Isolation, Cultivation, and Morphological Characteristics of Hair Follicle Adult Stem Cells in the Bulge Region in Mouse and Human

Skin contains various populations of stem cells (SCs). Among these are hair follicle stem cells (HFSCs) in the bulge region. The behavior of HFSCs de-serves to be widely studied due to the benefits to be derived from their identification, isolation, and amplification. Skin samples of newborn mice (n = 32) and human adults (n = 10) were used, and the bulge region was isolated and cultured. The isolation and characterization of cells were conducted through immunocytochemistry and immunofluorescence, using mainly CD34 and CD200 monoclonal antibodies. Initially, cells grew slowly from the explant around the bulge region, accruing cells with different morphology in both mouse and human, latter being mostly polygonal; the mouse cells reaching confluence faster (5 to 7 days) than the human (12 to 15 days). It was possible to isolate into subcultures cells with small size (10 - 13 μm diameter), round-shape, scant cytoplasm, central prominent nucleus and with nucleolus, which formed colonies, maintaining their phenotype in a high proportion (77% - 83% and 91% in mouse and human, respectively), without showing changes in their morphology during almost 7 months in the mouse cells, and a month and a half in the human. These results demonstrate that the selection, the isolation, and the conditioned mediums allowed population increas-es of bulge cells and indicate that cultured cells may retain their sternness in that they maintained their phenotypic characteristics, expressed


Introduction
Stem cells (SCs) are characterized by their capacity to self-renew and their ability to differentiate into various cell lineages. Stem cells are classified by stage of development into embryonic, and adult stem cells. Adult stem cells are able to differentiate into all cell types. Currently, the main sources of stem cells are bone marrow, umbilical cord blood, and adipose tissue [1]. There is a need to find new and better stem cell sources that could facilitate therapeutic research.
Adult stem cells, as skin stem cells, have emerged as a source of interest for study and, could provide a cell bank with potential utility in regenerative medicine; for this, identification and isolation are necessary. The skin contains different populations of stem cells [2], localized into the interfollicular epidermis, the hair follicle, and the sebaceous glands. The stem cells govern tissue homeostasis and wound repair; they reside in the niche, or microenvironment, that hosts and maintains stem cells [3] [4] [5]. The hair follicles are self-renewing structures that cycle and reconstitute themselves throughout life. Each hair follicle perpetually goes through three stages: the rapid growth stage (anagen), apoptosis or involution stage (catagen), and rest stage (telogen) [6] [7]. Hair follicle stem cells (HFSCs) reside in a structure within the outer root sheath (ORS) of the hair follicle known as the "bulge", which acts as a reservoir of multipotent SC and, extends from the insertion point of the sebaceous gland duct to the attachment site for the arrector pili muscle [8] [9] [10] [11]. Stem cells, including HFSCs, never lose their regenerative ability if their niche is not destroyed. SCs exit the bulge and proliferate downward, creating a long linear trail of cells, the ORS [12].
From the anatomical point of view, the hair follicle has proven to be a challenge in both mice and humans [13] due to the lack of obvious anatomical features that can be visualized using light microscopy, the relatively small size of the hair follicles, and there being no universally adopted method to identify and isolate stem cells as a pure and viable population in culture [14] [15]. For this reason, I have focused on stem cells in the region of the prominence of the hair follicle, with great potential for proliferation.
These properties make them ideal candidates to be used in the regeneration of tissues such as skin and hair, or in the treatment of diseases such as alopecia.
Our hypothesis states that a stem cell population resides in the bulge area and its maintenance is influenced by the microenvironment, if key factors are provided they can remain as undifferentiated adult stem cells with high potential for self-renewal. Therefore, determining the conditions that allow bulge cells to main-tain their phenotype and viability in vitro is necessary. The present investigation explores the in vitro isolation, culture and characterization of mouse and human HFSCs and specific markers; the differences between mouse and human hair follicles are also investigated.

Tissue Samples
Vibrissae follicles were obtained from the heads and bodies of newborn (2 to 3 day old) mice (n = 32). All animals were acquired from the animal facility of the Instituto Nacional de Higiene Rafael Rangel. Also, excess healthy human scalp skin from was collected from 30 to 58-year-old facelift patients (n = 10), with the approval of the ethical committee of the Faculty of Sciences of the Universidad Central de Venezuela and written informed consent.
Additionally, human hair follicles samples were obtained from various regions of the body (face, legs and arms) from two volunteer donors, a 32-year-old female and a 37-year-old male; several hairs with full hair follicles were plucked.
Anagen hair follicles and vibrissae were freshly dissected and divided into single hair follicle units under a microscope.

Tissue Isolation and Cultivation
The newborn mice were sacrificed, kept in 70% alcohol for 10 min, and washed 2× for 5 min with phosphate-buffered saline (PBS). The tissues were trimmed into small pieces (4 mm × 4 mm), and the skin fragments were incubated in 0.25% dispase II (Bacillus polymyxa, Gibco, BRL) in DMEM/F12 (1:1; Gibco-BRL) for 12 -18 h at 4˚C. Scalp tissues were first rinsed in lactated Ringer's solution, and excess adipose tissue was removed. The hair follicles were squeezed out carefully in anagen phase, identified by the visible bulb and intact ORS, and carefully selected under the dissecting microscope. After two rinses with F-12, the follicles were transferred into a 35-mm dish. Then the bulge region was amputated from the upper follicle by making two transverse cuts at the site of the enlargement spots of the ORS with a fine needle. All surgical procedures were performed in a sterile environment. After an additional two rinses, the bulges were transferred into a new dish at a density of 20 per dish, immersed in a Dulbecco's modified Eagle's medium (DMEM, Gibco) and Ham's F12 medium (3:1; Gibco), supplemented with 10% fetal bovine serum (FBS; Gibco), 10 ng/ml epidermal growth factor (Invitrogen), 5 g/ml hydrocortisone, 5 g/ml insulin (Sigma-Aldrich), 200 mmol/L L-glutamine (Gibco), and antibiotics (100 U/ml penicillin and 100 g/ml streptomycin). The culture was incubated at 37˚C and 5% CO 2 in air, and the medium was changed twice a week. Human scalp tissues were first rinsed in lactated Ringer's solution and Ham's F12 with a double concentration of antibiotics (penicillin G, 100 μg/ml and, streptomycin, 100 μg/ml).
This solution was used as a transport medium to maintain aseptic conditions and optimal physiological conditions. Next, the samples were rapidly washed in

Subculture and Amplification
Colony formation was performed, after 3 -4 days in primary culture; cells were collected by incubation with a 1:1 mixture of 0.125% trypsin (Sigma) and 0.02% EDTA (Sigma) for 5 -10 min at 37˚C. The dispersed cells were centrifuged for 5 -10 min at 2500 rpm and replated in 35-mm dishes coated with gelatin (mouse) and type I collagen (human) in tissue culture flasks with a medium change every 3 -4 days. Cells were routinely passaged every 7 days. Every week, the cells were trypsinized (Gibco), and mass cultures were serially passaged until growth capacity was exhausted. For the sake of preservation, the cells were digested from culture dishes as described above.
After one rinse with culture medium, the cells were resuspended in 1ml fetal calf serum containing 10% dimethyl sulfoxide (Sigma), transferred into a cryotube, and placed into a freezer in a 4˚C, −20˚C, and −80˚C container by turns. In addition, some cells, after 24 h., were transferred into a liquid nitrogen tank in cryotubes.

Determination of Growth Curve
Cells which were characterized as rounded were plated into 4 × 6-well plates at a density of 1 × 10 −4 cells/well. After 2 days in culture, three wells were trypsinized (Gibco) and counted manually, using a Malassez chamber, at days 2, 4, 6, and 8.
The growth curve was calculated from the mean cell number at each time point.

Immunohistochemical Staining
Cells, plated in 35-mm dishes, were washed 2× for 5 min with PBS and fixed in methanol for 5 min. Peroxidase and nonspecific antibody binding were blocked by incubation with 1% H 2 O 2 /methanol and serum for 10 min at room temperature, followed by incubation with primary antibodies (anti-mouse CK15, clone LHK15; Thermo Fisher) in mouse and human, anti-mouse cluster of differentiation CD34 IgG (eBioscience) in mouse and anti-human CD200 IgG (eBioscience) in human, diluted 1:100 overnight at 4˚C for 30 min. The cells were then washed 3× for 5 min to remove unbound primary antibody and incubated with a secondary antibody (kit) for 10 min at 37˚C; unbound second antibody was removed by washing 3× for 5 min and visualized by DAB kit (Dako). Cells whose cytoplasm appeared brown were taken to be positive cells. Counterstaining with hematoxylin was performed when it was necessary to identify the bulge area.

Immunofluorescence Staining
Cells plated in 35-mm dishes were washed 2× for 5 min with PBS and fixed in methanol for 5 min as above. Sections were blocked with 10% horse serum di- In general, a descriptive analysis was made of the morphology of the observed cells, a semiquantitative analysis in which the intensity of the fluorescence reaction was evaluated (intense, moderate, mild) and a quantitative analysis in which the cells positive for DAPI, CD34, and CD200 were counted.
The quantitative analysis of the immunoreactions for CD34, CD200, and DAPI in the culture was carried out in the following way: After immunostaining, the cells that expressed a positive reaction for CD34 in mouse and CD200 in human were counted twice by the same observer. To do this, it is needed to take into account the superposition of 400× images that represent a total area equal to 0.0243 mm 2 , using the ImageJ program, in an immunofluorescence micro-

Cellular Cryopreservation and Thawing
The cells of the hair follicle prominence were resuspended in the freezing medium composed of DMEM: F12, supplemented with 20% FBS and 10% glycerol and stored at 70˚C for later use. To thaw the cells, the cryotubes were placed in 37% distilled water until the sample was solubilized and found in the nutrient base medium. Then, it was centered for 5 minutes, and the cell pellet was resuspended in the base medium and seeded in 4-well plates (Nunc). The cells were counted in a Neubauer chamber, using Trypan blue exclusion dye.
Microcultures were seeded into 96-well plates at 0.5 to 1 cell/well, coated with 1% gelatin in (mice) and 1% collagen I (human) (BioCoat, BD Biosciences). Only wells containing a single cell were observed daily for proliferation. Culture medium was constituted as previously described. After 7 days, cultures were fixed with 70% ethanol and stained with eosin.

Statistical Analysis
For evaluation of flow cytometry data, the paired Student's t-test was applied to

Isolation of the Hair Follicle
Isolation of intact hair follicles was carried out through dermo-epidermal separation. The prominence region was evidenced better in human than in mouse     Enzymatic digestion was performed with trypsin to amplify and homogenize the population. Some colonies were formed, visible in the first days of culture.

Isolation of the Bulge Region of the Hair Follicle
These cells proliferated, and other cell types showed a tendency to decrease with time.
We also observed that subcultures considerably increased the cell population over generations and allowed the homogenization of the same, with a proliferative capacity greater in HFSCs (Figure 4

Analysis of the Cell Population of the Region of the Prominence of the Hair Follicle in Culture
As to the relationship between spherical cell size and the degree of fluorescence of CD34 + and CD200 + antibodies, statistical studies indicate that there is an inverse relationship between cell size and the intensity of fluorescence (p < 0.05) ( Figure 7). Cells of smaller diameter (10 -13 µm) were more frequent and more intensely fluorescent.
Also, insulation efficiency of spherical cells was determined by comparing the number of CD34 + and CD34 − mouse cells and CD200 + and CD200 − human cells,

Growth Curve
Mouse and human live cell number in the region of the hair follicle prominence

MTT Assays
After 48 hours of incubation, we used the MTT method to measure cell viability ( Figure 11). Moderate growth proliferation was observed for the first 4 days, followed by an exponential growth until the sixth day, after which a population decline and a decelerated tendency in proliferative speed was seen. The data indicate that viable cells behave similarly to the growth curve obtained above.
It is also known that there is a linear relationship between the absolute num- The proliferation assays were consistent with the characteristic proliferative pattern of the cells in culture, between lag phase, exponential, or log phase, and stationary growth phases ( Figure 10 and Figure 11) we found the time required to perform the subcultures in mice and in humans could correspond to 6 days.

Cell Cryopreservation and Thawing
Mouse cells, on the ninth and fifteen passage, were preserved by cryopreservation, maintaining morphology and proliferation capacity after 12 days of storage and subsequent thawing. They maintained their properties through recovery from storage, showing a homogeneous morphology (spherical) and testing positive for CD34 in mouse and CD200 in human cell expression (figure not shown).
However, the human cells showed a small core. Figure 11. MTT test. The graph shows the proliferation percentages in the course of 8 days of cultivation. The existence of significant changes (p < 0.05, indicated with asterisk) was observed through a two-way ANOVA (time and species). Mouse n = 9 and human n = 4, per day.

Culture of Isolated Clonal Cells in Prominence Region of Mouse Hair Follicle
As it has been possible to confirm the importance of knowing the in vitro behavior of the CRPFP, a thorough analysis is needed to determine the properties of the cells. In view of the fact that 100% pure cultures cannot be obtained by the methods used, a method based on the cloning of cells showing the phenotype of interest was developed for which a clonal microculture was carried out, from dilution tests, to check the capacity of self-renewal and differentiation of cells isolated from mouse and human RPFP. This was observed in some wells with a single cell, per well, similar to the spherical cells noted above, which proliferated

Discussion
The present study allowed isolation, characterization, and amplification in vitro of CD34 + and CD200 + stem cells from hair follicles of mice and humans, respectively, as mostly pure and viable populations in culture; starting from the identi-  [32] and in mouse follicles [9]. This distribution suggests that hair follicles in mice and humans represent an important repository of multipotent keratinocyte stem cells, more easily accessible in the human than in the mouse due to a tendency among the former to differentiate into keratinocytes, mainly.
Likewise, the presence of a heterogeneous population and the expressed proliferation potential may be due to the dermal tissue adhering to the mouse follicle, usually greater than in human, as well as culture medium and the age of the individuals, which are factors important in the growth and diversity of the cells present in the tissue and could justify these differences. To corroborate the results obtained, the ICQ determination of the primary culture of intact mouse hair follicle cells showed a moderate expression of CK15 in rounded cells in addition to expression of CK15 in other cell types with morphology different from the stem cells, which correlates with previous IHQ results [19] and may indicate that CK15 is not an antibody specific for CM and that the cells isolated from the follicle do not represent a pure population of CM. Likewise, the small rounded cells could correspond to CM with a certain degree of differentiation. In this sense, they coincide with the expression of CK15 in primary human epithelial progenitor cells in situ and in vitro, indicated by [33]. In relation to ICQ and immunofluorescence, positive reaction was observed for CD34 in most rounded cells derived from mouse follicles and vibrissae and CD200 in human of RPFP, revealing that they can correspond to cells with cha-

racteristics of CM similar to what has been reported by other authors [4] [34]
[35].
However, rounded cells that did not react to the antibody were observed, which could correspond to cells with a certain degree of differentiation. Additionally, positive expression was found for large cells of irregular contour and with possible vacuoles, which may correspond to macrophages [36] because CD34 antibody is also expressed in hematopoietic cells and its presence may be related to perifollicular macrophages contribution to the activation of skin epithelial stem cells [37].
Once we noted the origin of the proliferation of hair follicle cells in explants, we explored the growth potential and properties of the cells derived from the prominence region as a source of adult CM, performing a dissection of the region of the prominence as indicated by other authors [21] [29] [38], which al-  [34]. We consider that, to obtain the spherical cells, a meticulous and exhaustive isolation of the RPFP is required; otherwise, the heterogeneous population obtained will be predominantly of spindle cells similar to the mesenchymal cells. In contrast, mesenchymal stem cells (CMM) from the dermal papilla of the bulb region of human hair follicles have been cultured and described as fusiform [39], being very different from the rounded cells in the bulge region described in this work.
Stem cells showed an inverse relationship between the size of the cells and the degree of fluorescence, which indicates a greater expression with smaller diameter (10 -13 μm) in both mouse and human cells (p < 0.05), results that coincide with those described by Barrandon, and Green (1985) [40]. Likewise, these researchers point out that cells with a diameter between 10 and 12 μm have a greater efficiency in the formation of colonies than 20 μm cells [40], with colony formation efficiency being inversely proportional to the diameter of the cell and, with CK15 − and CD200 + cells smaller than CK15 + and CD200 + cells in humans [41].
On the other hand, in the subcultures, a remarkable increase of the cellular population was observed throughout the generations, which allowed their homogenization and the formation of colonies; these were characterized by a predominance of spherical cells, which maintained an undifferentiated phenotype.
Results suggest that mainly cells with high proliferative capacity and differentiation potential are found in the prominence region, which is consistent with pre-  [43].
From these results we consider that, in mice, the cell proliferation is greater than in human, which may be due to the age of the individuals, since this would influence the behavior of the cells in culture. It is known that, at a greater age of the patient, the ease of obtaining viable cells and their proliferation capacity decrease considerably [44]. In addition, the cumulative population is generally used as a biological age index, in which a greater proliferation potential could indicate that the cells are biologically younger than other adult cells [45].
Similarly, passages were made continuously, with a duration of 240 days in the mouse, an observation consistent with that of Nath et al. [46], who reported that these cells were cultivated continuously for more than 1 year (>100 passages) without changing their phenotype. In human cells, passages were made continuously, with a duration between 45 and 60 days, similar to the results obtained by Oh et al. [42], who demonstrated the possibility of cultivating CM from hu- Among the outstanding properties, we find that the cells of the prominence region can grow in suspension and form colonies similar to those pointed out by Yu, et al. [29]. In this regard, the International Society of Cell Therapy in 2006, proposed three criteria to define CMM [49]; however, criteria that define and classify the CMRPFP adequately have not yet been established.
Additionally, research on CM from the hair follicle indicates that most hair follicle samples have been derived from skins of the back of animals, or the human scalp by surgical means [43]. Therefore, human RPFP cell cultures, from male and female volunteer donors of 32 and 37 years of age, respectively, obtained by depilation of the chin, leg, and arm, were obtained, confirming that this method eliminates many of the problems associated with the isolation and manipulation shortening the procedure. These results were similar to those obtained by surplus tissue samples from plastic surgery procedures, achieving the isolation and amplification of the desired cell phenotype, preserving its potential for proliferation, and proving to be an easily accessible alternative.
In this context, in relation to the methodology, five key points were highlighted in the procedure: the dermo-epidermal separation; the manipulation and selection of hair follicles; adequate isolation of the prominence region; adequate adherence of the hair follicles to the plate and the use a culture medium that promotes the growth of cells.
During the dermo-epidermal separation, follicles and vibrissae were efficiently isolated. We recommend, the elimination of adipose and dermal tissue as much as possible because the thickness of the dermis influences enzymatic digestion, with disintegration being more effective if the biopsies are small, since the action of the dispase manages to penetrate better when acting at the level of the basement membrane to separate the bonds between the epidermis and the surrounding connective tissue; its cytotoxicity is also low in comparison to other enzymes allowing the follicles to be isolated [50].
Mouse follicles presented a prominent bulb, of smaller size that the vibrissae, and in both these regions, location is less evident. As it has been described in mice, it can be seen as a discrete protuberance, and in humans, it can be visualized as a clearly defined structure [8] [9] [19]. Human follicles, undoubtedly larger than those of mice, clearly showed the area of prominence, as described by A substrate was required that promoted a strong adhesion of the cells to the support surface and was sufficiently inert to not influence the induction of differentiation in low proportions. We suggest using plates coated with 1% gelatin and 1% type I collagen for explants, facilitating the adhesion of intact follicles of mice and humans, respectively, providing a good grip and support compared to

Conclusion
In summary, these results support our hypothesis, demonstrate that the selection, the isolation and the conditioned mediums allowed the population increases of bulge cells and indicate that cultured cells may belong to the stem cells because they maintained their phenotypic characteristics, and expressed specific markers for SC, proving a high proliferative capacity for long periods. Detailed characterization allows us to identify them among other types of cells and evaluate its degree of differentiation according to size, with greater proliferation potential in both mouse and human cells. Human hair follicle stem cells in the bulge region cell cultures obtained by depilation preserve its potential for proliferation and prove to be an easily accessible alternative with more advantage, for being a non-invasive or painful procedure. This suggests that bulge cells may furnish an alternative source of easily accessible, autologous stem cells for tissue engineering and regenerative medicine.