, 53, 54]
1.3. Studies of Use of Autologous Stem Cell in Hair Follicle Regeneration. Hair transplant has become a conventional treatment technique in androgenic alopecia (micrografts, follicular unit transplantation (both FUT an FUE), and individual follicular group harvesting (IFGH)) . Although an autologous transplant is regarded as the gold standard, its usability is limited, because of both a limited amount of material and a reduced viability of cells obtained in this way. Currently, methods are being developed which enhance the effectiveness of the use of autologous stem cells of the hair follicle.
Apart from cells of the "bulge," stem cells reside specifically in the HF mesenchyme and function to replenish the dermal papilla and connective tissue sheath. They are called self-renewing dermal stem cells (DSCs) [56, 57].When transplanted, DSCs integrate with mesenchymal cells and they act together with epithelial stem cells, participating in creating new hair follicles [56, 57]. In cultures, they form spherical, self-regenerating colonies. However, it is labour-consuming and ineffective. Therefore, methods have been developed for their simultaneous collection, isolation, and administration in vivo at acceptor sites with the use of the so-called stirred suspension bioreactors. They help to obtain cells of greater uniformity; increased cell density per volume; and control of the concentration of nutrients, metabolites, and growth factors [56, 58].
The findings of the study by Agabalyan et al. have confirmed that cells can retain their phenotype and an ability to form hair follicles even after five passages in bioreactors. Moreover, the productivity is five times higher compared to static cultures . This has given rise to the possibility of using this method commercially in the treatment of alopecia.
Gentile et al.  demonstrated the application of an innovative Rigeneracons[R] bioreactor (certificate CE, class I) in order to provide autologous micrografts and their immediate use in clinical practice. They proved that cells isolated from the bulge region can improve the thickness of hair in patients affected by androgenic alopecia using a new method of isolating human mature stem cells obtained from a patient self-biopsy, without culturing. Enhanced hair thickness was achieved in 11 men (aged 38 to 61 years) with androgenic alopecia even at the stage of 3-5 in the Norwood-Hamilton scale. After the biopsy and removal of unwanted remnants of fatty tissue, a medical device Rigeneracons (certificate CE, class I) was used to obtain cell suspension. The percentage of mesenchymal stem cells CD44+ from the dermal papilla was approx. 5% + 0.7% and CD200+ from the bulge was approx. 2.6%+ 0.3%. After 23 weeks of therapy, after the last administration of stem cells, the average number of hairs and their thicknesses increased by 29% [+ or -] 5% compared to the baseline for the hair thickness in the treated area and by less than 1% of hair thickness increase in the placebo area .
Furthermore, Nilforoushzadeh et al.  evaluated the regeneration potential of cultured mature dermal papilla to induce the growth of a hair follicle injected to the skin of bare mice. Initially, dermal papilla cells in the culture were observed to multiply with expression of CD200, and these fusiform cells tended to form colonies after three to five days. Subsequently, after two weeks, they acquired a passaging capability and they formed an extracellular matrix after the third passaging. Histopathological examination in mice which received 1.2 x [10.sup.6] of cells of dermal papilla revealed structures that transformed into hair follicles at sites of injection in the dermis .
Ibrahim et al. used autologous bone marrow mononuclear cells (BMMC) (including stem cells) to treat refractory patchy alopecia and androgenic alopecia, and the therapeutic effects were compared to the group treated with autologous stem cells of hair follicles. Cells were administered in a single application (1 millilitre in a density of 100,000 cell/ml was injected, using a 26-gauge needle, intradermally at per centimetre square of the treated site), and a significant improvement was observed in all patient groups under treatment . Interestingly, the effect of stem cells was similar despite the fact that they had been obtained from two different sources. The effect of intradermal injection of BMMC may result from the diversity of the cell mixture: progenitor, hematopoietic stem cells, various types of inflammatory cells, and mesenchymal stem cells. BMMC can stimulate hair growth as a consequence of the ability to differentiate into various cell types, the ability to secrete bioactive molecules which stimulate angiogenesis (VEGF) and anti-inflammatory molecules with an immunomodulatory and antiapoptotic effect .
1.4. Studies of the Use of Adipose-Derived Stem Cells. ADSCs (adipose-derived stem cells) seem an ideal cell population for use in regenerative medicine because of the absence of immunogenic properties, their ease of obtainment, multipotential character, ease of differentiating into various cell lines, and considerable potential for angiogenesis. ADSCs have been shown to originate from mural cells located in the perivascular niche, vascular smooth muscle cells and pericytes, both involved in the formation of normal vasculature and are responsive to VEGF . Naturally, hair follicles surrounded by subcutaneous adipose cells and by dermis form an interfollicular dermal macroenvironment, which is important in maintaining the proper growth of bulge and follicle cells [11, 61, 62]. ADSCs are indispensable in the activation of epidermal stem cells, which they do by secreting growth factors. The vascular endothelial growth factor (VEGF) regulates hair growth and the size of the hair follicle size by stimulation of angiogenesis. The hepatocyte growth factor (HGF) is involved in the duration of the hair cycle phases. The platelet-derived growth factor induces and maintains the anagen phase, and the insulin-like growth factor I (IGF-I) controls the hair growth cycle and hair cell differentiation [11, 63-67]. Another direction of their action is the stimulation of angiogenesis and an improvement of the blood supply to dermal papilla cells. They also have immunomodulatory and immunosuppressive properties through the direct interaction between cells and secretion of prostaglandin E2 (PGE2), leukaemia-inhibiting factor (LIF), and kynurenine [11, 62].
The paracrine activity of ADSCs is highly complex, and the factors secreted by stem cells have both a direct and an indirect effect on hair follicles. TB4 contributes to the activation of stem cells in a hair follicle, increasing their migration into the follicle and differentiation. SDF-1 acts through an increase in expression of EGR-1; it also increases the cell tropism towards the follicle and increases angiogenesis. The action of MCP-1 is less obvious; despite being an inflammatory factor, it has a proven tissue regenerative effect; also, a significant role of the microenvironment in the effect of paracrine factors in promoting the growth of the hair follicle has been emphasised . Huang et al., in a study on rats, found that an addition of ADSCs to a culture of dermal papilla cells or core cells, the inner and outer sheath, enhances their viability . A significant increase in the regenerative potential was recorded in a study by Huang et al., in which ADSCs were enriched with LL-37, which is an antibacterial peptide occurring naturally in wounds . That study showed a significant increase in the local regenerative factors (endothelial growth factor, thymosin beta-4, monocyte chemoattractant protein-1, and stromal cell-derived factor-1). A significant promotion of the growth of hair follicles, in both in vitro and in vivo animal models, was observed [60, 69-71].
Physiologically, adipose tissue surrounding hair follicles plays an important role in extending the anagen phase. Adipocytes progenitor cells have been observed to multiply during the transition from the telogen to the anagen phase, around the hair follicle [61, 64]. The thickness of the subcutaneous layer increases significantly during the intense hair growth phase (anagen) compared to their amount in the resting phase (telogen) [11, 59]. ADSCs stimulate hair follicle cells through peroxisome proliferator-activated receptors, whose three isoforms have been found on their surface (PPAR[alpha], PPAR[gamma], and PPAR[delta]) . Meanwhile, mature adipocytes have a negative effect on hair follicle cell proliferation and on proliferation of fibroblasts surrounding the hair follicle in simultaneous culture systems [11, 72].
Interestingly, a change in adipocyte cell line properties can cause skin and hair disorders. Lipid metabolism disorders can cause defects in the skin structure and functions. Over-expression of human apolipoprotein C1 (APOC1) with hyperlipidemia in transgenic mice causes hair growth disorders correlated with the level of expression of human APOC1 gene in the skin [11, 73].
Hypoxia, which is not toxic to mesenchymal cells, also increases the production of growth factors for ADSCs: vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), and insulin-like growth factor II (IGF-II) [74, 75]. The effect of hypoxia on ADSCs was examined in a study conducted by Park et al. . ADSCs passaged four times with C[O.sub.2] subsequently administered subcutaneously to mice to observe induction of the anagen phase and proliferation of human follicular cells of the dermal papilla and keratinocytes. Hypoxia resulted in increased secretion of insulin-like growth factor-binding protein-1 and protein-2 (IGFBP), macrophage colony stimulating factor (M-CSF), M-CSF receptor, PDGF receptor-[beta], and VEGF, whereas secretion of the epidermal growth factor was smaller .
Unfortunately, two-dimensional (2D) cultures of dermal papilla cells lose their hair formation capability in culture, which is why they require maintaining their spheroidal forms (3D) [76,77]. It is a challenge to develop methods that mimic in vivo conditions, which both maintain the 3D structure of cells and contain a special medium which imitates a natural niche rich in growth factors [56, 75].
Currently, there are no known tissue regeneration protocols applied for hair transplant with the use of ADSCs. Zanzoterra et al.  examined the capabilities of autologous cell suspension in the Rigenera system, which were obtained by mechanical fragmentation of subcutaneous and adipose tissue from the occipital area. The cell suspension was applied in the hair transplant area, thereby increasing the amount of growth factors. Microdamage has been observed to heal more quickly and to transplant hair to grow continuously even two months after the procedure, with the telogen phase shortened .
An ADSC-conditioned medium (ADSC-CM) was used in patients with alopecia (both male and female) in a study by Fukuoka and Suga . A commercial product containing a protein solution with ADSCs was used (AAPE, Prostemics, Seoul, Korea) with various growth factors (hepatocyte growth factor, fibroblast growth factor I, granulocyte colony-stimulating factor, granulocyte macrophage-colony-stimulating factor, IL-6, VEGF, and TGF). The preparation (0.02 ml/[cm.sup.2] of the solution) was administered intradermally every 3 to 5 weeks (4-6 sessions), and hair growth was monitored with trichograms. A significant improvement in hair thickness was achieved in patients of both sexes .
Shin et al.  used ADSC and conditioned media of ADSCs (ADSC-CM) in a retrospective, observational study in 27 women with a female pattern hair loss (FPHL). The application of ADSC-CM showed efficacy in treating FPHL after 12 weeks of therapy with increased hair density and thickness without severe adverse reactions . Won et al.  also showed that the application of ADSC-CM enhanced proliferation of cultured human dermal papilla cells (DPCs) by up to 130% .
Other studies have confirmed that enriching adipose tissue with a stromal vesicular fraction (SVF) supports adipocyte viability and yields better outcomes for a hair transplant procedure when they are present in grafts [62, 80]. Lipoaspirate obtained from abdominal fat (system Puregraft LLC, Solana Beach, CA, USA) was administered to the scalp at 1.0 ml/[cm.sup.2] in a Perez-Meza study. The amount of hair was found to increase by 23% after six months of the follow-up period .
1.5. Studies on Using Stem Cells from Wharton's Jelly
1.5.1. The Advantage of Stem Cells from Wharton's Jelly Compared to Other Mesenchymal Cells. Wharton's jelly has become a preferential source of stem cells due to its ready availability from a large pool of donors, noninvasive and painless acquisition, no risk to the donor, no ethical limitations, weak immunogenic potential, and high multipotential differentiation capability [81, 82]. Moreover, exposure to infectious agents occurs rarely, which guarantees safety to the donor .
Additionally, the decellularized Wharton's jelly matrix (DWJM) (fresh jelly was subjected to two cycles of osmotic shock, alternately with a hypertonic solution of NaCl, mannitol, Mg[Cl.sub.2], and KCl with the osmolarity of approx. 1.275 mOsm/l, and centrifuged at 5000 rpm at 4[degrees]C against a hypotonic solution of 0.005% Triton X-100) can provide a natural scaffolding for stem cells as a biocompatible matrix, which supports their viability, initiating aggregation of mesenchymal cells. DWJM contains TGF-[beta], collagen I, fibronectin, and tenascin, which may be responsible for condensation of added WJMSC in some areas of the DWJM. Hence, DWJM is a natural biocompatible 3D matrix which ensures adhesion, penetration, growth, and proliferation of cells--both in vitro and in vivo. To conclude, this paper presents DWJM as a new and natural 3D scaffolding which can be used in tissue engineering and regenerative medicine .
1.5.2. Neogenesis of Hair Follicles with Stem Cell Culture on Media and Grafting Them into the Skin: In Vitro Regeneration. In 2013, two researchers, demonstrated that it is possible to obtain cells with an expression of cytokeratin 19 and hair-like structures from WJMSC in in vitro conditions. Cytokeratin 19 (CK19) is a marker of bulge stem cells which determines the self-regeneration potential of modified skin [85, 86].
The Korean team of Yoo et al. [87-89] examined the effect of hWJSC on the acceleration of wound healing processes along with formation of hair follicles and other skin appendages. Enriched aggregates of hWJSC cells can form new hair follicles. The addition of growth factors to the culture medium is required: hepatocyte growth factor (HGF) stimulates growth of hair follicles in vivo and in vitro; basic fibroblast growth factor (bFGF) stimulates growth of dermal papilla cells in vivo; and vesicular endothelial growth factor (VEGF) stimulates growth of hair follicles and hair root in vivo. The hepatocyte growth factor (HGF) must be used at the stage of differentiation of the dermal papilla in culture, which is relatively expensive [87-89].
Moreover, Yoo et al.  compared effects of culturing bone marrow and umbilical cord stem cells to the spontaneous formation of dermal papilla-like tissues (DPLT). Isolated cells of the hair outer sheath were used for incubation: DPLT were recovered from 25T cell culturing plates and mixed with 1106 cells of hair sheath in 50 ml of physiological saline and injected into mice skin. The mice were examined after six weeks. Subsequently, the clinical effects of hair follicle formation in originally bare mice following their implantation in the skin were compared. No differences between the methods were observed .
Wu et al. (a Chinese study)  demonstrated the potential for differentiation of hMSC (from human embryos) to dermal papilla cells in cocultures of hMSC using dermal papilla cells previously obtained from patients. Expression of versican, Cd133, SCF (stem cell factor), ET-1 (endothelin-1), and bFGF (fibroblast growth factor) was observed during the process of differentiation .
1.5.3. Neogenesis of Hair Follicles from Isolated Cells (Stem Cells from Wharton's Jelly): In Vivo Regeneration. Intensive studies are being conducted on the commercial use of hWJSC in alopecia treatment at the University of Kansas Innovation and Collaboration, Kansas, USA (Dr. Omar Aljitawi) (no literature data) (Tables 2 and 3).
Maintaining a pool of stem cells is necessary for tissue homeostasis and damage repair. Their divisions are not frequent in mature organisms, and most of them are in a dormant state. Therefore, it is important to understand the mechanisms of their activation, which will allow for the use of multipotent cells in regenerative medicine . Their use is additionally complicated by the fact that expression of receptors on different growth factors and the effect of the microenvironment may vary. Moreover, not all target points in stem cell therapy have been identified. It requires further studies aimed not only at the use of stem cells and their various fractions and compositions with adjuvants but also at broadening of knowledge on the physiology and cytophysiology of the hair follicle .
Conflicts of Interest
The authors declare that they have no conflicts of interest.
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Agnieszka Owczarczyk-Saczonek [ID], (1) Magdalena Krajewska-Wlodarczyk [ID], (2) Anna Kruszewska, (1) Lukasz Banasiak, (3) Waldemar Placek, (1) Wojciech Maksymowicz, (4) and Joanna Wojtkiewicz (5,6,7)
(1) Department of Dermatology, Sexually Transmitted Diseases and Clinical Immunology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
(2) Department of Rheumatology, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland111
(3) Department of Plastic, Reconstructive and Aesthetic Surgery, L. Rydygier Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, Torun, Poland
(4) Department of Neurology and Neurosurgery, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
(5) Foundation for Nerve Cell Regeneration, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
(6) Department of Pathophysiology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
(7) Laboratory for Regenerative Medicine, Faculty of Medical Sciences, University of Warmia and Mazury, Olsztyn, Poland
Correspondence should be addressed to Agnieszka Owczarczyk-Saczonek; email@example.com
Received 8 April 2018; Revised 24 June 2018; Accepted 22 July 2018; Published 5 August 2018
Academic Editor: Shibashish Giri
Caption: Figure 1: Hair follicle anatomy.
Caption: Figure 2: The markers of hair follicle: (a) in human, (b) in mouse.
Table 1: The markers of hair follicle and their role. Author Signal Researched object Telerman et al., Blimp1 Transgenic mouse 2017  Kobielak et al., Bmpr1a Transgenic mouse 2007  Lei et al., 2014 DKK1 Transgenic mouse  Millar et al., 1999 Dvl2 Transgenic mouse  Lin et al., 2015 FGF1, FGF2, Transgenic mouse  FGF10 Kimura-Ueki et al., FGF18 Transgenic mouse 2012  Higgins et al., FGF5 DNA from families 2014  with long eyelashes Guo et al., 1993 FGF7 Transgenic mouse  Petiot et al., 2003 Fgfr2 Transgenic mouse  Ozturk et al., 2015 Gab1 Transgenic mouse  Milletal., 2003 Gli2 Transgenic mouse  Estrach et al., Jagged-1 Transgenic mouse 2006  Amalia Pasolli et LHX2 Transgenic mouse al., 2014  Ozturk et al., 2015 Mapk Transgenic mouse  Du et al., 2018 miR-214 Human scalp skin  tissue; in vitro Horsley et al., Nfatc1 Transgenic mouse 2008  Krieger et al., NF-kB Transgenic mouse 2018  Demehri and Kopan, Notch Transgenic mouse 2009  Lin etal., 2011 Pofut1 Transgenic mouse  Oro and Higgins, Ptch Transgenic mouse 2003  Hoi etal., 2010 Runx1 Transgenic mouse  St- Jacques et al., Shh Transgenic mouse 1998  Kadajaetal., 2014 Sox9 Transgenic mouse  Foitzik et al., TGF-[beta]1 Transgenic mouse 2000  Foitzik et al., TGF-[beta]2 Transgenic mouse 1999  Oshimori and Fuchs, TGF-[beta]RII Transgenic mouse 2012  Qiu et al., 2017 TPA Transgenic mouse  Lei et al., 2014 Wnt10b Transgenic mouse  Millar et al., 1999 Wnt3 Transgenic mouse  Donget al., 2017 Wnt7a Transgenic mouse  Kandyba and Wnt7b Transgenic mouse Kobielak, 2013  Enshell-Seijffers [beta]-Catenin Transgenic mouse et al., 2010  Author Conclusions Telerman et al., Ablation delayed HF morphogenesis, and growth 2017  and prevented new HF formation after wounding; role in promoting the dermal papilla inductive signaling cascade that initiates HF growth Ablation leads quiescent SCs to activate and Kobielak et al., to proliferate, causing an expansion of the 2007  niche and loss of slow-cycling cells; HFSCs are unable to terminally differentiate into hair DKK reduce hair follicle enlargement and Lei et al., 2014 decrease proliferation; injection of DKK1  during early anagen significantly reduced the width of prospective hairs Millar et al., 1999 Overexpression in the outer root sheath  causes the short-hair phenotype Topical application of FGFs induced an Lin et al., 2015 earlier anagen phase and prolonged the mature  anagen phase; FGFs promoted hair growth by inducing the anagen phase in telogenic mice Kimura-Ueki et al., Ablation causes telogen to become very short, 2012  giving rise to a strikingly rapid succession of hair cycles Higgins et al., FGF5 is associated with long-hair phenotype 2014  Guo et al., 1993 Overexpression causes marked suppression of  hair follicle morphogenesis Petiot et al., 2003 Lack of Fgfr2 leads to a decreased number of  HFs, and follicles were developmentally retarded Ozturk et al., 2015 Lack of Gab1 caused HF not to enter  catagen; instead HFSCs lose quiescence Lack of Gli2 causes arrest in HF development Milletal., 2003 with reduced cell proliferation and Shh-  responsive gene expression, but normal epidermal differentiation Deletion of Jagged-1 results in inhibition of Estrach et al., the hair growth cycle and conversion of hair 2006  follicles into cysts of cells undergoing interfollicular epidermal differentiation Ablation of LHX2 results in cellular disorganization and HFSC polarization within Amalia Pasolli et the niche. LHX2 loss leads to a failure to al., 2014  maintain HFSC quiescence and hair anchoring and progressive transformation of the niche into a sebaceous gland Ozturk et al., 2015 Activation of Mapk signaling can restore  quiescence of the SCs Downregulation of miR-214 promotes the Du et al., 2018 proliferation and differentiation of HFSCs;  overexpression of miR-214 led to decreased expression of EZH2, [beta]-catenin, and TCF4 Ablation causes stem cells to be activated Horsley et al., prematurely, resulting in precocious 2008  follicular growth Role in HF stem/progenitor cell activation Krieger et al., during anagen induction, involvement in hair 2018  fiber morphogenesis during HF cycling Absence of Notch signaling leads bulge stem cell descendents to retain their capacity to Demehri and Kopan, execute the follicular differentiation 2009  program but failing to maintain it owing to their genetic deficiency Disruption of Pofut1 in HF resulted in aberrant telogen morphology, a decrease of Lin etal., 2011 bulge SC markers; HF displayed a delay in  anagen reentry and dysregulation of proliferation and apoptosis during the hair cycle transition Oro and Higgins, Reduced Ptch is associated with tumor 2003  formation during anagen Hoi etal., 2010 Role in promoting anagen onset and  HFSC proliferation Shh signaling is not required for initiating St- Jacques et al., hair follicle development; however, it is 1998  essential for controlling ingrowth and morphogenesis of the hair follicle Sox9-deficient bulge HFSCs begin to differentiate into epidermal cells; as Kadajaetal., 2014 HFSC numbers dwindle, outer root sheath  production is not sustained, and HF down-growth arrests prematurely Foitzik et al., Injection ofTGF-beta1 induced 2000  premature catagen development Ablation causes delay of hair follicle Foitzik et al., morphogenesis, with a 50% reduced 1999  number of hair follicles TGF-[beta]2 signaling antagonizes BMP Oshimori and Fuchs, signaling in HFSCs with increased 2012  telogen length Refractory telogen hair follicles entered anagen prematurely after Qiu et al., 2017 TPA treatment, with the enhanced  proliferation of CD34-positive hair follicle stem cells Prolonged overexpression increased Lei et al., 2014 the size of regenerated hair follicles  and increased expression of CD34 in the bulge Overexpression causes a short-hair Millar et al., 1999 phenotype and cyclical balding resulting  from hair shaft structural defects Cultured human umbilical cord-MSCs (UC-MSCs) overexpressing Wnt7a Donget al., 2017 can accelerate wound repair and induce  hair regeneration via cellular communication in the wound microenvironment Underexpression causes shorter anagen, Kandyba and premature catagen onset with overall shorter Kobielak, 2013 hair production, and diminished  HF differentiation marker expression Inactivation in DP of HF results in reduced Enshell-Seijffers proliferation of the progenitors and their et al., 2010  immediate progeny that generate the HS and premature catagen HF: hair follicle; HS: hair shaft; DP: dermal papilla; SC: stem cell. Table 2: Researches on the use of stem cells in the regeneration of hair follicles. Authors Research object Indication Usage of hair follicle stem cell Agabalyan et al., Sprague Dawley rats/ Nude mice 2016  nude mice genetically mutated Nilforoushzadeh et Human/mice Nude mice al., 2016  genetically mutated Elmaadawi et al., Human Alopecia areata and 2018  androgenetic alopecia Gentile et al., Human Androgenetic 2017  alopecia Kalabusheva et al., Human In vitro study 2017  Hoffman et al., Human/mice In vitro study 2018  Usage of adipose-derived stem cells Park et al., 2010 Human/mice C (3)H/NeH nude  mice Zanzoterra et al., Human Androgenic alopecia 2014  Sabapathy et al., Rats In vitro study 2016  Yang et al., 2016 Human/mice C57BL/6 nude mice  Anderi et al., 2018 Human Alopecia areata  Usage of Wharton's jelly and embryo stem cells Yoo et al., 2010 Human In vitro study  Yoo et al., 2010 Human/mice Athymic nude mice  genetically mutated Wu et al., 2012 Human embryo Nude mice  MSCs/mice genetically mutated Authors Methods of obtaining Results Agabalyan et al., Bioreactors and Inducing de novo HF 2016  static cell cultures formation, with bFGF, PGF reconstituting the DP and connective tissue sheath Human scalp biopsy, Evidence of hair Nilforoushzadeh et isolation of only growth in mice al., 2016  papilla cells which received epithelial were cultured and and DP cells injected into nude mice Elmaadawi et al., Autologous bone Good clinical 2018  marrow-derived improvement in both mononuclear cells diseases compared to follicular stems cells (skin punch biopsy from unaffected areas) Gentile et al., Biopsies were A 29% [+ or -] 5% 2017  collected and increase in hair disaggregated by density for the Rigeneracons without treated area and culture condition, less than 1% in hair then injected to the density for the frontal scalp placebo area Aggrecan, biglycan, Human DP cells and fibronectin, and Kalabusheva et al., skin epidermal hyaluronic acid 2017  keratinocytes in a significantly hanging drop culture stimulated cell to develop an proliferation in a artificial HF germ DP cell monolayer culture without any effect on DP cell identity Hoffman et al., Hair follicle- Intensive hair 2018  associated- growth was observed pluripotent stem in the pieces of cells from human shaved mouse skin scalp skin and histocultured on transgenic mice with Gelfoam nestin-driven GFP Park et al., 2010 ADSCs in a ADSCs in a  conditioned medium conditioned medium injected increased the subcutaneously proliferation of induced the anagen human DP and human phase from telogen epithelial and increased hair keratinocytes; the regeneration in nude effect of hypoxia on mice ADSC function increased hair regrowth Zanzoterra et al., Injection of ADSCs After 2 weeks, the 2014  and growth factors healing of microwounds was complete and HF continued growing Sabapathy et al., ADSCs isolated from A core-shell 2016  rats were cocultured structure, outer with DP spheres ASCs shell, and an inner DP core exhibited superior potential to HF formation compared to a mixed sphere of ADSCs with DP cells Yang et al., 2016 Cocultured human The conditioned  ADSCs with LL-37 was medium of ADSCs topically applied preactivated with daily on the mouse LL-37 strongly skin promoted hair growth in vivo Anderi et al., 2018 Lipoaspiration, Increased hair  autologous ADSCs growth and decreased were injected into pull test, 3 and 6 the scalp of the months after ADSCs patient (4-4.7 x [10.sup.6] cells) Yoo et al., 2010 Cultivated umbilical Formation of  stem cells with EGF, aggregates similar HGF, and NGF to native DP in special media and reconstructed dermal papilla-like tissues Yoo et al., 2010 Isolated and Effect of inducing  cultivated stem new HF in mice cells from bone within 45 days marrow and umbilical cord, after obtaining a DP- forming medium, injected in skin of nude mice Wu et al., 2012 Three cultures: DP In fibroblast  cells cocultured injection to mice, with hMSCs; DP cells no HF was found cocultured with fibroblasts; hMSCs cultured single, next injected into skin of mice Authors Comments Agabalyan et al., Compared with static 2016  culture, stirred suspension bioreactors were significantly reduced, but they can generate larger numbers of autologous DSCs, maintaining their regenerative function The combination of Nilforoushzadeh et human cultured DP al., 2016  and epithelial cells could induce HF in nude mice Elmaadawi et al., Nonstatistically 2018  significant difference between the source of cells Gentile et al., 2017  No culture required, quick time of surgery (about 60 min) Most of the ECM compounds prevented Kalabusheva et al., the formation of 2017  cell aggregates while hyaluronic acid promoted the formation of larger organoids Hoffman et al., Model for 2018  chemotherapy- induced alopecia (observing a doxorubicin effect) Park et al., 2010 The secretion of  IGFBP, M-CSF receptor, PGF, and VEGF was significantly increased by hypoxia, while the secretion of EGF production was decreased Zanzoterra et al., Rigenera system for 2014  the automated mechanical disaggregation of cell population Sabapathy et al., PPAR[alpha] signal 2016  in ADSCs can induce the hair formation Yang et al., 2016 LL-37 treatment  significantly increased EGR-1 expression Anderi et al., 2018 Significant  variation was observed between men and women only for hair diameter, no differences with age Yoo et al., 2010 HGF is necessary in  the differentiation step Yoo et al., 2010  Wu et al., 2012 The expression in  vivo of HLA-I was confined to DP of the newly grown hair, and the survival time of hMSCs in mice is 1 month ADSCs: adipose-derived stem cells; DP: dermal papilla; DSCs: dermal stem cells; EGF: epidermal growth factor; bFGF: basic fibroblast growth factor; HF: hair follicle; HGF: hepatocyte growth factor; HFA-F human leucocyte antigen class I; hMSCs: human mesenchymal stem cells; IGFBP: insulin-like growth factor-binding protein; M-CSF: macrophage colony-stimulating factor; NGF: nerve growth factor; PGF: platelet- derived growth factor; VEGF: vascular endothelial growth factor. Table 3: Current studies of stem cell use registered on ClinicalTrials.gov . Number Study Kind of stem cells 1 "Stem Cell Educator Cord blood-derived Therapy in Alopecia Areata" multipotent stem cells (CB-SCs) 2 "The Effect of Allogeneic Allogeneic human ADSC Human Adipose Derived Stem component extract Cell Component Extract on Androgenic Alopecia" 3 "Adipose Tissue Derived Autologous MSC and Stem Cell Based Hair human platelet-rich Restoration Therapy for plasma Androgenetic Alopecia" 4 "Biocellular-Cellular High-density platelet-rich Regenerative Treatment plasma and adipose-derived Scaring Alopecia and tissue stromal vascular Alopecia Areata" fraction (AD-tSVF) 5 "AGA Biocellular Stem/ Adipose-derived tissue Stromal Hair Regenerative stromal vascular fraction Study" (AD-tSVF) and high-density platelet-rich plasma 6 "Point-of-Care Adipose- Stromal vascular fraction (SVF) cells Number Method Conditions 1 A closed loop system that Alopecia areata circulates a patient's blood through a blood cell separator, briefly cocultures the patient's lymphocytes with adherent CB-SCs in vitro, and returns the educated lymphocytes (but not the CB-SCs) to the patient's circulation 2 Applying 1.2 g of allogeneic Androgenic human adipose-derived stem alopecia cell component extract on their scalp for 16 weeks 3 MSCs derived from adipose Androgenic tissue with human platelet- alopecia rich plasma will be applied 4 Use of high-density platelet-rich Alopecia areata, plasma concentrates and cell- scarring enriched emulsified adipose- alopecia derived tissue stromal vascular fraction (AD-tSVF) via intravenous infusion 5 Biocellular mixture of Androgenetic emulsified AD-tSVF and high- alopecia, female density platelet-rich plasma pattern hair concentrate (HD-PRP) as loss compared with adipose- derived cell-enriched SVF (AD-cSVF)-i-AD-tSVF and HD-PRP using Healeon Centricyte 1000 system and intradermal injections 6 A single injection into the Androgenic scalp of autologous adipose- alopecia derived SVF cells Number Status Trial institution/sponsor NCT number and and country duration period 1 UKN The First Hospital of NCT01673789 Hebei Medical University 2012-2013 Shijiazhuang, Hebei, China 2 Completed Pusan National NCT02594046 University 2015-2017 Hospital, South Korea 3 Not yet King Edward Medical NCT02865421 recruiting University, Pakistan 4 Recruiting Regeneris Medical, NCT03078686 Global Alliance for 2017-2019 Regenerative Medicine, Healeon Medical Inc., USA 5 Recruiting Healeon Medical Inc., NCT02849470 Ministry of Health, 2016-2018 Honduras Irvine, California, USA 6 Recruiting University of Florida NCT02729415 Gainesville, Florida, 2016-2017 USA
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|Author:||Owczarczyk-Saczonek, Agnieszka; Krajewska-Wlodarczyk, Magdalena; Kruszewska, Anna; Banasiak, Lukasz;|
|Publication:||Stem Cells International|
|Date:||Jan 1, 2018|
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