Regulation of stem cell fate by HSPGs: implication in hair follicle cycling

Heparan sulfate proteoglycans (HSPGs) are part of proteoglycan family. They are composed of heparan sulfate (HS)-type glycosaminoglycan (GAG) chains covalently linked to a core protein. By interacting with growth factors and/or receptors, they regulate numerous pathways including Wnt, hedgehog (Hh), bone morphogenic protein (BMP) and fibroblast growth factor (FGF) pathways. They act as inhibitor or activator of these pathways to modulate embryonic and adult stem cell fate during organ morphogenesis, regeneration and homeostasis. This review summarizes the knowledge on HSPG structure and classification and explores several signaling pathways regulated by HSPGs in stem cell fate. A specific focus on hair follicle stem cell fate and the possibility to target HSPGs in order to tackle hair loss are discussed in more dermatological and cosmeceutical perspectives.


INTRODUCTION
Hair follicles (HFs) are mini-organs under the skin allowing to hair shaft growth 1 . A HF can be divided into three parts. The infundibulum is the upper portion between the skin surface and the sebaceous duct outlet. This duct connects the infundibulum of the hair follicle to the sebaceous gland, allowing the excretion of sebum along the hair shaft to hydrate the scalp. The middle part of the HF is the isthmus which extends from the sebaceous duct to the bulb. It is formed by different concentric layers forming the canal where the hair shaft grows 1 , from the outermost to the innermost layer: the connective tissue sheath, the outer root sheath (ORS), and the inner root sheath (IRS). This layer is in contact with the cuticle of the hair shaft until the level of the sebaceous canal where it disappears. The bulb, the deepest portion of the HF, is composed the hair matrix which surrounds the dermal papilla 2 .
Over the course of its life, hair follicle undergoes cyclic changes 3 . Forty to one hundred hairs are lost per day. Their renewal occurs during the hair growth cycle which is characterized by three main phases: anagen, catagen and telogen. An additional phase, called exogen phase, is controlled separately and leads to hair shaft loss 4,5 . The anagen phase allows the generation and growth of new hair shafts 6 . It lasts between three to 6 years on the average, divided into six stages. This phase is characterized by a remodeling of the HF morphology due to the activation (at the end of the telogen phase) and the intense proliferation of different cell types 7,8 . During the next catagen phase, the hair shaft stops growing and the transient segment of HF regresses 8 . It is divided into eight stages 8 and lasts between 15 and 20 days. During this phase, apoptosis of keratinocytes is observed in a localized area, particularly at the junction of the secondary hair germ (SHG) and the dermal papilla (DP) 8 . The telogen phase is characterized by a dormant state of the DP and hair follicle stem cells making it a resting phase 8 . During this phase, the hair shaft remains anchored in the hair follicle 6 . After several months, the HF will return to the anagen phase thanks to stimuli.
The hair growth cycle is centered on the activation of HF pluripotent cells to differentiate and to provide the different cell lineages of the HF. Indeed, the hair shaft formation and the HF remodeling involve hair stem cells, located at the bulge, which contribute to generate the different cell lineages of the sebaceous gland, epidermis, and HF 9 . The process of the hair stem cell differentiation is complex and its study has revealed different populations derived from the hair stem cells. Five major distinct cell populations are defined in the literature based on their location, provenance, cell fate during the cycle phases, and their cellular markers: bulge stem cells, ORS progenitor cells, SHG progenitor cells, matrix transit-amplifying (TA) cells, and terminally differentiating cells (of the hair shaft, IRS and ORS) [10][11][12][13] . Moreover, the process of the hair stem cell differentiation involves many other cell types and specific niches: keratinocytes, fibroblasts of the DP, endothelial cells, fat cells, and immune system cells. The set of possible interactions between these different cell types complicates the study of the regulation of hair stem cell differentiation during the hair growth cycle.
All these cellular interactions are still poorly characterized, but it is known that growth factors (GFs) regulate the passage between the different phases of the hair cycle 14 . In particular, for the telogen to anagen transition and the hair shaft growth, several GFs are involved (such as Wnts, bone morphogenic proteins (BMPs), hedgehogs (Hhs) and fibroblast growth factors (FGFs)). A fine regulation of the GFs involved in the hair shaft growth is essential for the process of the hair cycle. The mechanisms involved in the regulation of these growth factors are still poorly understood, but several studies suggest that heparan sulfate proteoglycans (HSPG) are involved. These studies have shown an evolution of HSPG expression and distribution on the HF according to the phases of the hair cycle [15][16][17][18] . It has been demonstrated that the morphogenesis of a correct hair shaft requires a complex control of HSPGs production and sulfation 19 . Moreover, HSPGs are known to regulate many GFs involved in 1 Université de Reims Champagne-Ardenne, SFR CAP-Santé (FED 4231), Laboratoire de Biochimie Médicale et Biologie Moléculaire, Reims, France. 2
The purpose of this review is to make an update of the pivotal role of HSPGs in stem cell fate. Moreover, a specific focus on hair follicle stem cell differentiation during hair shaft growth is reported. Further, applications of these findings in the context of alopecia are also discussed.
HSPG structure, synthesis, trafficking and location Structure of glycosaminoglycans and proteoglycans. In this section, the structure, the synthesis, the trafficking and the location are briefly described and summarized in Table 1. Proteoglycans (PGs), components of the extracellular matrix (ECM) and cell membranes, are produced by many cell types 26 . They are composed of a core protein to which one or more linear polysaccharide chains of glycosaminoglycans (GAGs) are covalently attached 27 . GAG chains are composed of a repeat of disaccharide units formed from a hexosamine (N-acetyl-glucosamine or N-acetyl-galactosamine) and an uronic acid (D-glucuronic acid or L-iduronic acid) or a single ose 27 . The detailed structure of the six different types of GAG has been already described [27][28][29][30] and is illustrated in Fig. 1.
The nature of the HS GAG chains, in addition to the amino-acid sequence of the core protein, defines the characteristics and properties of the HSPGs as well as their trafficking.
Biosynthesis and trafficking of HSPGs. HS biosynthesis takes place in the Golgi apparatus 38 . The HS GAG chain is formed in a stepwise manner by glycosyltransferases and sulfotransferases 39 . They use as substrate uridine diphosphate (UDP)-sugar and 3'-phosphoadenosine-5'-phosphosulfate (PAPS), respectively.
The first step of GAG chain initiation on core protein is the formation of the "linker" 38 . This step consists of the binding of a xylose to a serine of the core protein by xylose transferase. Then the addition of the other three monosaccharides forming the tetrasaccharide unit is catalyzed by galactosyl transferase I and glucuronic acid transferase I 39 . The next step consists of the elongation of the HS GAG chain by the addition of repeated disaccharide units 38,39 . Studies have shown that several factors influence this step and predetermine the type of GAG produced. For example, the amino-acid sequence surrounding the serine residue where the GAG chain covalent binding occurs; 40,41 the production and transport of UDP-sugars in the Golgi apparatus; 39 or the "linker" phosphorylation, epimerization or sulfation 42,43 .
Once the HSPGs have been synthesized, they are transported to the membrane or secreted to the ECM (except serglycin which is cytoplasmic). These two processes are influenced by different conditions (pH, specific amino-acid sequence, nature of sugars, glycosylphosphatidylinositol (GPI) anchor…). Studies, conducted on polarized cells, show that HSPGs are sorted to be secreted basolaterally to bind to the plasma membrane or apically to join the ECM 39 .
Classification of HSPGs according to their location. There are three main families of proteoglycans differing in their location. HSPGs are either cytoplasmic, either bound into the cell membrane, or composing the basement membrane or secreted in the ECM.
Cytoplasmic HSPGs. Only one PG belongs to the cytoplasmic PG family: serglycin. It is composed of a small core protein, about 16 kDa 44 , characterized by serine-glycine repeats 45 and different GAG chains depending on the cell type 46 . Most commonly, serglycin has GAG chains of HEP and/or CS 47 but can also rarely exhibit GAG chains of HS and CS 48 . Serglycin is found in different cell types of hematopoietic origin such as cytotoxic T lymphocytes, neutrophils, and eosinophilic polymorphs 49 . It is also detected in macrophages 50 , endothelial cells 51 and in embryonic stem cells 52 . When associated with HEP or HS chains, this PG is predominantly present in mast cells rich in secretory granules 50,53 .
Serglycin plays an important role in hematopoietic cell protease activity 54 . Indeed, it regulates the storage of proteases in the secretory granules as well as their protease activity during their intracellular release. It also plays a role in the immune response, in particular in the inflammatory response, by interacting with many components of the immune system 54 .
Moreover several studies have shown the pro-tumorigenic functions of the serglycin 55 and its ability to induce epithelial to mesenchymal transition 56,57 .
Membranes HSPGs. Membrane HSPGs are composed of several members grouped into two major families: transmembrane and GPI-anchored HSPGs 45 .
The transmembrane HSPG family is largely represented by syndecans (SDCs), and three other members: betaglycan, neuropilin-1 and CD44 58,59 (Fig. 2). The three latter's can occur in two forms: with or without GAG chains that may be HS and/or CS or DS 58 . They regulate many cell signaling pathways as GF coreceptors 58,59 .
The SDC group is composed of four members: SDC1 to 4, in order of discovery. They differ from each other largely by their extracellular domain, which is the most variable area of the core protein, with only 10-20% similarity 60 . Within the highly conserved amino-acid sequence of SDC family, there is a specific motif, close to the transmembrane domain, that is recognized by proteases responsible for cleaving the extracellular domain of SDCs and sequences involved in the attachment of GAG chains 61 . All four syndecans have HS chains at their N-terminus. In addition, SDC1 and 3 also carry CS chains (Fig. 2). The transmembrane and cytoplasmic regions of the syndecans are highly homologous, with 60 to 70% sequence homology 60 . The cytoplasmic domain is characterized by a   SDCs are present on the surface of most of the cells and their expression is finely regulated. Depending on the stage of cell, tissue or organ differentiation, the four syndecans are expressed differently and selectively, reflecting their distinct function 62 . SDCs regulate many cell processes such as adhesion, cytoskeletal remodeling, and migration 63 . They act as co-receptors for several cell signaling pathways such as FGF2, vascular endothelial growth factor (VEGF), granulocyte-macrophage colonystimulating factor (GM-CSF) or hepatocyte growth factor (HGF) 63 . They are also known for their implication in tissue repair and regeneration such as wound healing, vascular or neuronal repair [64][65][66][67] .
Glypicans (GPCs) form the GPI-anchored proteoglycan family 68 . It is characterized by a unique motif of 14 cysteine residues, which is conserved in all GPCs, including those of Drosophila, Dally and Dally-like-protein (Dlp) 23 . In human, six GPCs have been identified, GPC1 to 6 ( Fig. 3). They are divided into two subfamilies: GPC1, 2, 4 and 6 (related to Dlp) and GPC3 and 5 (related to Dally). These two subgroups present 25 % of similarity including the sequences of the insertion site of HS type GAG chains 23 . These sites are composed of highly conserved serine/glycine repeats located near the C-terminus 23,69 . The GAGs of the glypicans are exclusively HS type except for GPC5 which can also have CS-type GAG chains. All GPCs have a core protein of~60 kDa. They are expressed by many tissues and cells. Usually in polarized cells, GPI-anchored proteins are found in lipid rafts located at the apical pole 70 , involved in various cell signaling 71 . In these cells, GPI-anchored GPCs can also be present in large quantities at the basolateral region 72 . Interestingly, it has been shown that unglycosylated GPCs are localized at the apical pole, demonstrating that HS chains play a role in the trafficking of GPCs 72 . GPCs can be cleaved by different molecules such as Notum 73 , disintegrin and metalloproteinase 17 (ADAM17) 74 or phospholipase C (PLC) 75 . GPCs play a role in modulating cell signaling during tissue or organ development and regeneration by regulating many cell signaling pathways such as Wnt, Hh, BMP, and FGF 23,59,76 .
Pericellular HSPGs. There are four pericellular PGs or basal membrane PGs: perlecan, agrin, type XVIII and type XV collagens. These PGs exhibit HS chains but, the type XV collagen can also have CS chains. They are associated with laminin or type IV collagen allowing the integrity of basement membranes 77,78 . They can also be associated with the cell surface via integrins 45 ensuring signal transmission 79 , an essential function of the basement membrane. Moreover they are essential for embryogenesis and tissue maturation 80 . For example, perlecan promotes the chondrocyte differentiation 81 and agrin plays a crucial role in the hematopoietic niches 82 and monocyte maturation 83 .
ECM HSPGs. The ECM HSPGs are represented by the testican family. They are also named secreted protein acidic and cysteine rich (SPARC)/Osteonectin and Kazal-like domain proteoglycans (SPOCKs). This family is composed of three members: testican 1, 2 and 3. Their other name, SPOCK, is due to their characteristic protein domains. Their core protein is composed of five domains 84 . The N-terminal domain I is specific to the testican family, hence the name testican-specific domain, and corresponds to a signal peptide 84 . The domain II is called follistatin domain and is a module rich in cysteine. The domain III is characterized by binding sites of low affinity to calcium 85 , earning it the name of calcium binding domain. The domain IV, or thyroglobulin domain, contains a tetrapeptide sequence CWCV stabilized by three disulfide bridges corresponding to a thyroglobulin-like domain 45 . The domain V, also specific to this family, contains the two potential binding sites for GAG chains.
The GAG chains of SPOCKs are predominantly of the HS type 45 . They are expressed almost exclusively in the brain 84,86,87 and studies suggest that they play a regulatory role during central nervous system development [88][89][90] . A recent study has shown that SPOCK1 induces adipocyte differentiation and adipose tissue maturation through the up-regulation of adipogenesisrelated genes 91 .
Role of HSPGs in the regulation of stem cell differentiation during morphogenesis and regeneration of organs. In this section, the role of HSPGs in the regulation of major signaling pathways known to be involved in embryonic and adult stem cell differentiation are presented ( Table 2). The stem cell differentiation needs to be finely regulated during the development or the regeneration of organs. Several studies have shown the role of HSPGs in this orchestrated process. The mechanism of action of HSPGs is well described in numerous studies conducted on Drosophila. Indeed, it is a powerful model to understand the complex processes involved in human stem cell fate because they present very high homology with human HSPGs (Fig. 4).
Implication of HSPGs in Wnt signaling pathways. The human Wnt family is composed of 19 members that bind to Frizzled transmembrane receptors (7 members) involving a coreceptor recruitment 92,93 . The Wnt signaling pathways were demonstrated to regulate the stem cell differentiation process occurring during embryogenesis and development/regeneration of numerous organs [94][95][96][97][98][99][100][101][102][103][104][105] . Wnts are able to induce different signaling pathways 93 and to exhibit different effects on the stem cell fate, depending on the coreceptor with which Frizzled receptors are associated.
The regulation of Wnt signaling pathways is crucial for a correct differentiation of stem cell and thus for a correct morphogenesis or regeneration of organs. Some studies have shown the regulation of Wnt signaling pathways by HSPGs during the stem cell differentiation.
Regulation of Wnt signaling by HSPGs during Drosophila embryogenesis. The two first examples highlight the role of HSPGs during Drosophila embryogenesis. Kreuger and collaborators have studied the regulation of dorso-ventral axis establishment during Drosophila embryogenesis 24 . They were able to show that the glypican Dlp is involved in the formation of the Wingless (Wg) gradient necessary for the establishment of the dorso-ventral axis (Fig. 4a). Drosophila perlecan homologue Trol was shown to have   an important role during Drosophila nervous system development 106,107 . Indeed, by regulating Wg signaling, it was demonstrated to regulate the formation of pre-and postsynaptic structures 106 and a second brain instar 107 .
Regulation of Wnt signaling by HSPGs modulates vertebrate embryonic stem cell fate. In the case of vertebrate development, several studies have demonstrated the roles of HSPGs in the regulation of embryonic stem cells and neuroepithelial cells by modulating Wnt signaling. In particular, GPC4 regulates Wnt/ β-catenin signaling inhibiting the differentiation of mouse embryonic stem cells and promoting their self-renewal 108 . GPC4 was also shown to play a role during zebrafish development 109 . Indeed, it regulates Wnt/β-catenin signaling to promote the essential migration of lateral line collective cells during embryogenesis. Moreover, Sasaki and collaborators have demonstrated the crucial role of HS sulfation for the regulation of selfrenewal and differentiation of mouse embryonic stem cells 110 . A study conducted in vivo on mouse cortical neurogenesis has demonstrated that SDC1 promotes the activation of Wnt signaling pathways during neural progenitor cell differentiation 111 . This activation permits to maintain their phenotype and their proliferation potent. As last example, it has been shown that a collaboration between Wnts and agrin promotes cell differentiation for the formation of vertebrate post-synaptic neuromuscular junctions 112 .    132,133 . For example, Ayers and collaborators have been able to demonstrate the involvement of the glypican Dally in the establishment of the Drosophila anterior-posterior axis thanks to the regulation of the Hh gradient (Fig. 4b). Future cells of the posterior zone secrete Hhs, two gradients are then formed: a short-range gradient at the basolateral level of the cells by limited diffusion and a long-range gradient at the apical pole due to the sequestration of Hhs by Dally and its release by Notum, allowing to transport Hhs to act over a long distance 20 . Drosophila perlecan homologue Trol was shown to induce the activation of neural stem cell 134 and their differentiation into a specific neuroblast population by regulating Hh pathway 107 . Some studies have demonstrated the implication of Hh signaling modulation by HSPGs during vertebrate development in particular in the case of brain formation. For example, perlecan has been shown to regulate mouse neurogenesis by mediation of Shh concentration gradient 135,136 and agrin has been demonstrated to control neuron development in zebrafish brain by regulating Shh 137 .  21 . Indeed, GPC6 binds the Hh ligand with its core protein and the Hh receptor with its HS chains to promote their interaction and the long bone growth. SDC3 was also shown to be involved in chick chondrocyte proliferation and maturation by regulating Ihh signaling 142 .
Implication of HSPGs in BMP signaling pathways. The 20 secreted BMPs composed the human BMP family. They bind to transmembrane BMP receptors (BMPR) 143,144 . The signaling pathways of BMPs are involved in the regulation of vertebrate embryogenesis and vertebrate organs development/regeneration 145,146 and they are particularly known to regulate bone formation and osteoblast differentiation 147,148 .
The regulation of the activation of BMP signaling pathways is crucial for a correct differentiation of stem cell and thus for a correct morphogenesis or regeneration of organs.  Drosophila embryos have shown the regulation of these pathways through the interaction between HSPGs and BMPs 149-151 . More precisely, as shown in Fig. 4c, Dally could regulate Dpp (BMP2 and 4 Drosophila homologous) through the stabilization of the interaction between Dpp and its receptor or the sequestration of Dpp 151 . Moreover, Dally could participate to the internalization and the degradation of the Dpp-receptor complex 149 during wind development. Trol is known to play a role during Drosophila second brain instar formation by regulating Dpp in a Trol dependent manner 107 .
In the case of vertebrate embryogenesis, few evidence show the implication of HSPGs in the regulation of BMP signaling. In particular, the HS chain and its sulfation are crucial for a correct regulation of mouse embryonic stem cell differentiation 110 and for mesoderm formation from mouse embryonic stem cells 152 induced by BMP.
Regulation of BMP signaling by HSPGs modulates adult stem cell fate. Some studies have been conducted on adult stem cell differentiation to understand how HSPGs regulate the BMP signaling pathways in the context of organ homeostasis or regeneration. As first example, two studies conducted on Drosophila adult stem cells have demonstrated that Dally is the co-receptor of Dpp in the germline stem cell niche and it regulates the number of these stem cells 150,153 .
In the case of vertebrate adult stem cells, several studies performed on osteogenic and chondrogenic progenitors have revealed the role of HSPGs in the regulation of BMP signaling involved in cell maintenance or differentiation. HS chains have been shown in vitro to potentiate the BMP2-induced bone repair by prolonging BMP-2 half-life, reducing interactions between BMP-2 with its antagonist noggin, and modulating BMP2 distribution on the cell surface 154 . SDC3 has been demonstrated in vitro to impair the interaction between BMP2 and its receptors leading to an inhibition of chondrogenesis during cartilage differentiation 155 . Similarly, GPC1 and 3 were able to inhibit BMP signaling and the osteogenesis mediated by BMP2 in human primary cranial suture mesenchymal cells 22 . BMP2 is also known to be regulated by perlecan. In contrast, perlecan has been shown in vitro to stimulate chondrogenic differentiation by modulating BMP2 156 and to improve osteogenesis by increasing BMP2 signaling 157 . Finally, agrin has been demonstrated to play a role in osteoblast differentiation by regulating BMP signaling pathways 158 .
Implication of HSPGs in FGF signaling pathways. The human FGF family is composed of eighteen secreted proteins that can induce various different actions by binding one of the four FGF receptors (FGFR) 159 . The FGF signaling is known to regulate stem cell pluripotency and differentiation 160,161 . Numerous studies have demonstrated the implication of FGF in embryogenesis 105,162 and in development/regeneration of various organs 161 such as bones 163,164 , spinal cord 165 or lung 166,167 .
The FGF signaling pathways need a fine regulation for a correct morphogenesis or regeneration of organs and numerous studies have proven the role of HSPGs in these processes. Other studies, carried out on neuroepithelial cells have proven evidence of the role of different HSPGs on FGF signaling regulation during embryogenesis. In the case of neural stem cell proliferation, survival and differentiation, it has been shown that the HS chains of HSPGs and their sulfation are essential for the regulation of FGF distribution and binding and thus for the correct neuroepithelial tissue development [170][171][172][173][174] . The regulation of FGF signaling by GPCs seem to be highly implicated in the brain development processes. For example, GPC1 was shown to regulate the interaction between FGF17 and its receptor during early neurogenesis controlling the size of the mouse brain 175 . In mouse neuroepithelial cells, the HS chains of GPC4 are able to sequestrate FGF2 to prevent the binding with its receptor leading to the maintenance of their proliferative stem cell phenotype 176 . In contrast, in the case of Xenopus neurulation, GPC4 was demonstrated to bind FGF2 to facilitate the binding with its receptor regulating the dorsoventral forebrain patterning 177 . As last example for GPCs, a study conducted on mouse cerebral cortical development suggests a role of GPC6 in the regulation of FGF2 signaling during this process 178 . Perlecan was also shown to be important in the regulation of the brain development modulated by FGF signaling pathways. In the case of Drosophila, Trol is able to mediate FGF signaling to activate neural stem cell division 134 . In the case of vertebrate, FGF2 signaling is modulated by perlecan to regulate proliferation and differentiation of neural stem cells 135,179,180 . Agrin was demonstrated to regulate GABAergic and glutamatergic neuron development in zebrafish forebrain by the modulation of FGF signaling 137 .
Regulation of FGF signaling by HSPGs modulates vertebrate adult stem cell fate. Numerous studies have provided evidence on the role of HSPGs in vertebrate adult stem cell differentiation modulated by FGF signaling pathways. SDC3 was shown to induce the proliferation of chick chondrogenic progenitors by modulation of FGF2 signaling 181,182 . The regulation of FGF2 signaling by the HS chains of HSPGs was demonstrated to be essential for the regulation of mouse muscle satellite cells and myoblasts differentiation 183,184 . SDC3 is able to facilitate the interaction between FGF2 and its receptor leading to the repression of myogenic differentiation of murine skeletal myoblasts 185 . In contrast, GPC1 was reported to sequester FGF2, preventing its binding to its receptor, to promote mouse muscle differentiation 186 . The last following example highlights the role of HSPGs on skin progenitor cell fate modulated by FGF signaling. GPC1 is expressed by the epidermis keratinocytes mainly in the basal layer where the progenitors are present. In parallel, GPC1 cleavage is able to decrease human keratinocyte proliferation induced by FGF2 187 . These results tend to indicate a role of GPC1 in skin precursor cell proliferation during skin regeneration.
In case of adult stem cell fate, the role of HSPGs on chondrogenesis and cartilage formation was confirmed by other studies conducted on HS chains 200 and perlecan 201 . Moreover, the HS chains have been shown to potentiate the TGFβ3 signaling promoting chondrogenic differentiation of human mesenchymal stem cells 202 .
The sulfation of HS chains is crucial for a correct mouse colonic epithelial cell differentiation 203 . SDC3 interacts with Notch and regulates mouse adult myogenesis 204 .
As last examples, the role of HSPGs on the regulation of skin progenitor cell differentiation are presented. The first evidence was proven by the HSPG distribution within the epidermis which depends on the state of keratinocyte differentiation 205,206 . For example, SDC1 is weakly detected in the basal layer of epidermis and highly expressed in the suprabasal layers 207 . On the contrary, GPC1 is distributed throughout the epidermis but preferentially in the basal layer 187 . Few studies have been conducted on the pathways regulated by HSPGs involved in epidermis progenitor cell differentiation (see FGF subsection). Another example is the regulation of adhesion and early differentiation of keratinocytes progenitor cells by the formation of a complex between transient receptor potential canonical channel 4 (TRPC4) and SDC, demonstrating in Caenorhabditis elegans 208 .
All these examples demonstrate the role of HSPGs on the stem cell fate via the regulation of growth factor distribution, sequestration and downstream signaling pathway. Moreover, they highlight the opposing effects that HSPGs can exhibit on stem cell behaviors, either by favoring the maintenance of pluripotency or by promoting differentiation. Finally, these examples also show the different mechanisms of action of HSPGs. Indeed, they can sequester growth factors to prevent or facilitate the interaction with the receptors thanks to their HS chains or they can promote the interaction by their core protein.

Importance of HSPG in hair follicle stem cells differentiation
Major signaling pathways involving in hair follicle stem cell differentiation. In the case of hair follicles, the major signaling pathways of growth factors (Wnt, Shh, BMP and FGF) are particularly important to regulate stem cell maintenance or differentiation during hair cycle and hair shaft growth (Fig. 5).
The platelet-derived growth factor (PDGF) secreted by fat cells activates the Wnt pathway within the DP 209 . In parallel, an inhibition of the BMP pathway is observed in the adipose macro-environment and in the dermal papilla 210,211 . The BMP pathway is active throughout the telogen phase and allows the maintenance of quiescence of SHG progenitor cells.
At the end of the telogen phase, various BMP inhibitors are secreted from the dermal papilla, particularly Noggin 211 (Fig. 5). With the BMP pathway inhibition, secretion of Wnts from the dermal papilla has an activating effect on SHG progenitor cells and bulge stem cells at early anagen phase. Then, the Wnt/β-cat pathway will be activated in SHG progenitor cells and in bulge stem cells 211 .
The activation of these cell types leads to the secretion of different growth factors regulating the hair stem cell differentiation and the formation of a new hair shaft. For example, the production of insulin-like growth factor 1 (IGF1) by the DP controls the proliferation and the differentiation of SHG progenitor cells to regenerate the hair matrix 212 . The keratinocyte growth factor (KGF or FGF7), produced by the DP, stimulates the hair matrix cells, which then produce keratinocytes to form the new hair shaft 212 . The secretion of HGF by the DP promotes the elongation of the hair shaft 213 . Hedgehog (Hh), secreted by the hair matrix cells, stimulates the bulge stem cells to provide a new pool of SHG progenitor cells necessary for the next growth cycle 214 . In parallel, angiogenic pathways are also involved. The fibroblasts of the DP as well as the keratinocytes of the matrix and the ORS secrete VEGF which induces the formation of new blood vessels which provide the nutrient supply necessary for the formation of the hair shaft 212,215,216 .
Other growth factors are very important for the anagencatagen transition. For example, epithelial growth factor (EGF) and FGF5 are necessary for this transition 217 . TGFβ and brainderived neurotrophic factor (BDNF) inhibit HGF expression and VEGF secretion, respectively 14 . Moreover, the inhibition of Noggin and Wnt pathway signaling, as well as FGF18 and BMP2, 4 secretion by the dermal papilla, promote the quiescence of bulge stem cell 14,211 .
This brief summary provides an overview of the intense complexity of the crosstalk required for the maintenance and regulation of the hair cycle. This level of regulation is permitted by multiple growth factors, several signaling pathways and includes numerous different cell types. These regulations are still poorly understood but some studies provide evidence of the implication of HSPGs in these regulatory processes.
Involvement of HSPGs in hair follicle stem cell differentiation. In this section, the roles of HSPGs in hair follicle stem cell fate are presented ( Table 2).
Studies have been conducted on the distribution of HSPGs in the different hair structures during hair growth cycle. During the anagen phase, HS chains are detected in the basement membrane, connective tissue sheath and the dermal papilla of hair follicle 17,[218][219][220][221] . Experiments conducted on specific HSPGs have demonstrated that perlecan is expressed in basement membrane and the dermal papilla; 17 SDC1 is expressed in the ORS, in the hair shaft and lower in the IRS and dermal papilla; 15,17 GPC1 is expressed in the hair matrix (more strongly in the differentiation zone) and less in the hair shaft 16 .
Moreover, it has been shown that HSPG distribution in hair follicle evolves during hair growth cycle. For example, it has been demonstrated that perlecan is still expressed in basement membrane and connective tissue sheath during catagen phase but in dermal papilla a decrease of its expression is observed in late catagen 17 . Bayer-Garner and collaborators have shown that SDC1 expression in ORS decreases in telogen phase 15 . In contrast, the distribution of GPC1 in the hair matrix and hair shaft seems to be the same all along the hair cycle 16 . Moreover, it has been demonstrated that a fine regulation of HSPG sulfation is necessary for a correct formation of hair shaft 19 . It is interesting because the type and/or the degree of sulfation vary during hair cycle 16 . These studies suggest a role of HSPGs on hair follicle stem cell fate. Indeed, the tissular or cellular distribution of growth factors is associated with the expression of HSPGs during Drosophila embryogenesis 20,24,151 , bone formation 222 or skin regeneration 187,208 where HSPGs are demonstrated to regulate the pathways involved during these processes.
One another evidence is the fact that other proteoglycans, such as CSPG or DSPG regulate the signaling pathways involved in hair follicle stem cell fate. In particular, versican expressed in the dermal papilla is well described. Several studies showed the ability of versican to induce anagen phase and hair inductivity 219,223 via the Wnt/β-cat pathway 224 . In addition, decorin was shown to be an anagen inducer probably by downregulating TGFβ signaling 225 . The similarity between the mechanisms of action of HSPGs and other sulfated proteoglycans emphasizes the role of HSPGs in the regulation of signaling pathways involved in hair follicle stem cell differentiation.
To conclude this section, the key role of HSPGs in the regulation of hair growth cycle and hair shaft formation is well established. Unfortunately, despite several studies carried out on the distribution of HSPGs on hair follicles, only few studies have investigated the mechanism by which HSPGs regulate these processes and which growth factor and signaling pathways are involved. Further works are necessary to better understand HSPG mechanisms of action and to develop HS proteoglycan-based therapies for hair disorders.
HSPGs as therapeutic targets for androgenetic alopecia. Androgenetic alopecia accounts for 90% of alopecia cases and affects 50% of women and 80% of men in their lifetime 226 . In men, it manifests as hair loss in localized areas 227 and diffused loss in women 228 . According to Grand View Research, Inc., the global alopecia market size was valued in 2020 at USD 7.6 billion. From 2021 to 2028, it is expected to expand at a Compound Annual Growth Rate of 8.1% and to reach USD 14.2 billion. Androgenetic alopecia is due to an excessive supply of androgen to the dermal papilla causing physiological disruptions in hair follicles 229 . As described in the previous chapter, the Wnt signaling is crucial for hair stem cell differentiation and for the growth of the new hair shaft. In vitro, androgens were shown to inhibit the production of Wnt by dermal papilla cells 230 . This type of inhibition could explain the deregulation in vivo of the various growth factors secreted during the anagen phase. These deregulations during the anagen phase promote hair follicle miniaturization 231 . For example, the inhibition of IGF1 and KGF promotes hair thinning 231 . Shh inhibition disrupts the SHG formation and VEGF inhibition disrupts perifollicular revascularization. In addition, it has been shown that androgens stimulate the secretion of TGFβ 231 and IL-6 232 that induces premature transition to the catagen phase. In other context, androgens modulate the HSPG expression. For instance, the SDC1 expression decreased in the mouse mammary tumor cells after incubation with testosterone 233 . In addition, the steroid hormone estradiol has been demonstrated to modulate SDC3 expression and distribution playing a role in rat uterine growth 234 .
Currently, two major drug treatments exist, Minoxidil (lotion) and Finasteride (oral tablet) as well as surgical and low-level laser treatments 227,235 . Several cosmetic active ingredients, such as Stemoxydine, have also been developed and packaged in the form of shampoo, hair lotion, etc. These active ingredients reduce inflammation and/or improve micro-vascularization to promote the growth phase of the hair shaft and stop hair loss (Table 3). These drug treatments act to restore cellular signaling pathways dysregulated in androgenetic alopecia. For example, Minoxidil was demonstrated to activate the Wnt pathway in the mouse dermal papilla in vivo and in human dermal papilla cells in vitro 236 . This activation may be related to a stimulation of adipocyte precursors to secrete PDGF that activates the dermal papilla 237 . The stimulation of Wnt signaling in dermal papilla is able to restore the secretion of IGF1, HGF, and VEGF 238 . Furthermore, during the catagen phase, Minoxidil was shown to inhibit TGFβ-induced apoptosis in matrix TA cells and to increase the Bcl-2/Bax ratio protecting cells from apoptosis 239 .
Some studies have highlighted the link between hair growth disorder and the alteration of the expression and/or the distribution of proteoglycans. In particular, it has been shown that the dermal papilla of hair follicle isolated from a bald area presents a lower gene and protein expression of versican compared to those isolated from a hairy area 240 . The alteration of the expression and/or the distribution of HS proteoglycans in case of hair loss could be explained by the fact that androgens are able to modify the HSPG expression. Finally, evidence demonstrates the role of HSPGs in hair follicle stem cell fate during hair growth cycle. Altogether, these data highlight the role of HSPGs is the hair follicle physiopathology and make HSPGs as interesting targets for treatment of androgenetic alopecia. Despite this fact, few studies have been conducted to develop proteoglycan-based treatments for hair loss (and no one on HSPG-based treatments). After many years of proteoglycans related studies on their potential therapeutic development, Wadstein and Thom were the first to demonstrate the effect of proteoglycan concentration or synthesis in hair follicle growth 18 . Based on a clinical study, a cocktail of proteoglycans (oral administration) was shown to induce changes in hair follicle structure 241 . A specific cocktail of proteoglycans (Nourkrin), rich in lectican and decorin, was formulated. Clinical studies, conducted on patient with hair loss, have proven its efficacy on hair growth in monotherapy 242,243 or in add-on treatment 244 by increasing the hair count and reducing hair loss. Nourkrin is developed as oral treatment. It seems that the oral ingestion of proteoglycans leads to an increase of proteoglycan concentration in the hair follicles by direct deposition and/or synthesis of proteoglycans without secondary effect 18 . Nourkrin is a drug-free bioactive proteoglycan formula, based on natural ingredients. Studies are required to develop topical HS proteoglycan formula. However, some studies highlight possible vehicle or formula to induce the HS proteoglycan skin penetration such as polymersomes, vesicular nanocarriers, liposomes, nanoparticles, topical solutions and gels 245 . For example, in the case of wound healing, proteoglycans were topically applied using 30% glycerol formula 246 . Minoxidil was shown to present long term benefits although it has been demonstrated to induce a transient posttreatment hair shedding 247 . Interestingly, Nourkrin treatment was shown to do not induce post-treatment hair shedding with an uncharacterized molecular mechanism 18 . Nevertheless, it has been demonstrated that proteoglycans purified from salmon cartilage promote the wound healing of dermal fibroblasts 248 and induce their proliferation, by the MAPK/ERK signaling pathway activation 249 . Similarly, a versican treatment on fibroblast stimulates their proliferation 250 . Interestingly, the application of proteoglycans purified from salmon cartilage on hematopoietic progenitor cells induce their differentiation into progenitor cells for granulocyte-macrophages, erythrocytes and/or megakaryocytes 251 . All these studies provide evidence that application or oral administration of proteoglycans can modulate the cell fate by the regulation of signaling pathways. Moreover, the first studies conducted on proteoglycan-based therapy for hair loss are promising. The mechanism of action of HSPGs on signaling pathway involved in hair follicle stem cell fate remains to be elucidated in order to develop HSPG-based treatment for hair loss.

CONCLUSION
It is clear that our knowledge on the role of HSPGs on stem cell fate becomes greater by the day. Several publications and reviews have reported the capacity of HSPGs to modulate signaling pathways in stem cells in various different ways. Indeed, they can interact directly with the growth factors and/or their associated receptors by their core protein or their HS GAG chains. This interaction can facilitate the binding between receptor and growth factor or can inhibit the pathway by the sequestration of the growth factor. Moreover, membrane HSPGs can be cleaved to favor long-range activity of a growth factor. The understanding of the molecular mechanisms of action of HSPGs on stem cell fate highlights their key role on the regulation of organ morphogenesis, homeostasis and regeneration. In the case of hair follicle stem cells, there is still a long way to characterize the role of HSPGs. Indeed, no studies have reported a link between HSPGs and growth factors/signaling pathways involved in hair follicle stem cell fate. Moreover, few studies have shown a regulation of hair follicle stem cells by other proteoglycans (versican, decorin). Evidence on the role of HSPGs on the regulation of signaling pathways involved in hair follicles stem cell differentiation are proven by several studies that have demonstrated changes in the distribution of HSPGs during hair growth cycle and by the fact that HSPG sulfation is necessary for hair shaft growth. Currently, the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have only approved the topical Minoxidil and the oral Finasteride as treatment of androgenetic alopecia. These two drugs have demonstrated their ability to induce hair regrowth. However, both present side effects limiting their efficacy in a still unclear mechanism. In particular, the Finasteride is known to induce sexual disorder when the oral use is prolonged 245 . The Minoxidil topical application required specific formulation known to induce inflammatory skin reaction, such as eczema and allergy in case of repetitive application 247 . The fact that the treatment of alopecia requires long periods of application or oral administration represents the greatest limitation of these treatments. The Nourkrin oral tablet administration has shown good results on hair growth. Drug-free, it does not induce any side effect even during long period of application 243 . The intake of proteoglycan to the hair follicle leads to re-establishment of the hair follicle metabolism. The studies of the role HS proteoglycans in the regulation of the hair follicle metabolism will contribute to mastering the mode of action of the treatment based on HSPG delivery or to develop therapies targeting HSPGs. Proteoglycanbased treatment by oral delivery of proteoglycans appears to be a promising method to tackle androgenetic alopecia. Indeed, it has been shown to increase the hair counts on scalp. This kind of treatment can be applied with HSPGs oral delivery or topical application on scalp. Another way of investigation would be a treatment able to regulate the cellular expression of HSPGs to modulate their local distribution in hair follicles.