Hair Follicle: A Novel Source of Multipotent Stem Cells for Tissue Engineering and Regenerative Medicine

Bulge and hair germ

Due to the complex architecture of the HF, the location of the stem cell reservoir remained elusive for many years. Initial studies reasoned that stem cells resided in the bulb, ¹⁰ but this hypothesis was abandoned, as removal of the bulb did not inhibit generation of new HFs. ¹¹ In the early 1990s, Cotsarelis et al. were the first to propose that stem cells reside in the bulge area of the HF. They took advantage of the fact that in vivo stem cells cycled very slowly so that long time after administration of tritiated thymidine, only the cells that retained the label (label retaining cells) were the slow cycling stem cells.^12,13 Several years later, this finding was verified using transgenic mice that were engineered to express the fusion protein histone H2B-GFP under the Krt5 promoter in a tetracycline regulatable manner. As a result, skin cells expressed GFP except when the mice were fed Doxycycline, which suppressed GFP expression. Once GFP expression stopped, the protein disappeared from the fast cycling cells, but was retained in the slow cycling stem cells that were localized in the bulge region of HFs. ¹⁴ Additional tracing studies with transgenic mice expressing the lacZ transgene under the control of either the Krt5 or Lgr5 promoter further supported the bulge activation hypothesis, which states that during anagen, stem cells from the bulge migrate in the bulb region where they are induced to proliferate and differentiate to all epithelial cell types of the HF.^15,16

Notably, transplantation of KRT15⁺ or LGR5⁺ cells along with dermal fibroblasts in the dermis of nude mice generated new HFs with high efficiency.^15,16 On the other hand, damage of the bulge from the autoimmune disease Lichen planopilaris resulted in permanent hair loss, ¹⁷ further highlighting the importance of bulge-derived stem cells for hair regeneration. Finally, experiments with single-cell clones showed that single bulge cells were highly clonogenic and after expansion and transplantation they were capable of generating intact follicles in vivo,^18,19 providing strong evidence that the bulge harbors true stem cells and not a collection of progenitors.

Although it is widely accepted that the bulge harbors stem cells, the exact stem cell population is still under debate. Jaks et al. challenged the notion of label-retaining cells as the true stem cell population in the HF, as LGR5⁺ cells can regenerate the whole follicle, but do not coincide with the label-retaining cells of the bulge. The same study reported that LGR5⁺ cells were found in the hair germ, a region between the DP and the bulge, which remains discrete during telogen, but overlaps with the matrix during anagen. ¹⁶ In a recent review, Li and Clevers argue that the bulge contains both active and quiescent (label retaining) stem cells. ²⁰ The former express CD34 and LGR5, whereas the latter are CD34⁺ LGR5⁻. ¹⁶ LGR5⁺ cells seem to be the first to respond to the inductive signals in early anagen and start proliferating and differentiating. ¹⁶ On the other hand, the slow cycling stem cells located in the bulge may not contribute to hair cycling, but get activated only if needed (e.g., injury).

Others believe that the hair germ originates from the bulge and contributes to the generation of the new HFs in the beginning of anagen. ²¹ In agreement, Greco et al. showed that the transcriptional profile of hair germ cells resembles that of bulge cells. They also found that hair germ cells proliferate faster than bulge cells and respond first to the DP signals at the late telogen. However, they also lose their proliferative capacity faster than bulge cells during long-term expansion in vitro. ²² Using a sophisticated tracing method, Hsu et al. reported recently that during anagen some stem cells exit the bulge, but remain slow cycling, retain the stem cell properties, and become the reservoir of stem cells for the next cycle. The same study also demonstrated that although some cells are committed to differentiate, they still return to the bulge. Surprisingly, these lineage-committed cells expressed stem cell markers, but failed to regain stemness; instead, they appeared to regulate the activity of bulge stem cells by secreting key factors. ²³

Additional studies showed robust multipotency of bulge stem cells in vivo, where they were found to participate in angiogenesis, and in vitro, where they were coaxed to differentiate into neurons, glial cells, melanocytes, keratinocytes, and mesenchymal cells.^24–28 In addition, Yu et al. reported that single bulge cells can differentiate into mesenchymal as well as neuronal lineages in vitro. ²⁸

In addition to hair regeneration, bulge stem cells were found to contribute to wound healing following skin injury by migrating and differentiating into epidermal keratinocytes.^29,30 However, they are not necessary for the maintenance of the epidermis (ablation of the cells does not affect the homeostasis of the epidermis) and in the long run they fail to stay at the sites of injury. ³⁰ Others showed that HFs also harbors melanocytes, which differentiate and produce melanin during each hair cycle stimulating hair pigmentation. Nishimura et al. identified melanocyte stem cells in the bulge and sub-bulge areas. These cells were slow cycling, undifferentiated cells that were activated during anagen to produce melanocytes. ³¹

Isthmus/infundibulum

Cells located above the bulge are believed to retain multipotent properties. Studies have reported that they can differentiate not only into the epithelial lineages of the HF, but also into the sebaceous gland and the epidermis. However, it is not yet known whether these cells represent a unique stem cell population, a subset of bulge stem cells, or even progenitors with limited differentiation capacity. Isolated cells from the area between the bulge and the sebaceous gland were found to be distinct from the bulge-derived stem cells since they did not express bulge-specific markers, such as KRT15 and CD34. Although they maintained high clonogenic potential in vitro, they were also actively proliferating in vivo—in contrast to the notion that in vivo stem cells are the slow cycling, label-retaining cells. ³² Similarly, Jensen et al. reported that cells of the upper bulge region were not quiescent in vivo, but they could generate new follicles after implantation in vivo, suggesting that quiescence might not be a requirement for maintaining multipotency. ³³ Several other studies showed that cells derived from a region above the bulge can give rise to epidermis and persist there for a long time following injury.^33–35

Sebaceous gland

There are two theories with regard to the origins of the sebaceous gland. The first asserts that stem cells residing in the bulge region migrate and give rise to resident gland cells. This theory is supported by transplantation studies showing that bulge cells generated functional sebaceous gland in vivo.^15,16 The second theory suggests that stem cells located above the bulge differentiate into sebocytes.^34–36 Horsley et al. bridged these two theories as they identified a unique cell population in the region of the sebaceous gland that expressed the transcription factor BLIMP1 and exhibited a unipotent differentiation potential into sebocytes. Loss of BLIMP1 in HFs resulted in activation of bulge cells, suggesting a possible connection between the bulge and sebaceous gland. In support of this hypothesis, the same study also showed that implanted bulge stem cells could give rise into BLIMP1⁺ cells. ³⁶

DP and dermal sheath

DP and dermal sheath (DS) are cell populations within the HF that are believed to contain stem cells. Whereas bulge cells originate from ectoderm, DP and DS cells are derived from mesoderm and they are known to regulate hair cycling by exchanging signals with the bulge. ² Multiple studies showed that DP and DS cells have a broad differentiation potential. In a pioneering study, Lako et al. demonstrated that DP and DS cells could reconstitute multiple lineages of the hematopoietic system in lethally irradiated mice. ³⁷ A more recent study showed that DP/DS stem cells are the precursors of dermal stem cells and contribute to dermal maintenance and wound healing. ³⁸ Several laboratories, including ours, showed that rat and human HF-derived DP and DS cells could also be induced to differentiate toward the myogenic, osteogenic, chondrogenic, and adipogenic lineage resembling bone marrow MSCs.^3–5,39,40 Notably, we showed that single human DP/DS-derived cell can differentiate into all mesenchymal lineages with high efficiency, suggesting these are truly multipotent stem cells and not a collection of unipotent progenitors. ⁵

The HF stem cell niche

The interaction of HF-SCs with the microenvironment is critical for the maintenance, expansion, and differentiation of the HF stem cell pool. In contrast to the other stem cell population located in the HF, the microenvironment of bulge-derived stem cells is extensively studied because of its significance in regulating hair cycling. Stem cells located in the bulge are surrounded by cell populations derived from diverge developmental origins, for example, follicular epithelium-ectoderm, melanocyte stem cell-neural crest, DP/DS-mesoderm that all together interact, coordinate and exchange signals to generate a pigmented hair shaft.

Several studies have been demonstrated that the Wnt pathway plays a critical role in HF development, the activation of the bulge niche and consequently the telogen to anagen transition. Activation of the canonical Wnt pathway results in stabilization of beta-catenin by inhibiting its phosphorylation and subsequently degradation. Stabilized beta-catenin accumulates in the nucleus where it makes a complex with TCF/LEF1 and regulates transcription. Indeed, transgenic mice expressing the WNT inhibitor DKK1 under the Krt14 promoter were hairless, as DKK1 inhibited the onset of HF development. ⁴¹ In addition, ectopic expression of a truncated stabilized form of beta-catenin by an epidermal promoter resulted in the formation of new HFs and tumors in adult mice. ⁴² In agreement, transient activation of beta-catenin was sufficient to activate entry of telogen HF into the anagen phase and induce de novo hair formation.^43,44

Interestingly, beta-catenin is important in mediating the mesenchymal–epithelial interactions between the DP and bulge cells, as ablation of beta-catenin in the DP reduced proliferation of the adjacent matrix cells and subsequent premature entry into catagen. ⁴⁵ Interestingly, the nuclear beta-catenin ²² and W/s (a gene responsible for Wnt secretion) appear first in hair germ during the early stages of anagen, suggesting that secretion of the WNT ligand by the follicular epithelium precedes its expression by the follicular mesenchyme. Indeed, deletion of Wntless in the follicular epithelium led to decreased nuclear beta-catenin in both the epithelial and mesenchymal compartments and ultimately, hair cycle arrest. ⁴⁶

In addition to the WNTs, the TGFB family also regulates stem cell activation and HF cycling. Bone morphogenetic proteins (BMPs) are secreted from both the dermal ⁴⁷ and bulge cells ²³ during telogen and maintain the quiescence of bulge stem cells. Indeed, conditional ablation of the bone morphogenetic protein receptor 1α (Bmpr1a) gene resulted in beta-catenin stabilization and and stem cell activation; conversely, overexpression of Bmpr1a induced differentiation of HF-SCs. ⁴⁸ In agreement with the inhibitory role of BMP in hair cycling, NOG (NOGGIN) (BMP inhibitor) expressed by the mesenchymal cells of the follicle, was reported to promote hair induction.^49,50 Whereas BMP signaling is mediated through SMAD1/5/8, TGFBs signals through SMAD2/3, suggesting a possible differential role in hair cycling. Indeed, a recent study reported that TGFB2 (secreted by DP) decreased the activity of BMP signaling during telogen and this effect was mediated by the TGFB target TMEFF1. ⁵¹ Collectively, these studies suggest that there is a fine balance within the bulge niche between stimulatory and inhibitory signals that regulate HF-SC quiescence/activation and hair cycling.

Murine HFs

Human HFs

Murine HFs have been largely explored with respect to stem cell markers, however, human HFs remain relatively unexplored. In contrast to the murine bulge, the human bulge cannot be identified as a distinct anatomic projection, rendering isolation of bulge cells very challenging. Screening a number of markers in vivo, Kloepper et al. identified CD200, KRT15 and KRT19 as putative bulge stem cells markers, although their location is not restricted to the bulge, but extends to a wider area of isthmus as well. In contrast to mouse, human bulge cells do not express CD34, NES, or LHX2. ⁶⁹ CD200 expressing cells that were isolated from a population of label-retaining cells using laser capture microdissection showed an increased clonogenic potential in vitro, but the multipotency of CD200⁺ cells was not examined. ⁷⁰ On the other hand, KRT15^high/CD200⁺/CD34⁻/CD271⁻ bulge-derived cells showed an increased clonogenic potential as compared to KRT15^low/CD200⁺/CD34⁻/CD271⁻ cells. ⁷¹ More recently, our laboratory reported that human DP/DS cells displayed a cell surface profile characteristic of MSCs being positive for CD90, CD44, CD49b, CD105, and CD73.^4,5 These cells were clonally multipotent as individual clones could be coaxed to differentiate into fat, bone, cartilage, and smooth muscle with high efficiency. ⁵

Extensive studies on murine models provided a better understanding of the HF biology and identified many putative stem cell markers. Unfortunately, very few studies on human HFs exist and a reliable marker is still under quest. However, due to the apparent differences between human and mice, the conclusions derived from experiments with mice do not necessarily apply in the human HF. Therefore, further studies have to be conducted on human HFs to verify the results. In this direction, engineering-reliable in vitro models that can recapitulate the in vivo environment of the human HF are absolutely necessary. The most prevalent approach has been the isolation and culture of intact, anagen HFs. Using this model, the effect of several growth factors and cytokines on the HF growth and morphology have been examined.^72–76 Although the culture of intact follicles is a well-established system, still many issues need to be resolved so that it can better mimic the in vivo environment. The main challenges facing this model are the absence of hair cycling and consequently, the limited culture time in vitro (9 days).

On the other hand, engineering the HF from its constituent cellular components that is, epithelial stem cells and dermal stem cells in the presence of the appropriate functional scaffold—might provide the appropriate microenvironment to support cycling and sustain longer culture times. To this end, Krugluger et al. injected human DP and outer root sheath cells into an in vitro skin organ culture model and observed induction of velus-like HFs, demonstrating that engineering HFs in vitro is feasible. ⁷⁷ Recent studies showed that biomaterials can be decorated with various biomolecules to provide appropriate growth or differentiation signals as necessary.^78–83 It might be interesting to examine whether this approach could be applied in the context of HFs to overcome the current limitations and facilitate studies of human HF biology.

Microdissection

Microdissection is a technique that has been commonly applied for the isolation of cells from DP^{11,37,39,84,85} or the bulge.^70,86 This technique requires use of fine forceps and blades for the isolation of the area of interest, which is subsequently transferred onto tissue culture plates, where the cells migrate out of the tissue and proliferate in the presence of an appropriate culture medium. For DP cell isolation, application of pressure on the suprabulbar region by forceps was shown to compress the bulb and facilitate removal of the connective tissue sheath surrounding the DP, which is subsequently detached from the epithelium using a scalpel blade. ⁸⁷ Finally, a highly reliable technique that has been used for isolating human bulge cells is laser capture microdissection. ⁷⁰ A thermolabile membrane is placed on top of the sample and the area of interest is targeted by laser, which melts the membrane locally marking the cells that are subsequently separated. ⁸⁸ The major advantage of microdissection is that it preserves the whole tissue, thereby increasing the efficiency of cell isolation. However, it is quite laborious and requires experienced technicians.

Enzymatic digestion

Another approach that has been employed for isolation of HF stem cells involves enzymatic digestion of the follicle from the surrounding dermis, usually with dispase or collagenase. The incubation time and concentration of enzymes used vary depending on the amount of extracellular matrix present around the follicle. Generally, collagenase treatment requires few hours of incubation at 37°C, whereas dispase requires overnight treatment.^3–5,27,70 Others use a combination of enzymes to isolate DP cells. Specifically, dispase was employed initially to remove follicles from the cutaneous fat, followed by collagenase D to digest the DS and isolate the DP. The remaining DS fibroblasts could be removed by low-speed centrifugation of the DP. ⁸⁹ Enzymatic digestion is a simple method of HF stem cells isolation, but with little control over the type of cells that are obtained leading to possible variation between isolations.

Fluorescence-activated cell sorting

FACS is a common method for isolating stem cells, especially from the murine HF.^{15,16,24,33,35,63,66,67} Fluorescently labeled antibodies are used to tag the cell surface and cells are sorted based on the fluorescence intensity, which is proportional to the expression level of the particular target receptor. FACS can also be applied for isolating cells based on markers that are not expressed on the cell surface. Our group made use of the smooth muscle alpha-actin (ACTA2) promoter-driven GFP to isolate a homogeneous population of SMCs from ovine and human HF-MSCs.^3,4 FACS yields highly purified cell populations that can be further expanded or directly analyzed for mRNA or protein expression. Regrettably, lack of reliable stem cell markers hampers use of this method in sorting human HF stem cells.

Tissue-engineered vascular grafts

A functional arterial graft should contain both endothelial cells (ECs) and SMCs. ECs line the lumen of a vessel, endow it with thromboresistant properties, and are selectively permeable to substances circulating in the blood. SMCs form the medial layer of an artery and are mainly responsible for the dilatation and constriction of the vascular wall in response to vasoactive agonists. Our laboratory showed that DS cells of ovine and human HF stained positive for ACTA2, a marker of SMC (Fig. 2). This finding prompted us to hypothesize that functional SMCs can be derived from the HF. To this end, HFs were transduced with a lentivirus encoding for GFP under the control of the ACTA2 promoter and GFP⁺ cells were sorted out using flow cytometry. We found that both ovine and human HF-derived SMCs exhibited a significantly higher proliferation and clonogenic potential compared to vascular SMCs. In addition, tissue-engineered vascular grafts prepared from HF-derived SMCs displayed high reactivity in response to vasoactive agonists and generated significant mechanical force as shown by compaction of fibrin hydrogels.^3–5,101 More recent studies in our laboratory showed that these vascular grafts could be implanted into the arterial circulation of an ovine animal model where they remained patent for at least 3 months ¹⁰² (and unpublished data), suggesting that the HF may be a readily accessible source of stem cells for cardiovascular tissue regeneration and cell therapies.

OPEN IN VIEWER

FIG. 2.

Cells comprising the DS of hair follicle are positive for smooth muscle alpha-actin (ACTA2). (A) H&E staining from neonatal ovine dermis. (B) Immunohistochemistry showing ACTA2⁺ cells in the DS of hair follicles. Image taken from Ref. ¹⁰¹ Color images available online at www.liebertpub.com/teb

Tissue engineering of cartilage, bone, and fat

In addition to myogenic differentiation, rodent DP/DS cells have the capacity to differentiate into the osteogenic, chondrogenic, and adipogenic lineage, similar to bone marrow-derived MSCs.^39,40 Extending these studies, we demonstrated that human DS/DP cells also possess a multilineage differentiation potential. ⁴ We also showed that single clones gave rise to all four lineages, strongly indicating that human HF cells represent true mesenchymal stem cells—termed HF-MSC—and not a mixed population of unipotent progenitors. ⁵ These results suggest that the human HF can be an easily accessible source of true MSCs that could be employed for regeneration of bone and cartilage for the replacement of joints or for meniscus repair.

Skin regeneration

Several studies suggested that HF cells migrate to the epidermis during homeostasis and to a larger extent following skin injury,^{29,30,35,38,103} suggesting that HF cells could be used to generate the epidermis and enhance wound healing. Indeed, Hoeller et al. reported generation of bioengineered skin by introducing fibroblasts and HF tissue into the dermis. Interestingly, epidermal keratinocytes migrated out of the HF and developed multiple layers of epidermis and stratum corneum. ¹⁰⁴ In addition, HF-derived melanocytes have been used to develop a pigmented skin substitute. ¹⁰⁵ Most important, transplantation of tissue-engineered skin from HF-derived stem cells was shown to enhance healing of ulcers and burns significantly.^106–108 Notably, when hair buds were introduced into bioengineered skin before implantation, they sped up and guided nerve regeneration, suggesting that the HF may recover the lost sense of touch. ¹⁰⁹

Nerve regeneration

Mouse HF-derived NES⁺/KRT15⁻ stem cells have the capacity to differentiate into neurons in vitro suggesting a possible application to nerve regeneration in a variety of central and peripheral nervous system diseases. ²⁶ Indeed, Amoh et al. transplanted mouse HF Nestin⁺ stem cells into a severed sciatic nerve or spinal cord, where they differentiated into Schwann cells and promoted nerve regeneration.^59,110 The same group also reported that human HF stem cells have the capacity to restore the function of injured nerves.^111,112 HF-derived neuronal and Schwann cells have also been introduced into an acellular sciatic nerve conduit, where they exhibited long-term survival and significant electrophysiological properties in vitro, but failed to induce repeated potentials. ¹¹³

Engineering functional HF

An important application of HF stem cells is the bioengineering of HFs to restore hair loss (alopecia). Common forms of alopecias include (1) the androgenetic alopecia, which results from the miniaturization of the hair; (2) the alopecia areata, which results from an autoimmune response that damages the HF; and (3) permanent alopecia, which can be caused, for example, from severe trauma. ¹

Bioengineering HF has been a topic of intense scientific research over many years. To date, two strategies have been developed to achieve this goal. The first approach includes the transplantation of intact HFs from a HF-rich area into the bald area. This technique requires initially the surgical excision of a thin strip of scalp that contains dense HFs and subsequently the isolation of the individual follicles and implantation to the hairless scalp. ¹¹⁴ Although transplantation of whole follicles is considered as the gold standard for hair restoration, studies demonstrated that segments of the HF can also induce hair growth after transplantation.^{85,100,115–119} Transplantation of a truncated human HF after amputating the bulb resulted in hair regeneration, suggesting that the bulb compartment can be regenerated from the DS compartment.^115,116,119 Interestingly, transplantation of intact DS into mouse^85,117 or human dermal compartments ¹¹⁸ led to hair induction, possibly as a result of interactions between mesenchymal cells with the host epithelium. However, in contrast to human DS when human DP was transplanted into human skin, it failed to induce hair regeneration. ¹⁰⁰

In severe cases of alopecias, the number of available HFs is not sufficient to restore the bald site. On the other hand, HF stem cells can be expanded in culture into large numbers that may be sufficient to cover the whole area and result in hair restoration. Jahoda et al. were the first to report that implantation of DP cells resulted in hair growth in mice. ⁶² Although the hair-inductive properties of DP cells were lost after long-term expansion in vitro, coculture with keratinocytes or in the keratinocyte conditioned medium could maintain the inductive properties of DP cells for almost 70 passages. ¹²⁰ Similar to DP, DS cells were also found to induce HF growth. ¹²¹ Finally, HF restoration was enhanced by the mixture of bulge/hair germ stem cells from adult HFs with neonatal dermal cells.^{15,16,18,34,35} Notably, when mixed with embryonic mouse dermal and epidermal cells, mouse bone marrow-derived cells differentiated into HF cells, suggesting hair-inductive properties of bone marrow cells. ¹²² Although the results with mouse models are very encouraging, the significance of these findings in large animal models or humans has yet to be demonstrated.

Drug delivery through the HF

Skin is an easily accessible organ that has been widely considered as a unique target for drug delivery. In contrast to the conventional delivery methods (oral, injections), the transdermal route allows drug administration to the circulation through the dermal vasculature and may increase drug bioavailability while avoiding painful injections. However, the presence of stratum corneum, the outermost layer of the skin, severely limits the penetration of hydrophilic and high molecular weight substances. ¹²³ To bypass this drawback, microscale devices have been developed to enable transdermal delivery, including liquid jet injectors, microneedles, and thermal ablation devices. ¹²³

Alternatively, scientists have focused on drug administration via the follicular route. The HF disrupts the stratum corneum and provides an opening to the epidermis. In certain areas, such as the scalp or the face, the total area of pores can reach up to 10% of the skin area, contributing significantly to solute permeation.^124–126 In addition, the dense network of blood vessels that are associated with the HF suggests that drug release to the circulation may be feasible. ¹²⁷ The heterogeneity of the HF resident cells (stem cells, gland cells, immune cells, etc.) may enable cell-specific drug targeting for treatment of skin diseases or vaccination.^128–130 Last, but not least, the relatively large volume of infundibulum renders the HF a reservoir for sustained drug release to the circulation, further highlighting the importance of follicular delivery. ¹²⁷

Several studies highlighted the contribution of follicular penetration during drug delivery through the skin. Mitragotri modeled the permeability of hydrophilic and hydrophobic compounds in skin, assuming that the solutes can transport through one or more of the following mechanisms: free-volume diffusion, lateral diffusion of the lipids, diffusion through pores, or diffusion through shunts (HFs and glands). The model predicted that high molecular weight and highly hydrophilic molecules penetrate the skin preferentially through the shunts. ¹³¹ Others suggested that there is a critical value of octanol/water partition coefficient beyond which the flux through the follicle is greatly diminished. ¹³² However, most studies omit the significance of sebum (a lipophilic product of the sebaceous gland) during drug delivery due to lack of representative experimental models. The presence of sebum in the HF and its upward flow may hinder the delivery of hydrophilic compounds and may favor the delivery of hydrophobic compounds. Indeed, apart from molecular weight and molecular orientation, diffusion through the sebum was found to be affected by compound lipophilicity. ¹³³

To further improve tissue targeting and drug delivery via the HF, studies have incorporated particle-based formulations. Lademann et al. demonstrated that nanoparticle containing dye could penetrate up to 1400 μm into the follicle of porcine skin, whereas the nonparticle formulation reached only 500 μm. Interestingly, the nanoparticles prolonged the storage of the dye into the follicle. ¹³⁴ The nanoparticle size was shown to play a critical role in follicular penetration, which was optimal for particles between 750–1500 nm and decreased for larger particles. ¹³⁵ In addition, Vogt et al. demonstrated that the particle size affected cellular uptake. They reported that only the 40-nm size nanoparticles could enter Langerhans cells that were localized around the HF, indicating that size-specific particle formulations can be designed to target antigen-presenting cells and deliver vaccines via the follicular route. ¹²⁹ Finally, systemic delivery of a chemical through the HF has also been examined in vivo.^127,136 Caffeine was introduced into a shampoo formulation and its delivery into the circulation via the skin was examined in human subjects. Interestingly, the follicular route not only accelerated the delivery, but it also prolonged detection of caffeine in the blood indicating that HFs may act as a reservoir of chemical compounds.

Cell and gene therapy using HF stem cells

The goal of gene therapy is to restore the lost tissue function by introducing the correct gene copy at the sites where the gene is missing or mutated. ¹³⁷ Application of gene therapy for hair restoration has been attempted and showed promising results. Transduction of rat bulge-derived HF stem cells with lacZ-encoding retrovirus showed stable expression of the transgene in the HF epithelial compartments for at least 6 months after implantation of transduced cells in an immunodeficient mouse model. ¹³⁸ Retroviral gene transfer of the streptomyces tyrosinase gene was used to treat albinism. Specifically, transduction of ex vivo cultured skin from albino mice restored melanin production from the skin HF. ¹³⁹ Direct gene transfer into the skin in vivo has also been reported to restore hair growth. Intradermal administration of the Sonic Hedgehog gene into C57BL/6 mice using an adenovirus resulted into anagen induction and subsequently enhanced hair growth. ¹⁴⁰ More recently, in vivo transfection of the human telomerase reverse transcriptase DNA complexed with polyethylenimine induced telogen to anagen transition in the rat dorsal skin. ¹⁴¹ In addition to the treatment of hair- or skin-related disorders, gene transfer to the HF could be used for delivery of proteins into the systemic circulation through the vascular plexus surrounding the follicles. As we demonstrated previously, human bioengineered skin could be engineered to produce insulin in a regulatable manner by administration of a small molecule (doxycycline), reversing hyperglycemia in a diabetic mouse model. ¹⁴² Engineering HFs into the dermal compartment of bioengineered skin might increase insulin production and facilitate transport to the system circulation further enhancing the potential of this technology.

Reprogramming of HF stem cells

In a breakthrough study in 2006, Yamanaka and colleagues demonstrated that introduction of four transcription factors (OCT4, SOX2, KLF4, and MYC) into mouse embryonic fibroblasts or adult fibroblasts endowed them with enhanced proliferation capacity and potential for differentiation into all three germ layers, similar to ESCs.^143–146 The Thomson group demonstrated that two of the transcription factors (KLF4 and MYC) could be replaced by NANOG and LIN28 with a similar outcome. ¹⁴⁷ The resulting cells were designated as induced pluripotent stem cells (iPSCs). An explosion of studies that followed demonstrated that iPSCs could be generated from many human cells, including blood cells,^148,149 MSCs, ¹⁵⁰ fetal ¹⁵⁰ and neonatal fibroblasts,^147,150 adipose-derived stem cells, ¹⁵¹ adult testis, ¹⁵² β-pancreatic cells, ¹⁵³ and T lymphocytes. ¹⁵⁴ Interestingly, HF-derived primary keratinocytes could be reprogrammed with 100-fold higher efficiency than fibroblasts. ¹⁵⁵ Finally, DP cells were shown to reprogram using only two factors (OCT4, KLF4), ¹⁵⁶ suggesting the presence of endogenous factors that might facilitate reprogramming.

A higher reprogramming efficiency with fewer transcription factors suggests that HF cell-derived iPSCs may be useful for regenerative medicine applications as well as for development of models to study the genetics and pathophysiology of human disease. Indeed, HF-derived MSCs were also reprogrammed and used to understand the feedback loops that sustain self-renewal using global genomic and proteomic strategies. ¹⁵⁷ In our laboratory, we developed a robust, stage-wise strategy to differentiate human HF-MSC-derived iPSCs (HF-iPSCs) into functional SMCs through an intermediate stage of multipotent and highly proliferative MSC that could be propagated for more that 45 population doubling. Interestingly, vascular constructs engineered from HF-iPSC-derived SMCs exhibited high levels of contractility in response to receptor- and nonreceptor-mediated agonists. ¹⁵⁸ These results are encouraging and suggest that our strategy may facilitate acquisition of large numbers of cells required for regenerative medicine or for engineering vascular tissues that could serve as models for studying mechanisms of vascular development or disease.