Hair Follicle Stem Cells for Tissue Regeneration

Abstract

With the positive outcomes of various cell therapies currently under preclinical and clinical studies, there is a significant interest in novel stem cell sources with unique therapeutic properties. Studies over the past two decades or so demonstrated the feasibility to isolate multipotent/pluripotent stem cells from hair follicles. The easy accessibility, high proliferation, and differentiation ability as well as lack of ethical concerns associated with this stem cell source make hair follicle stem cells (HFSCs) attractive candidate for cell therapy and tissue engineering. This review discusses the various stem cell types identified in rodent and human hair follicles and ongoing studies on the potential use of HFSCs for skin, bone, cardiovascular, and nerve tissue engineering.

Impact statement

Hair follicle stem cells are an autologous stem cell source, and recent preclinical and clinical studies demonstrated its unique properties to support and accelerate tissue regeneration, making it an attractive candidate for cell therapy and tissue engineering.

Introduction

The human body is composed of a vast array of tissue types, each of which have specialized cells unique to each tissue as their building blocks. In addition to these, tissue-specific stem cells or adult stem cells are present in various tissues throughout the body. These stem cells are undifferentiated multipotent cells with self-renewing capabilities and play a key role in tissue renewal and repair of resident tissues.¹ In areas that undergo a higher level of impact in the body such as bone marrow and the gastrointestinal tract, stem cells are quite active. Even though stem cells are found in niches throughout the body, they mostly exhibit the ability to differentiate into lineages within their own germ layer. Most of the adult stem cells currently under investigation are derived from the mesoderm of the embryo. The well-studied adult stem cells include bone marrow-derived mesenchymal stem cells (MSCs), adipose-derived MSCs, hematopoietic stem cells, and cells from placenta, umbilical cord, and dental pulp.

Unlike the adult stem cells, developing embryo can serve as another source of stem cells and has the capabilities to give rise to cells of any of the three germ layers. While research is ongoing to understand the potential use of embryonic stem cells in a clinical setting, many ethical and safety barriers need to be appropriately addressed for that to happen.² Induced pluripotent stem cells (iPSCs) is another class of pluripotent stem cells that are raising significant interest lately. Here, differentiated cells such as fibroblasts are reprogrammed to an embryonic state. These stem cells also exhibit the ability to differentiate into many different cell types across multiple germ layers.³ While these cells have tremendous clinical potential, they still face significant technological and biological hurdles. The need for genetic manipulation and the tumorigenic properties of these cells in vivo are some of the current challenges, and comprehensive studies are required to address these biological issues before clinical translation. The sources and properties of different types of stem cells have already been reviewed.⁴

The self-renewal and differentiation capabilities of stem cells make them attractive candidates for a variety of clinical applications including cancer and regenerative medicine. By now, hematopoietic stem cell transplantation is successfully used in therapeutic oncohematology. Moreover, stem cells are also used as rescue therapy after high-dose chemotherapy.⁵ Due to their ability to proliferate and differentiate, adult stem cells can provide functional contributions in regenerating damaged tissues or organs. Moreover, MSCs are attractive candidates for cell therapy since these cells do not express significant histocompatibility complexes, have immunomodulatory properties, and exhibit paracrine effect to promote regeneration. The ability of MSCs to support tissue regeneration has been demonstrated in a wide range of preclinical models, and several clinical trials are currently underway.⁶

Bone marrow-derived mononucleated cells represent a heterogeneous cell population consisting of hematopoietic stem cells, MSCs, and endothelial progenitor cells. Even though bone marrow-derived MSCs are the most extensively studied stem cell populations so far, it only forms 0.01–0.001% of the mononucleated marrow cells, highly invasive and painful procedure is required for its isolation, and the yield and differentiation potential of these cells varies with donor age. In early 2000, adipose tissue was recognized as a rich source of MSCs in addition to its established function of energy storage in the body. Adipose tissue is an attractive MSC source as it is easily accessible and can be obtained through minimally invasive techniques. The MSCs derived from adipose tissue express cell surface markers that are similar to bone marrow-derived MSCs. Various preclinical studies have demonstrated the potential of adipose-derived stem cells, and comprehensive clinical trials are underway to determine its clinical efficacy.⁷

Even though adult stem cells are present throughout the body in various tissues, studies have shown that the proliferation and differentiation capabilities of the stem cells vary widely, depending on the source. Moreover, the ease and cost of extraction, quantity, and quality of stem cells play a key role in identifying clinically translatable stem cell sources. Recently, hair follicle is getting significant attention as another potential and plentiful source of stem cells for autologous cell therapy. Both multipotent and pluripotent stem cell sources have been identified in hair follicle, making them an unique stem cell source for a range of therapeutic applications. This review will briefly discuss the anatomy of hair follicle, various types of stem cells identified in rodent and human hair follicles, and ongoing studies on the potential use of hair follicle stem cells (HFSCs) for skin, bone, cardiovascular, and nerve tissue engineering.

Hair Follicle Anatomy

Hair follicle is developed by the tightly coordinated prototypic ectodermal–mesodermal interactions and is considered as a mini-organ with its unique structure and functions.⁸ Hair growth is a cyclic regeneration phenomenon, and during this process, the hair follicle undergoes involution and regeneration. These include cycles of repeated growth (anagen), involution or regression (catagen), and rest (telogen). Figure 1 shows the anatomy of the hair follicle.⁹ It consists of a central hair shaft, which is the fully keratinized nonliving section of the hair. The hair shaft is composed of three concentric layers. The core or medulla is formed from transparent cells and surrounded by the cortex, which in turn is covered by the cuticle. The cortex is made of 50–60% macrofibrils and plays a key role in determining the mechanical and physical properties of the hair. The cuticle consists of sublamellar structures with flat overlapping cells. The cross section of the hair follicle shows concentric epithelial layers with distinct patterns of keratins (Kr). These include the outer root sheath, the companion layer, the inner root sheath, and the hair shaft. The outer root sheath contains the bulge region, which is considered as the major repository of HFSCs.

FIG. 1.

Schematic illustration of the menagerie of stem cells and their individual locations in the resting (telogen) adult hair follicle. The stem cell populations are depicted by their distinct gene/protein expression or promoter activity: Lgr5 (green, hair germ and bulge), CD34 (orange, bulge), LRC (yellow, bulge), Lgr6 (pink, lower isthmus), Lrig1/MTS24 (blue, isthmus), Blimp1 (violet, sebaceous gland opening), and K15* (a truncated version of the K15 promoter, restricted in its activity to the bulge area). IFE, interfollicular epidermis; LRC, label retaining cell. Color images are available online.

The hair follicle is generally divided into three segments: the infundibulum, isthmus, and the lower follicle.¹⁰ The infundibulum is the upper funnel-shaped portion, which generally begins at the surface of the epidermis to the opening of the sebaceous duct and is filled with sebum. The upper portion of the infundibulum has shown to have stem cell population that expresses Sca-1 (stem cell antigen 1, expressed by uncommitted progenitor cells) and has the ability to regenerate infundibulum.¹¹ The isthmus is the middle portion between the sebaceous duct and the bulge. The bulge is a distinct area of the outer root sheath, an epithelial compartment, and has cells with stem cell characteristics capable of differentiating into hair follicle lineages.¹²

Several markers have been identified in these cells isolated from rodents, including Gli 1, MTS24, Lgr6, and Lrig1.¹³ These cells were originally considered as a source of epithelial cell pool for hair regeneration. In vivo lineage tracing studies in mice demonstrated the presence of multipotent Keratin 15-positive stem cells (Kr15+) with high proliferating ability in the bulge area.¹⁴ In addition to keratinocyte lineage, the presence of melanocyte stem cells has also been identified in the bulge area. Even though the ability of bulge cells to differentiate into epithelial lineages has been demonstrated, recent studies indicate the presence of nestin (neuroepithelial stem cell protein expressed by nerve cells) positive cells in the bulge area, which are capable of differentiating into non-epithelial tissues such as neural cells.^15,22

The lower follicle extends from the bulge to the base of the follicle and consists of hair germ and the mesenchymal dermal papilla (DP). The infundibulum, isthmus, and bulge are of ectodermal origin; however, the DP is derived from the mesoderm. Hair germ has shown to have distinct stem cell population, which expresses high levels of P-cadherin (a transmembrane glycoprotein involved in cell–cell adhesion).¹⁶ The DP cells of cranial hair derive from neural crest cells, and DP cells play a very crucial role in hair growth and serve as a signaling center during hair regeneration. Additionally, a layer of mesenchymal fibroblasts, dermal sheath (DS), has also been identified, which lines the outer surface of the hair follicle. The gene expression profile of DS cells shows some similarity to DP cells.

Hair Follicle Stem Cells

HFSCs were first located in the bulge region of the hair follicle, and the high colony-forming efficiency of bulge cells was reported in early 1990s.¹⁷ One of the earliest markers used to identify these cells is keratin, keratin 15 (Kr15) and keratin 19 (Kr19). The slow cycling cells in the bulge area have been identified to be Kr15+. Melanocyte stem cells derived from the neural crest also reside in the bulge area and supply melanocytes to the hair matrix during each hair cycle. The interactions between these two cell types are not completely understood. Tanimura et al. showed that COL17A1, a hemidesmosomal transmembrane collagen, is highly expressed in mouse HFSCs and is required for their self-renewal.¹⁸ Moreover, the study demonstrated that HFSCs serve as a functional niche for melanocyte stem cell maintenance via transforming growth factor beta (TGF-β) signaling.

Morris et al. performed a comprehensive study using transgenic mice model for the lineage analysis and isolation of Kr15+ HFSCs.¹⁹ The study demonstrated the multipotency of the cells and their ability to give rise to all the epithelial cell types of the hair follicles and even sebaceous glands and epidermis. The study confirmed that HFSCs are epithelial stem cells expressing β1 integrin and cluster of differentiation 34 (CD34), a well-known hematopoietic stem cell marker, with higher proliferative potential than non-bulge keratinocytes and can reconstitute all epithelial cell types within the skin. In addition, transcriptional profiling of bulge HFSCs demonstrated that of 97 upregulated genes, 34 were upregulated in hematopoietic stem cells, 20 in neural stem cells, and 22 in embryonic stem cells demonstrating their similarity to stem cells present in other organs.

Li et al. demonstrated the colocalization of green fluorescent protein (GFP)-labeled nestin and keratins 5/8 and 15 in HFSCs indicating a possible relation between bulge HFSCs and neural stem cells.²⁰ Importantly, the study showed that the location and number of nestin-expressing cells are hair cycle-dependent. Cell fate mapping studies in transgenic mice confirmed the presence of neural crest-derived cells in the bulge area and DP.²¹ Amoh et al. using nestin-driven GFP transfected mice demonstrated the unique ability of hair follicle bulge cells to form endothelial cells and to support skin vascularization.²² Interestingly, these nestin (intermediate filament protein marking neural progenitor cells) positive cells also express the stem cell marker CD34; however, these cells did not express keratinocyte marker, Kr15. These cells are considered relatively primitive as they remained Kr15– and CD34+ even after 4 weeks of in vitro culture in Dulbecco's modified Eagle's medium (DMEM)-F12 media. The nestin-GFP cells showed the ability to differentiate into neurons, astrocytes, glia, keratinocytes, smooth muscle cells, adipocytes, and melanocytes in vitro demonstrating pluripotency.^23,24 These cells also showed the ability to form neuronal cells in vivo. This is further supported by a previous study demonstrating the ability of hair follicle cells to differentiate into neurons, smooth muscle cells, rare Schwann cells, and melanocytes.²⁵ These studies indicate the presence of a unique pluripotent stem cell source in hair follicle.

Although the studies so far indicate the bulge area as the major stem cell source, the exact stem cell population and location are still under debate. Transcriptional profile indicates that the hair germ cells resemble closely to bulge cells, even though there are differences in their proliferation potential.²⁶ Cells isolated from area between the bulge and sebaceous gland were found to be distinct from bulge-derived stem cells and did not express Kr15 and CD34 markers, even though the cells maintained clonogenic potential in vitro.²⁷ Another region is the DP and DS, both shown to have stem cell population. Pluripotent neural crest stem cells were identified in the DP of adult hair follicles.²⁸ Their cells resemble bone marrow-derived MSCs with the ability to differentiate into myogenic, osteogenic, chondrogenic, and adipogenic lineage, making it a potential source for cell therapy.

It is to be noted that most of the studies so far were performed using mouse/rat models, and significant differences exist in hair cycle, the microanatomy of hair follicle, and properties and location of HFSCs in mouse/rat compared with human. Further studies are therefore required to fully understand the location, nature, and differentiation potential of human HFSCs. Even though Kr15 and CD34 are by now considered as specific markers of mouse bulge HFSCs, they are not characteristic of human bulge cells. CD200 is expressed at high levels in human bulge cells and CD34 at very low levels.¹⁴

Even though previous studies indicated that human HFSCs may be located in the lower hair follicle well below the bulge, Lyle et al., by grafting human scalp to immune-deficient mice, demonstrated that keratinocyte stem cells are present in the bulge area of the human hair.²⁹ These keratin 15-positive (Kr15+) keratinocyte stem cells showed high levels of β1 integrin expression. A distinct population of cells has also been identified in the outer root sheath and lower third of human anagen follicle, which are Kr19+ and may be considered as a second pool of stem cells in the human hair follicle.³⁰ Yu et al. showed the feasibility to isolate stem cells with neural crest characteristics from the bulge area of human hair follicles.³¹ The isolated cells showed gene expression profile similar to murine skin immature neural crest cells. The cells showed the ability to differentiate to myogenic, melanocytic, neuronal, and mesenchymal cell lineages indicating their potential for tissue engineering applications.

Bajpai et al. performed comprehensive characterization of human HFSCs isolated from scalp hair follicles.³² The study showed the high proliferative capacity of human HFSCs when cultured in DMEM supplemented with 10% fetal bovine serum and 1 ng/mL basic fibroblast growth factor (bFGF). The cells can be maintained in culture for ∼45 population doublings without undergoing cellular senescence. The myogenic, osteogenic, adipogenic, and chondrogenic differentiation potential of these cells were also confirmed demonstrating the clinical potential of this autologous stem cell source. Xu et al. showed the unique advantage of low oxygen tension culture of human HFSCs from the outer root sheath to enhance cell proliferation and function. Under low oxygen tension, these cells showed enhanced colony-forming efficiency with significantly higher total and holoclone colony numbers.³³

Wang et al. recently reviewed various approaches to isolate and culture human HFSCs.³⁴ Different growth factors such as bFGF and epidermal growth factor (EGF) were investigated to support self-renewal, high proliferation rate, and multilineage differentiation of human HFSCs. Furthermore, a recent study demonstrated the feasibility to cryopreserve human hair follicle while maintaining the differentiation potential of the cells. The pluripotent stem cells were shown to produce keratinocytes, smooth muscle cells, cardiac muscle cells, neurons, and glial cells.³⁵ The tremendous translational potential of HFSCs for cell therapy is also evident from a recent study showing that hair follicle-derived keratinocyte can be reprogrammed to become iPSCs with 100-fold higher efficiency than fibroblasts.³⁶ In summary, hair follicles contain various pools of stem cells such as epithelial, melanocyte, and MSCs with broad differentiation capabilities and hence could serve as a novel autologous stem cell source for tissue engineering applications.

HFSCs for Tissue Regeneration

With the positive outcomes of various cell therapies currently under clinical and preclinical studies, there is a significant interest in novel stem cell sources with unique therapeutic properties.³⁷ Studies so far indicate that HFSCs have a variety of unique advantages over the currently used stem cell sources, making them a potential alternative to the use of bone marrow-derived stem cells (BMSCs), embryonic stem cells, or fetal stem cells for tissue engineering applications. The easy accessibility with minimum tissue damage and lack of ethical concerns associated with HFSCs makes it an attractive candidate for tissue engineering and cell therapy. The ease of isolation of HFSCs, and their high proliferative properties in vivo, is considered as a unique advantage compared with BMSCs.^34,38 It is reported that ∼10¹⁵ HFSCs can be obtained from a hair follicle before senescence occurred indicating the potential for long-term in vitro expansion.³⁴ A recent study also reported the feasibility of large-scale expansion of human adult neural crest-derived stem cells from hair follicle.³⁹ Approximately 10⁴ neural crest-multipotent stem cells can be isolated from a single hair follicle. This allows for the feasibility to isolate therapeutic number of cells (40–200 × 10⁶) for clinical usage from minimum number of hair follicles. Another valuable property of HFSCs is their broad differentiation potential into cell lineages across different germ layers. The translational potential of HFSCs is further evident from recent studies showing their ability to downregulate major histocompatibility complexes and produce immunosuppressants implying the non-immunogenic nature of the cells.⁴⁰

Several methods can be used to isolate HFSCs from hair follicle. These include microdissection, enzymatic digestion, and fluorescence-activated cell sorting.^34,37 Understanding the HFSC niche is very crucial to appropriately culture the cells to maintain their functionality. Several culture media and culture conditions have been developed to support in vitro expansion while maintaining multipotency.³⁴ Another area of significant interest lately is to develop substrates of cellular microenvironments that are suitable for expanding HFSCs without adversely affecting its proliferative and differentiation potential. Three-dimensional (3D) culture condition was found to be more suitable compared with two-dimensional (2D) substrates to support cell functions. Microcarrier culture, spheroid culture, as well as constructs that can provide enhanced cell–cell interactions by closely mimicking the in vivo niche of hair follicle are being studied.³⁴ Chen et al. used layer-by-layer (L-b-L) self-assembly technology to develop a nanoscale microenvironment for HFSC culture.⁴¹ The study also showed the feasibility to encapsulate TGF-β2 in the layers. The TGF-β2-loaded substrates induced transformation of CD34+ cells into highly proliferating Lgr5+ stem cells, thereby demonstrating the feasibility to achieve quiescent and activated states of hair follicle (HF) cells.

Nanofiber matrices are attractive substrates for in vitro cell culture due to their extracellular matrix-mimic architecture, and higher surface area to volume ratio to support cellular attachment. Hejazian et al. investigated the potential of aligned biodegradable nanofiber scaffolds as substrates for HFSCs.⁴² Aligned polycaprolactone (PCL) nanofiber matrices were prepared by electrospinning. The nanofiber matrices supported significant increase in rat bulge HFSC proliferation compared with tissue culture plate. The cells cultured on the nanofiber matrices also showed the ability to express βIII-tubulin and differentiate into neural lineages. Sarkovic et al. studied the potential of electrospun PCL fiber mesh to serve as a biomimetic substrate for human epidermal melanocytes as well as hair follicle-derived human melanocytes from the outer root sheath.⁴³ The PCL fiber mesh maintained the melanotic properties of the cells. The cells showed reduced mitochondrial activity and PAX-3 gene expression showing that the 3D scaffold supported differentiation rather than proliferation of the cells. Higher melanotic expression was observed in cells cultured on PCL fiber mesh compared with 2D adherent culture.

Even though HFSCs were initially studied in the context of hair growth, recent studies point to the broader applicability of these cells. Many studies are currently underway to understand its potential as an alternative cell source for the tissue engineering of a wide range of tissues. Recent studies in the area of skin, bone, cardiovascular, and nerve regeneration are discussed below (Table 1).

Table 1.

Summary of Preclinical Studies Using Hair Follicle Stem Cells

Cell source	Application	In vivo model	Reference
Nestin+, CD34+, and Kr15– cells	Epidermal regeneration and wound healing	Rat model	47
Bulge HFSCs	Skin regeneration during expansion	Rat model	48
Chitosan sponge matrices with dermal fibroblasts co-cultured with HFSCs	Treatment of full-thickness wounds	Rat model	49
Acellular amniotic membrane seeded with HFSCs	Treatment of full-thickness skin defects	Mouse model	50
VEGF-165 gene-modified HFSCs	Enhancement of neovascularization for skin tissue regeneration	Rat model	52
Human HFSCs from bulge area and DP	Skin tissue engineering	Mouse model	53
HFSCs in EpiDex^®	Epidermal regeneration	Human clinical trial	58
HFSCs and VEGF-165 in Matrigel	Vascular tissue engineering and treatment of ischemic diseases	Mouse model	79
SIS-supported HFSC alignment	Vascular graft engineering	Ovine model	82
Bulge HFSCs	Formation of neuronal cells	Developing nervous system of chick	89
Nestin-positive, keratin-negative, CD34+ bulge HFSCs	Neural tissue engineering	Mouse model	15
Human HFSCs	Sciatic nerve repair	Mouse model	90
Mouse bulge HFSCs	Sciatic nerve regeneration	Mouse model	93
Nestin-positive mouse bulge HFSCs	Thoracic spinal cord reattachment	Mouse model	94,96
Rat HFSCs	Spinal cord regeneration	Rat model	100
Rat bulge HFSCs with PLLA substrates	Sciatic nerve regeneration	Rat model	103

DP, dermal papilla; HFSCs, hair follicle stem cells; PLLA, poly-L-lactic acid; SIS, small intestinal submucosa; VEGF, vascular endothelial growth factor.

Skin tissue engineering

Studies have shown that following full-thickness wounds, cells from interfollicular epidermis and hair follicles migrate to the damaged area.⁴⁴ Moreover, there is close developmental and structural similarities between hair follicle epithelial outer root sheath and the DS. The possibility of HFSCs serving as progenitor fibroblast populations in response to wounding is therefore highly likely. Interestingly, studies have shown that skin containing large numbers of anagen follicles heals more rapidly than skin with predominantly telogen follicles.⁴⁵ Moreover, the contribution of Kr15+ bulge cells to re-epithelialization in full-thickness wounds in mice implies the possible role of HFSCs in epidermal regeneration and wound healing.⁴⁶ Babakhani et al. used nestin+, CD34+, and Kr15– cells to treat deep partial-thickness burn wounds in a rat model.⁴⁷ The cells were injected around the wound bed. The study showed significant acceleration in wound closure rate in the cell-treated group. Moreover, the cell-treated group showed faster re-epithelialization and collagen deposition. The quality of healing is further evidenced by the enhanced vascular density and tensile strength of the regenerated skin demonstrating the therapeutic efficacy of HFSCs for deep partial-thickness burn wound healing. Skin expansion is commonly used in reconstructive surgery. Cheng et al. recently investigated the potential of bulge HFSCs on skin regeneration during expansion.⁴⁸ Injection of HFSCs in a rat skin expansion model led to increased skin area, tissue weight, epidermal and dermal thickness, collagen content, blood vessels, and lower retraction rates. The transplanted HFSCs were also shown to differentiate into vascular endothelial cells, epidermal cells, and outer root sheath cells of hair follicle. In addition, the cells upregulated the expression of growth factors such as EGF, vascular endothelial growth factor (VEGF), bFGF, and TGF-β indicating the paracrine effect of HFSCs in regeneration.

Studies have also shown that as in other cell therapeutic approaches, combining HFSCs with biomaterials can further improve the efficacy of tissue regeneration. Hilmi et al. combined cell-seeded chitosan dermal substitute to treat full-thickness wound created in a rat model via irradiation.⁴⁹ The chitosan dermal substitute was prepared by seeding chitosan sponge matrices with dermal fibroblast, followed by co-culturing with HFSCs. The study showed the highest re-epithelialization, longest epithelial tongue, and shortest migratory tongue distance achieved with chitosan dermal substitute compared with DuoDERM^®, a commercially available dressing. Moreover, the scar size was significantly decreased in chitosan dressing demonstrating the advantage of combining HFSCs with biomaterials in treating full-thickness dermal wounds.

Acellular amniotic membrane was investigated as another bioactive substrate for the localized delivery of HFSC to treat full-thickness skin defects in nude mice.⁵⁰ The HFSCs attached and proliferated well on acellular amniotic membrane. The wound healing rate of the group treated with acellular amniotic membrane seeded with HFSC was found to be much higher than that of acellular amniotic membrane alone. By 28 days post-implantation, the cuticular layer of the wound treated with the HFSC-seeded membrane showed significantly increased thickening along with hair follicle formation compared with the acellular membrane. The study demonstrated the potential of combining amniotic membrane and HFSCs for skin tissue engineering. A recent study by Liu et al. further confirmed the potential of the approach and that the combination approach supported neovascularization, thereby promoting better wound healing.⁵¹

Another study investigated the potential of VEGF-165 gene-modified rat HFSCs to enhance neovascularization for skin tissue regeneration.⁵² The transduced cells were seeded on gelatin-chondroitin-6-sulfate-hyaluronic acid sponge and implanted onto full-thickness skin wound in a rat model. The sponge seeded with transfected cells showed the highest healing rate as well as the highest microvessel density. The number of new blood vessels formed was found be highest in the sponge seeded with transduced HFSC cells, followed by sponge seeded with HFSC cells. The sponge-alone group showed only trace amounts of mature subcutaneous blood vessels, indicating the regenerative capability of the cell-seeded sponges.

Wang et al. investigated the potential of human HFSCs derived from the bulge area and DP for skin tissue engineering.⁵³ Poly(glycolic acid)–collagen scaffolds were seeded with the cells and used as the bioengineered dermis. A keratinocyte sheet was placed on the surface, which served as the bioengineered epidermis. In vivo study using a full-thickness skin wound model in nude mice showed the efficacy of the bioengineered HFSC-composite scaffold to effectively heal the full-thickness skin defect, demonstrating the therapeutic efficacy of the approach. The potential of electrospun nanofiber scaffold seeded with HFSC as a skin substitute was also investigated.⁵⁴ The fiber diameter and pore size of the trilayered electrospun nanofiber construct made of gelatin and poly(ethylene glycol) methacrylate mimicked the collagen structural gradation of native skin. Moreover, the electrospun matrix showed mechanical properties suitable for skin grafting and supported the adhesion and proliferation of HFSCs, demonstrating its applicability for skin tissue engineering. Another study demonstrated the potential of randomly oriented PCL nanofiber matrix as a substrate for HFSC delivery to support skin tissue regeneration.⁵⁵ Similar to aligned nanofibers, the randomly aligned fibers also supported HFSC adhesion and showed higher cellular proliferation compared with tissue culture plate.

Many epidermal substitutes are commercially available as autologous or allogenic keratinocytes seeded on biomaterials or as aerosol cell spray.^56,57 Among these, EpiDex^® is a fully differentiated stratified and minified autologous epidermal equivalent from cultured outer root sheath keratinocyte cells of anagen hair follicles. The efficacy of this tissue-engineered graft was studied using a randomized, parallel group multicenter study in 77 patients with recalcitrant venous or arteriovenous leg ulcers. The study showed that EpiDex is as efficacious as in-patient split-thickness meshed autograft, which is considered the gold standard treatment for hard-to-heal skin ulcers.⁵⁸ The study demonstrated the significant clinical efficacy of this noninvasive tissue-engineered alternative strategy.

A retrospective study investigated the efficacy of EpiDex treatment in 68 patients with chronic wounds, which are unresponsive to best conservative standard of care. The primary endpoint of the study was complete wound closure within 9 months post-transplantation; secondary endpoints were change of wound surface area, reduced pain, and overall patient satisfaction. The study showed that 75% of the patients showed complete wound healing by 9 months, 15% showed reduction in wound surface area by >50%, and 12% did not respond to EpiDex treatment. Moreover, the wound pain completely subsided in 78% of the patients and 13% reported reduced pain. The study concluded that EpiDex can effectively heal up to three quarters of patients with recalcitrant chronic leg ulcers.⁵⁹

Navsaria et al. micrografted autologous hair follicles into a clinically used dermal regeneration matrix (Integra^®)—composed of a porous collagen–chondroitin-6-sulfate covered with a thin sheet of silicone, onto the scalp of a 26-year-old man.⁶⁰ By day 28 post-grafting, growth of epithelium and restoration of the hair growth in the scalp was evident. One year post-grafting, keratins 1 and 10, which are markers of mature epidermis, were present in the regenerated tissue. Similarly, keratins 6 and 16, which are markers of epithelial hyperproliferation, were not expressed demonstrating the feasibility to achieve mature and normal epidermal phenotype via this approach. Moreover, the study also showed that the regenerated epidermis was derived from the implanted hair follicles demonstrating the role of HFSCs in regeneration.

Bone tissue engineering

Several studies investigated the osteogenic differentiation of HFSCs to understand its usefulness as a novel stem cell source for bone tissue engineering. Urano-Morisawa et al. demonstrated the efficacy of bone morphogenetic protein-2 (BMP-2) on osteogenic differentiation of HFSCs obtained from the whisker follicle of mice.⁶¹ The presence of BMP-2 elevated alkaline phosphatase activity, osteocalcin and osterix expression, as well as mineral deposition by the cells. Moreover, the cells expressed macrophage colony-stimulating factor and osteoprotegerin and showed the feasibility to generate multinucleated osteoclasts upon co-culture with bone marrow cells. Wu et al. demonstrated the feasibility of mice follicle dermal papilla MSCs to differentiate into adipocytes and osteoblasts.⁶² The study also demonstrated the ability of these cells to form odontoblasts in vivo demonstrating its applicability for dental tissue engineering in addition to bone tissue engineering. Zaki et al. compared the osteogenic and adipogenic differentiation potential of stem cells derived from rat hair follicle and bone marrow.⁶³ Upon in vitro culture, the HFSC clusters expressed CD34, CD73, and CD200 and negatively expressed CD45. The BMSCs, however, expressed CD73 and CD200 and negatively expressed CD34 and CD45. Even though both the cell types supported osteogenic differentiation, BMSCs showed higher alkaline phosphatase expression and calcium deposition.

Jahoda et al. demonstrated the feasibility of human hair follicle DP and DS cells to differentiate toward osteogenic lineage.⁶⁴ Yu et al. demonstrated the feasibility of isolating stem cells with neural crest characteristics from the bulge area of human hair follicles.³¹ The cells showed the ability to differentiate toward mesenchymal lineages and were able to form chondrocytes and osteocytes. Liu et al. performed a comprehensive characterization of human hair follicle cells and their multilineage differentiation potential.⁶⁵ The isolated cells from the hair follicle expressed MSC markers such as CD44, CD49b, CD73, CD90, and CD105 and did not express hematopoietic markers such as CD45 and CD34. Under osteogenic conditions, these cells showed the ability to differentiate toward osteogenic lineage. Gao et al. demonstrated the ability of human HFSCs to differentiate into osteogenic lineage along with differentiation into adipocytes and chondrocytes confirming its multipotency.⁶⁶

Aran et al. investigated the ability of HFSCs to differentiate into osteogenic lineage on a natural collagen scaffold.⁶⁷ HFSCs were isolated from the bulge area of rat whiskers and cultured on collagen scaffolds using osteogenic media. The study demonstrated the significant potential of HFSCs to differentiate into osteoblasts and collagen scaffold served as a good substrate for cellular growth and osteogenic differentiation. The potential of 3D osteogenic differentiation of human HFSCs is confirmed by another study where in hair follicle DP cells were encapsulated in 3D self-assembling peptide scaffold RAD16–1.⁶⁸ The human HFSCs upon encapsulation in the self-assembled gel maintained their native phenotype characterized by markers such as alkaline phosphatase, versican, and corin. Moreover, the 3D environment supported osteogenic differentiation of the cells in the presence of osteogenic media. These studies confirm the osteogenic potential of rodent and human HFSCs, indicating them as a potent cell source for bone tissue engineering.

Cardiovascular tissue engineering

Cardiovascular diseases are considered some of the leading cause of mortality and morbidity. Cell therapy for cardiovascular diseases is raising significant interest lately, and search is ongoing for novel stem cells sources with ability to differentiate into functional cardiomyocytes, endothelial cells, and smooth muscle cells. Several studies investigated the potential of HFSCs to differentiate into these lineages. Yashiro et al. demonstrated that HFSCs, particularly those from the upper part of the hair follicle, have the highest efficacy to differentiate into cardiac smooth muscle cells.⁶⁹ The presence of isoproterenol significantly increased the spontaneous beating rate of the differentiated cells, which in turn was inhibited by propranolol. Moreover, tissue sheets of beating heart muscle cells were formed by the addition of activin A, BMP4, bFGF along with isoproterenol.⁷⁰

Tohgi et al. demonstrated the feasibility to isolate similar pluripotent cells from the upper part of the human hair follicles. The cells showed the ability to differentiate into cardiac muscle cells along with neurons, glial cells, keratinocytes, and smooth muscle cells.⁷¹ One of the major drawbacks of the differentiation protocols discussed above is that along with cardiomyocytes other cell types such as neurons, glial cells, and smooth muscle cells are also formed. Studies are currently ongoing to address this issue, and a recent study reported that hypoxic culture conditions can significantly increase the efficacy of mouse HFSC differentiation to cardiac muscle cells compared with normoxic conditions.⁷² Kim et al. investigated the feasibility of developing a cardiomyocyte-specific differentiation media for mouse HFSCs.⁷³ Among the different media tested, cells cultured on OP9 feeder cells in KnockOut-DMEM/B27 in the presence of VEGF (known to promote cardiomyocyte differentiation) showed the ability to differentiate into cardiomyocytes and did not express non-cardiac lineage markers. Moreover, the differentiated cells showed morphological similarities to cardiac muscle cells and exhibited the ability to spontaneously and continuously beat for over 3 months.

Another study investigated the age as well as site of isolation of hair follicle on its ability to undergo cardiomyocyte differentiation.⁷⁴ Study showed that whiskers located near the ear have higher efficiency to differentiate toward cardiac muscle cells compared with whiskers located near the nose. Moreover, the differentiation potential of the cells isolated from 4-week-old mice was significantly higher compared with those isolated from 10-, 20-, and 40-week-old mice, and differentiation potential significantly reduced after 10 weeks. The study points to the possible need to bank these cells at an early age or rejuvenate stem cells using molecules such as MTOR inhibitors.

Engineering large diameter vascular grafts still present challenges due to the need of large number of cells and the biomechanical need. Several studies investigated the feasibility of developing smooth muscle cells and endothelial cells from HFSCs. Liu et al. showed the ability to obtain functional smooth muscle cells from ovine hair follicles using smooth muscle actin-driven EGFP expression and fluorescence-activated cell sorting.⁷⁵ The study demonstrated that hair follicle can serve as a rich source of highly proliferating smooth muscle progenitor cells with contractile functions as demonstrated by compaction of fibrin gels. Moreover, the study demonstrated the ability of the progenitor cells to differentiate into mature smooth muscle phenotype in the presence of TGF-β1.

A follow-up study investigated the feasibility of isolating smooth muscle cells from multipotent human HFSCs.⁶⁵ Culturing cells in bFGF supported cell proliferation and prevented myogenic differentiation demonstrating the possibility of obtaining large numbers of undifferentiated stem cells for clinical application. Smooth muscle progenitors from HFSCs were then encapsulated in fibrin gel and cultured in TGF-β1 to promote smooth muscle cell differentiation. The cells showed significant ability to compact the gel and exhibited remarkable contractibility in response to receptor and non-receptor agonists. The construct also showed superior mechanical properties compared with construct seeded with human vascular smooth muscle cells.

Xu et al. investigated the hypothesis that the cross talk between platelet-derived growth factor BB and TGF-β1 will enhance human HFSC differentiation toward contractile smooth muscle cells for vascular tissue engineering.⁷⁶ The study demonstrated the efficacy of in vitro-expanded human HFSCs to differentiate and express SMC markers such as α-smooth muscle actin, α-calponin, and smooth muscle myosin heavy chains (MHC) when cultured in low serum media. Moreover, the differentiated cells upon encapsulation in collagen matrix showed contractile functions. A recent study demonstrated that TGF-β1-mediated differentiation of human HFSCs to SMCs is regulated by microRNA (miR-128) via targeting SMAD-2, a main transcription regulator.⁷⁷ microRNAs are non-coding RNAs that are known to play crucial roles in cell differentiation. The study showed that miR-128/SMAD2 can serve as a unique target in vascular tissue engineering using HFSCs.

Studies also showed the feasibility to differentiate human HFSCs to vascular endothelial cells, another critical cell population for vascular tissue engineering. Xu et al. showed the efficacy of differentiating human HFSCs to endothelial lineages by culturing in the presence of VEGF and bFGF.⁷⁸ The differentiated cells showed endothelial cell markers similar to human umbilical vein endothelial cells (ECs) such as von Willebrand factor (vWF), vascular endothelial cadherin, and CD31 and showed capability for endothelial tube formation. Using rat HFSCs, another study demonstrated the ability of VEGF-165 in promoting differentiation of HFSCs to vascular endothelial cells as well as the role of Notch signaling pathway in modulating the differentiation efficiency.⁷⁹ In vivo transplantation of HFSCs and VEGF-165 in Matrigel into the subcutaneous tissue of nude mice showed the ability of the cells to promote vascularization via host-derived neovascularization. The study showed the potential of HFSCs not only for vascular tissue engineering but also for treating ischemic diseases.

Sarkovic et al. studied the feasibility of differentiating human outer root sheath-derived HFSCs to differentiate into endothelial cells and smooth muscle cells as potent cell source for tissue engineering vascular grafts. The study demonstrated the higher efficacy of HFSCs to differentiate compared with adipose-derived MSCs. The differentiated HFSCs expressed higher levels of VEGF genes compared with human umbilical vein ECs. Moreover, differentiated HFSCs showed higher ability to build anastomotic tubular networks superior to adipose-derived stem cells. Importantly, the study showed the feasibility to form a co-cultured cell membrane complex by attaching endothelial cells onto the smooth muscle cell sheet, demonstrating the unique advantage of the approach for developing engineered vascular grafts.⁸⁰

Peng et al. demonstrated the utility of HFSCs to develop engineered vascular grafts. HFSCs were seeded on hydrated small intestinal submucosa (SIS).⁸¹ The construct was then subjected to uniaxial strain for 24 h or 2 weeks. SIS supported HFSC alignment and allowed development of vascular contractility in response to receptor or non-receptor-mediated constriction by day 1 post-seeding. By 2 weeks, cells migrated into the matrix and laid down collagen and elastin and the contracting response increased three- to fivefold. These positive outcomes were further confirmed using an ovine model wherein the tissue-engineered vascular graft remained patent for at least 3 months.⁸² These studies demonstrated the unique advantage of combining HFSCs with SIS to form mechanically robust and biologically functional constructs for arterial implantation.

Another study demonstrated the feasibility to culture differentiated HFSCs on poly(glycolic acid) sheets to form large diameter tissue-engineered vascular grafts. Histological evidence indicates significant increase in matrix synthesis when seeded with differentiated HFSCs compared with undifferentiated cells.⁸³ Gao et al. used acellular umbilical arteries as scaffolds for culturing HFSC to develop tissue-engineered vascular grafts.⁶⁶ The engineered arterial grafts showed close similarity in the structure and functions of native blood vessels and exhibited vasoreactivity in response to humoral constrictors demonstrating its potential to be used as small diameter arterial grafts for cardiovascular regeneration. These studies showed the unique potential of HFSCs for vascular tissue engineering.

Neural tissue engineering

With the studies showing the feasibility to obtain nestin expressing and neural crest-like cells from HFSCs, there is a significant interest in using these stem cells for neural tissue engineering. Several studies investigated different culture media to optimize HFSC differentiation toward neuronal lineages. While retinoic acid, serum-free media containing EGF, bFGF, and neural differentiation media are all culture conditions that support neuronal differentiation of CD34+ HFSCs, the serum-free medium significantly increased the number of glial fibrillary acidic protein (GFAP)-positive glial cells.^84–86 The protocol to isolate neural crest stem cells from human hair follicle is also reported.⁸⁷ Ni et al. demonstrated the ability of microRNA (miR-21) to promote the differentiation of neural crest stem cells from human HFSCs into Schwann cells.⁸⁸ The study also showed that the differentiation was through the downregulation of SOX protein expression in the cells, which is a protein that plays a role in inhibiting neural crest cells from differentiating into Schwann cells.

Mignone et al. demonstrated the multipotent nature of nestin-expressing stem cell population from bulge HFSCs.⁸⁹ The study demonstrated the ability of the cells to form neurosphere in vitro and ability to differentiate into various cell types such as neurons, astrocytes, oligodendrocytes, smooth muscle cells, and adipocytes. The in vivo efficacy of the cells to form neuronal cells was demonstrated by implanting them into the developing nervous system of chick. The cells injected into the dorsal part of the neural tube were able to migrate along the pathways of migration of neural crest cells and differentiate into neuronal cells. Wu et al. isolated nestin-negative human outer root sheath HFSCs and evaluated their efficacy to differentiate into neurons in vitro.⁹⁰ The neural induction media consists of DMEM-high glucose medium containing 10 ng/mL of NT-3 (a regulator of neural survival and differentiation), nerve growth factor, and 50 ng/mL of brain-derived neurotrophic factor. The cells exhibited sphere-forming ability and expressed nestin upon neuroinduction. Under the culture conditions, the cells expressed neuronal (GAP-43, NTR-3, and P75^NTR) and gliocyte (GFAP and S100) markers, however, did not express mature neuron markers (NF-M, NeuN, and NSE) indicating their ability to differentiate to neural stem cells rather than mature neurons or neurogliocytes.

Amoh et al. demonstrated the ability of nestin-positive, keratin-negative, and CD34+ bulge HFSCs to form neurons in vivo upon transplantation to the subcutaneous tissue of nude mice. The transplanted cells show the ability to migrate within the subcutaneous tissue at 7 days, and by 14 days post-implantation, the cells differentiated into neurons. The study showed the efficacy of bulge HFSCs to serve as an autologous source of multipotent stem cells for neural tissue engineering.¹⁵ The nestin-expressing mouse HFSCs also showed the ability to differentiate into neuronal and glial cells upon transplantation to injured peripheral nerve and spinal cord. The cell therapy led to injury repair and increased locomotor recovery demonstrating its ability to repair peripheral nerve and spinal cord injury.⁹¹ The ability of human HFSCs in nerve regeneration was demonstrated by injecting the cells around impinged sciatic nerve of ICR nude mice.⁹² Upon transplantation, the cells differentiated into GFAP-positive Schwann cells and promoted the recovery of the pre-existing axons. The restoration of the sciatic nerve function was confirmed by measuring the contraction of the gastrocnemius muscle upon electrical stimulation. Yamazaki et al. encapsulated mouse bulge HFSCs on polyvinylidene fluoride membrane cylinders and transplanted them to the severed sciatic nerve of immunocompetent and immunocompromised mice.⁹³ The ability of the transplanted cells to differentiate to neurons and glial cells was evident by 8 weeks. Moreover, the cells promoted rejoining of the sciatic nerve ends leading to sciatic nerve regeneration in both immunocompetent and immunocompromised mice and the mice recovered the ability to walk normally.

A subsequent study used the same approach of encapsulating nestin-positive mouse bulge HFSCs in poly(vinylidine fluoride) membranes and transplanted into severed thoracic spinal cord of nude mice.⁹⁴ By 7 weeks, the ability of the transplanted cells to form neurons and glial cells were evident. The cell therapy supported complete reattachment of thoracic spinal cord. Moreover, the treated mice showed significant increase in the motor functions. The ability of nestin-positive HFSCs to differentiate into motor neurons was previously demonstrated.⁹⁵ The nestin-positive cells were produced by culturing mouse HFSCs in the presence of serum and retinoic acid. The cells were encapsulated in Matrigel and transplanted into the transected distal sciatic or tibial nerve stump of transgenic nude mice. The transplanted cells expressed neuronal markers Isl1/2 and EN1. By 2 weeks post-transplantation, the nerve fibers in the distal sciatic nerve stump showed higher expression of motor neuron markers and neurotrophic factor-3. Moreover, the muscle fiber areas of the transplanted group were significantly higher than the control demonstrating the ability of HFSC cell therapy to reduce muscle atrophy.

Amoh et al. demonstrated the ability of nestin-positive HFSCs to treat severed thoracic spinal cord in immunocompetent mice.⁹⁶ The transplanted cells differentiated into Schwann cells, which in turn facilitated the spinal cord repair and reestablishment of high-limb performance. Amoh et al. further explored the translational potential of this approach by directly transplanting the nestin-positive mouse HFSCs without in vitro culture into the gap region of severed sciatic nerve in mice. The transplanted cells predominantly differentiated to form glial cells and promoted axonal growth and functional recovery.^97,98

Even though nestin-positive cells are found in the bulge and DP of mouse hair follicles, cells from the bulge showed higher sphere-forming efficiency.⁹⁹ When transplanted into injured spinal cord, both cells differentiated into neuronal cells and accelerated spinal cord repair. Both showed similar effects toward locomotor recovery. However, the more constant expression of nestin in the bulge cells suggests it as a major source of pluripotent stem cells. In addition to the mouse models, several rat models were also studied. Najafzadeh et al. evaluated the efficacy of rat HFSCs in spinal cord regeneration using a rat compression-induced spinal cord lesion model.¹⁰⁰ The differentiation of cells into oligodendrocytes and neuronal cells 3 weeks post-transplantation was demonstrated. Moreover, cell therapy promoted motor function recovery by 8 weeks post-transplantation.

Liu et al. studied the feasibility of developing tissue-engineered nerve conduit by seeding rat HFSC-derived neurons on beagle acellular nerve grafts for repairing peripheral nerve injury with long distance defects.¹⁰¹ Another study explored the potential of poly-L-lactic acid (PLLA) nanofiber scaffolds on rat bulge HFSC neuronal differentiation.¹⁰² PLLA substrates significantly increased cell viability and when cultured in the presence of NT-3, the cells differentiated into neuronal lineage. Lin et al. used a cell-scaffold approach to evaluate the potential of rat HFSCs in sciatic nerve regeneration using a rat sciatic nerve defect model.¹⁰³ HFSCs were first differentiated into neurons and Schwann cells. The cells were then implanted using acellular nerve xenograft. The study demonstrated the ability of transplanted cells to survive even up to 52 weeks and support nerve regeneration. These studies show the significant advantages of HFSCs as a unique autologous stem cell source for nerve tissue engineering.

Conclusions

Studies in the past few decades demonstrated hair follicle as a unique source of different types of cells with multi- and pluripotent differentiation capabilities. The high proliferation rate of these stem cells makes them an attractive autologous stem cell source. HFSCs demonstrate an impressive spectrum of differentiation capabilities ranging from epithelial cell types, endothelial and smooth muscle cells, cardiomyocytes, neurons, astrocytes, glia, osteoblasts, adipocytes, and chondrocytes. Moreover, the ability of HFSCs to grow and undergo differentiation when seeded on biomaterial constructs makes them potential candidates for cell therapy and tissue engineering. Skin, bone, cardiovascular, and nerve tissue engineering are just a few among many promising clinical applications for this versatile cell source.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by the National Institutes of Health (Grant No. R01 AR075143) and Health Research Program at the University of Connecticut.

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