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Stem Cells in the Face: Tooth Regeneration and Beyond

Jeremy J. Mao1,* and Darwin J. Prockop2 1Center for Craniofacial Regeneration, Columbia University Medical Center, 630 West 168 Street – PH7E, New York, NY 10032, USA 2Institute for Regenerative Medicine at Scott and White, Texas A&M University Health Science Center, Temple TX 76502, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2012.08.010

The face distinguishes one person from another. Postnatal orofacial tissues harbor rare cells that exhibit stem cell properties. Despite unmet clinical needs for reconstruction of tissues lost in congenital anomalies, infec- tions, trauma, or tumor resection, how orofacial stem/progenitor cells contribute to tissue development, pathogenesis, and regeneration is largely obscure. This perspective article critically analyzes the current status of our understanding of orofacial stem/progenitor cells, identifies gaps in our knowledge, and high- lights pathways for the development of regenerative therapies.

Introduction natally from orofacial tissues, have been shown to exhibit stem/ The face consists of vastly diverse tissues, which not only are progenitor cell properties such as self-renewal, clonogenicity, vital for esthetics, but also exert several indispensable func- multilineage differentiation, and the ability to induce tissue tions including breathing, chewing, speech, sight, and smell. formation in vivo. However, how orofacial stem/progenitor cells Orofacial tissues are lost in congenital anomalies, infections, contribute to patterning in prenatal development, pathogen- trauma, or tumor resection. There is a tremendous and unmet esis, or tissue regeneration remains largely a mystery at this clinical need for reconstruction of lost orofacial tissues and time. restoration of both function and esthetics. Postnatally, orofacial This review discusses two types of orofacial stem/progenitor cells can be readily isolated, for example, from surgically cells: (1) stem/progenitor cells that are present in orofacial con- removed gingiva or teeth, without undue trauma to the patient. nective tissues including dental pulp, jaw bone, periodontal liga- Among extracted primary orofacial cells, there are rare cells ment, and lamina propria of oral mucosa, and (2) epithelial stem that possess stem/progenitor cell properties, as shown by cells in oral epithelium, salivary glands, and the developing tooth work in the past few decades. Also demonstrated in previous organ (Figures 1A and 1B). Rather than an exhaustive review, we work are examples of how orofacial stem/progenitor cells choose to identify, in broad strokes, what is known and what might be used for regeneration of orofacial tissues. However, needs to be known about orofacial stem/progenitor cells, and enthusiasm for harnessing the presumed therapeutic power translational pathways for the development of putative regener- of orofacial stem/progenitor cells must be matched with suffi- ative therapeutics. cient scientific rigor to study their potency and limitations, and ultimately in randomized clinical trials that determine Connective Tissue Stem/Progenitor Cells in Orofacial whether/how orofacial stem/progenitor cells might be used in Structures patients. Defining Orofacial Connective Tissue Stem Cells Facial development, including that of the tooth and oral Bone marrow stromal cells frequently serve as a reference for the cavity, is a classic act of interactions by stem cells of the characterization of stem/progenitor cells that reside in orofacial epithelium, craniofacial mesoderm, and neural crest-derived connective tissues, given that both are of mesenchymal and/or mesenchyme (Thesleff, 2006; Cordero et al., 2011). For mesodermal origins. Hematopoietic stem cells reside in bone example, tooth enamel derives from oral epithelium, whereas marrow niches that are formed by stromal cells and osteoblasts the remaining dental structures, including tooth pulp, dentin, (Sacchetti et al., 2007; Me´ ndez-Ferrer et al., 2010; Song et al., and cementum, originate from neural crest-derived mesen- 2010; Bianco, 2011). Colony-forming unit fibroblasts (CFU-Fs) chyme (Thesleff and Tummers, 2008). Endoderm makes little were first identified as nonhematopoietic bone marrow cells contribution to orofacial development with the exception of that readily adhere to tissue culture polystyrene and, importantly, taste buds and small glands of the tongue (Rothova et al., generate bone with marrow sinusoids upon in vivo heterotopic 2012). Salivary glands are generated by epithelial stem cells transplantation (Friedenstein et al., 1974; Owen and Frieden- growing into the underlying mesoderm that gives rise to glan- stein, 1988). They were named as bone marrow stromal cells dular stromal cells, similar to invagination of oral epithelial cells (BMSCs) to indicate their residence in bone marrow stroma, into the underlying mesenchyme during tooth development their primary function to support hematopoiesis and their (Tucker, 2007). Even some of the seemingly simple flat bones ability to generate heterotopic bone (Friedenstein et al., 1974; of the skull are formed by a patchwork of mesodermal cells Prockop, 1997; Robey, 2000; Sacchetti et al., 2007). The term and neural crest-derived mesenchyme cells (Jiang et al., ‘‘mesenchymal stem cells’’ (MSCs) was later coined to suggest 2002). During the past few decades, certain cells of ecto- their potency to generate or regenerate multiple connective dermal, neural crest, or mesodermal origin, when isolated post- tissues (Caplan, 1991; Caplan and Correa, 2011). However,

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Figure 1. Diagrams of Human and Mouse Orofacial Tissues from which Stem/Progenitor Cells Have Been Studied (A) Epithelial stem cells reside in the developing tooth germ, oral epithelium, and salivary gland. Connective tissue stem/progenitor cells (of mesenchyme/ mesoderm origin) have been isolated from calvarial bone, tooth pulp, dental papilla, the periodontal ligament, and marrow of alveolar bone. (B) The developing rodent incisors have been the most prevalent model for studying orofacial epithelium stem cells. Rodent incisors undergo continuous growth and eruption throughout life. The cervical loop of the developing incisor harbors both epithelial and mesenchyme stem cells. Epithelial stem cells are known to give rise to transient amplifying cells that propagate and migrate anteriorly and differentiate into ameloblasts that produce enamel matrix. Strikingly, enamel is produced only on the labial side in rodents (red). In contrast, mesenchyme stem cells migrate anteriorly to differentiate into odontoblasts that produce dentin (green), in addition to giving rise to interstitial fibroblast-like cells in dental pulp.

evidence is lacking at this time to support the concept that prog- surface, in addition to abundant interstitial fibroblasts that are enies of a single MSC can generate an entire connective tissue located among a web of blood vessels and nerve endings. (Bianco et al., 2008; Keating, 2012). Regardless of the name, Dental pulp is highly cellular in the young, but its cellularity one must recognize that commonly studied MSCs isolated decreases with age (Smith et al., 1995; Nanci, 2007). Develop- from bone marrow, adipose or orofacial tissues as mononucle- mentally, Cranial neural crest cells are multipotent stem cells ated and adherent cells are each highly heterogeneous cell pop- and give rise to dental mesenchyme in a structure known as ulations (Gronthos et al., 2002; Guilak et al., 2006; Marion and the dental papilla (Chai et al., 2000). Dental papilla is the recog- Mao, 2006; Lee et al., 2010a; Keating, 2012). Given that nized origin of postnatal dental pulp stem/progenitor cells mesenchyme only exists prenatally, we use ‘‘connective tissue (Smith et al., 1995; Nanci, 2007; Chai et al., 2000). Mesen- stem (CTS) cells’’ to refer to stem cells in postnatal orofacial chymal cells in the developing E13.5 mouse tooth germ are connective tissues. For practicality, CTS cells also refer to multipotent and readily differentiate into nondental lineages all putative stem/progenitor cells that have been studied in including chondrocytes and osteoblasts, in addition to odonto- orofacial connective tissues including dental pulp, jaw bone, blasts (Yamazaki et al., 2007). Some, but far from all, of the periodontal ligament, apical papilla, calvaria and lamina propria mononucleated and adherent cells isolated from postnatal of oral mucosa. Developmentally, orofacial CTS cells arise dental pulp demonstrate stem/progenitor cell properties includ- from (1) neural crest derived mesenchyme and/or (2) orofacial ing colonogenecity and differentiation into a limited number mesoderm. of cell lineages ex vivo (Gronthos et al., 2000; Batouli et al., Currently, mononucleated cells that are isolated from orofa- 2003). At a clonal level, about two-thirds of dental pulp CTS cial connective tissues and adhere to tissue culture polystyrene cells generate ectopic dentin when transplanted heterotopically are deemed to be stem cells (Table 1). Ex vivo differentiation of in vivo, but not the remaining one-third (Gronthos et al., mononucleated and adherent cells into osteoblasts, chondro- 2002). The spatial distribution of dental pulp CTS cells has cytes and/or adipocytes is considered as evidence that they been recently explored by in vivo cell tracing, showing that are stem cells (Table 1). However, mononucleated and adherent odontoblasts in dental pulp may originate from two different cells isolated from orofacial connective tissues, even if they sources: perivascular and nonperivascular cells, both of which differentiate into multiple lineages ex vivo, are far from pure are capable of migrating towards trauma and potentially replen- stem cells. Additional rigor is essential to characterize orofacial ishing odontoblasts upon pulp injury (Feng et al., 2011). Impor- CTS cells, including colony formation and clonogenecity, in vivo tantly, few cells in dental pulp undergo migration in postnatal cell lineage tracing and/or orthotopic cell infusion and tissue homeostasis (Feng et al., 2011). To date, few studies have regeneration (Table 1). focused on molecular signaling of orofacial CTS cells. Notably, Dental Pulp CTS Cells Notch signaling has been shown to maintain the stemness of The bulk of the tooth in humans and many other mammalian dental pulp CTS cells and attenuate their differentiation (Zhang species is formed by highly mineralized dentin. Dentin is et al., 2008). However, little else is known about the contribution covered by the enamel in the crown of the tooth and cementum of other molecular signaling pathways that regulate orofacial in the root in humans. Dental pulp is the only soft tissue in the CTS cells. tooth, and functions primarily to maintain its own homeostasis Jaw Bone CTS Cells and that of dentin. Dental pulp is a heterogeneous cell reservoir, Tissues in dental pulp are connected via the root apex with and consists of odontoblasts that reside on mineralized dentin both the periodontal ligament and bone marrow in the maxilla

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Table 1. Existing and Rigorous Approaches for Characterization axis (Seo et al., 2004). When transplanted ectopically into of Orofacial CTS Cells immunocompromised rodents, human periodontal ligament Existing Approaches Additional Rigor CTS cells yield cementum/periodontal ligament-like structures in porous calcium hydroxyapatite (Seo et al., 2004). However, colony formationa cologenecity and clonal analysisb in comparison to tendinopathy in which adipose tissue accumu- multilineage differentiation in vivo cell tracing, lineage tracing, lates in tendons, there is no report of adipose tissue accumula- in vitroc label retention, and functional assaysd tion in the periodontal ligament, suggesting that native f in vivo ectopic tissue in vivo orthotopic tissue regeneration periodontal ligament CTS cells are perhaps incapable of adipo- formatione genesis. a Sparsely seeded cells each forming a colony. Oral Mucosa CTS Cells bA single cell, when plated, yields a progeny. Oral mucosa consists of oral epithelium and the underlying cMultilineage differentiation ex vivo: frequently into osteoblasts, adipo- connective tissue, the lamina propria. Mononucleated and cytes, and chondrocytes. dTransplanted cells are tagged with fluorescent marker or nanoparticles adherent cells isolated from postnatal lamina propria of gingival and traced in vivo. and alveolar mucosa are highly proliferative and contain putative eFrequently heterotopic implantation such as the dorsum or omentum. stem/progenitor cells (Marynka-Kalmani et al., 2010). Oral mucosa fDetermines the fate of in vivo transplanted cells. CTS cells differ from dental pulp and periodontal ligament CTS cells by their high expression of CD49d (Integrin a2orVLA-4) and weak expression of osteogenic transcriptional factors or mandible. Given that bone marrow MSCs were initially iso- such as Runx2 (Lindroos et al., 2008). Compared to our marginal lated from the marrow of appendicular bones such as the iliac understanding of lamina propria CTS cells in oral mucosa, next crest, one would assume that the marrow of jaw bone also to nothing is known about oral epithelial stem cells (e.g., Izumi harbors stem/progenitor cells. Indeed, CTS cells have been et al., 2007). isolated from jaw bones of both humans and rodents (Matsu- Despite the original tenet that MSCs participate in regenera- bara et al., 2005; Akintoye et al., 2006; Yamaza et al., 2011). tion as tissue builders, recent data show that MSCs interact Like iliac crest MSCs, stem/progenitor cells from the jaw with inflammatory cells and immune cells that infiltrate the bone are clonogenic and have potent osteogenic potential wound. Similarly, gingival CTS cells prompt macrophages to in vitro and in vivo (Matsubara et al., 2005). However, a number acquire an anti-inflammatory M2 phenotype when cocultured of differences exist between these two cell types. Compared to in vitro (Zhang et al., 2010). In vivo, systemically infused gingival iliac crest MSCs, mandibular CTS cells appear to proliferate CTS cells improve wound repair by homing to skin wound more rapidly, exhibit delayed senescence, express alkaline sites and promoting macrophage polarization toward an M2 phosphatase more robustly and accumulate more calcium phenotype (Zhang et al., 2010). The M2 polarized macrophages when cultured in vitro (Akintoye et al., 2006). When trans- play important roles in resolving inflammation by releasing planted heterotopically in vivo, MSCs from long bones yield trophic factors and suppressing the secretion of pro-inflamma- greater bone marrow area than mandibular CTS cells (Yamaza tory cytokines (Sica and Mantovani, 2012). Periodontal ligament et al., 2011), while mandibular bone marrow CTS cells yield CTS cells also suppress inflammatory cells and peripheral greater bone volume than appendicular marrow MSCs (Akin- blood monocytes, independent of cell contact (Wada et al., toye et al., 2006; Yamaza et al., 2011). Interestingly, jaw bone 2009), similar to bone marrow MSCs (Lee et al., 2009a). These CTS cells are far less chondrogenic and adipogenic than findings endorse the general concept that transplanted orofacial MSCs from the iliac crest (Matsubara et al., 2005). The CTS cells, similar to appendicular MSCs, primarily serve as underlying mechanisms for the observed differences between signaling cells in wound healing, rather than as tissue replace- jaw CTS cells and appendicular bone marrow MSCs are elusive ment cells (Wagner and Ho, 2007; Lee et al., 2009a; Prockop, at this time. Interestingly, MSCs isolated from the iliac crest and 2009). vertebral body are also known to differ (McLain et al., 2005). A Orofacial CTS Cells and Appendicular Bone Marrow meaningful reference is perhaps whether the differences MSCs: Are They Different? between jaw CTS cells and appendicular bone marrow MSCs Table 2 provides such a comparison, with the caveat that few are more pronounced than differences of MSCs isolated from studies have been performed with donor-matched samples. the iliac crest and vertebral body. Additionally, molecular markers expressed by either orofacial Periodontal Ligament CTS Cells CTS cells or appendicular bone marrow MSCs are sensitive to The periodontal ligament connects tooth roots to the perturbation by a multitude of factors such as passaging, incu- surrounding alveolar bone, and primarily functions to maintain bation medium, medium lot selection, plating density, and its own homeostasis and that of the cementum, in addition to freezing and thawing (Sekiya et al., 2002; Smith et al., 2004; transmitting mechanical stresses. Dental follicle cells, which Lee et al., 2009b). Bearing these caveats in mind, orofacial originate from neural crest derived mesenchyme, differentiate CTS cells and appendicular bone marrow MSCs indeed overlap into cells that form the periodontal ligament and are present in many molecular markers but nonetheless have several impor- in the developing tooth germ prior to root formation (Yao tant differences. For example, CTS cells from either deciduous or et al., 2008). Cells isolated from the periodontal ligament of ex- adult dental pulp undergo more rapid proliferation ex vivo than tracted teeth differentiate into cementoblast-like cells, adipo- appendicular bone marrow MSCs for reasons that are not well cytes, and collagen-forming cells under permissive conditions understood (Gronthos et al., 2000; Miura et al., 2003). When in vitro, and express markers including Stro1, CD146, and scler- transplanted heterotopically in vivo, dental pulp CTS cells from

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Table 2. Comparison of Orofacial CTS Cells with Appendicular Bone Marrow MSCs Orofacial CTS Cellsa Appendicular Bone Marrow MSCsb tissue origin; negative markers (nonexclusive)d dental pulp, periodontal ligament, marrow of marrow of appendicular bones or vertebrae; jaw bones, lamina propria of oral mucosa;c CD14, CD31, CD34, CD45 CD14, CD31, CD34, CD45 positive markers (nonexclusive)e CD29, CD44, CD49d, CD73, CD90, CD105, CD29, CD44, CD73, CD90, CD105, CD106, CD106, CD146, Stro1, Oct4 and Nanogf, CD146, Oct4 and Nanogf, Stro1, nestin, etc. hTERT, endostatin, Stro1, nestin, scleroxis, etc. heterotopic transplantationg dental pulp CTS cells yield dentin-like tissues; heterotopic bone with marrow sinosuidsf periodontal ligament CTS cells yield fibrous tissue and bone orthotopic transplantationh yields mineralized tissue in tooth root scaffolds promote bone fracture healing although cell fate is uncertain aNonepithelium orofacial CTS cells. bNonhematopoietic stem cells of bone marrow or bone marrow stromal cells. cSee Huang et al., 2009 for detailed catalog of markers of orofacial CTS cells. dThese markers are typically less than 1%–2%. eThese markers may vary from overwhelming expression (e.g., >90%) to definitive presence but not dominance (e.g., 10% or less). fOct4 and Nanog expression in orofacial CTS cells or appendicular bone marrow MSCs is present but is thousands-fold less than that in embryonic stem cells. gHeterotopic transplantation of orofacial CTS cells is exemplified by subcutaneous implantation in the dorsum or omentum. hOrthotopic transplantation refers to delivery of cells to the very location of their origin, such as bone marrow MSCs to a fracture site.

both deciduous and permanent teeth yield dentin nodules on the promote cardiac infarct healing likely due to their ability to surface of dentin substrate or porous calcium phosphate (Gron- secrete proangiogenic and antiapoptotic factors (Gandia et al., thos et al., 2000; Batouli et al., 2003; Yu et al., 2007). A subset of 2008). Implanted adult human dental pulp CTS cells from bone marrow MSCs have the ability to generate orthotopic bone wisdom teeth promote the migration and sprouting of avian in vivo (Mankani et al., 2006). Importantly, dental pulp CTS cells trigeminal ganglion via CXCL12/SDF1 and its receptor, lack the capacity of appendicular marrow MSCs to form hetero- CXCR4, in vivo (Arthur et al., 2009). Similarly, untreated rhesus topic bone (Robey, 2011). dental pulp CTS cells delivered into the hippocampus of Can Orofacial CTS Cells Participate in the Regeneration immune-suppressed mice recruit endogenous nestin+ cells of Nonorofacial Tissues? and b-3-tubulin+ neurons to the grafting site (Huang et al., Dental pulp CTS cells have been differentiated, mostly in vitro, 2009). Transdifferentiation of orofacial CTS cells, as outlined into putative hair follicle cells, hepatocyte-like cells, neuron-like above, has been pursued as isolated examples and needs to cells, myocyte-like cells, islet-like cells, and cardiomyocyte-like be considered in the context of in vivo functional assays and cells (Reynolds and Jahoda, 2004; Iohara et al., 2006; Ishkitiev perhaps also cellular programming/reprogramming in order to et al., 2010; Yang et al., 2010; Sugiyama et al., 2011; Govindas- convincingly demonstrate a direct role in regeneration of nonor- amy et al., 2011), thus raising the possibility that they could ofacial tissues. participate in the regeneration of nonorofacial tissues. However, What We Already Know about Orofacial CTS Cells ex vivo differentiation, especially of heterogeneous orofacial CTS Orofacial CTS cells have been intensely studied in the past cells, is of limited value. In vivo functional and lineage tracing decade or so. However, most studies have relied on in vitro studies are necessary, as in Table 1, to appreciate whether cultures of mononucleated and plastic-adherent cells that have wild-type and/or selected fractions of orofacial CTS cells indeed been isolated from various orofacial structures. At best, these transdifferentiate into nonorofacial lineages. In one study, only studies have extended our understanding of cells of orofacial two out of dozens of clonal progenies of deciduous dental pulp tissues, including stem/progenitor cells that are rarely studied CTS cells spontaneously fused into multinucleated myocyte- separately from heterogeneous cell populations. like cells that produce myosin heavy chain ex vivo (Yang et al., 2010), underscoring the rarity of cells in dental pulp with the d Postnatal orofacial CTS cells are rare cells that remain ability to transform into natively unintended lineages. Nonethe- quiescent or undergo slow cycling in vivo at most times. less, when transplanted into injured skeletal muscle, myocyte- It is virtually impossible to identify true stem cells without prone dental pulp clonal progenies successfully engraft and in vivo label retention, lineage tracing and/or serial trans- express human dystrophin, a protein that is missing in muscular plantation experiments. dystrophy (Yang et al., 2010). Injection of GFP+ human dental d Typical cultures of isolated orofacial CTS cells as mononu- pulp stem/progenitor cells into acute cardiac infarct sites in clear and adherent cells from dental pulp (regardless nude rats improves cardiac function with efficacy similar to of deciduous or permanent teeth), lamina propria of appendicular marrow MSCs (Gandia et al., 2008). Interestingly, oral mucosa, periodontal ligament and mandibular bone GFP tagged dental pulp CTS cells fail to differentiate into cardi- marrow are each heterogeneous, and far from uniform omyocytes in vivo, suggesting that dental pulp CTS cells ‘‘stem cell’’ cultures.

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d Orofacial CTS cells express a broad array of molecular Epithelial Stem Cells in Orofacial Tissues: The Tooth as markers that are also ascribed to as yet incompletely a Model defined bone marrow MSCs, but nonetheless express little Tooth development is a classic model of epithelial-mesenchymal CD14 (innate immune marker), CD31 (PECAM-1), the hem- interactions. Rodent incisors continue to grow and erupt atopoeietic markers CD34 and CD45. Thus far, no single throughout life, providing a unique and powerful model for (or combination of) cell-surface marker has been identified studying stem cells of the epithelium and mesenchyme. Epithe- to mark stemness in CTS populations or to differentiate lial stem cells in the developing rodent incisor reside in the distinguish between CTS cells types isolated from different cervical loop (Figure 1B) and are surrounded by dental mesen- orofacial tissues. chyme, somewhat analogous to the hair follicle bulge and the d Orofacial CTS cells from dental pulp, lamina propria of intestinal crypt (Moore and Lemischka, 2006; Hsu et al., 2011; oral mucosa, periodontal ligament, and mandibular bone Thesleff, 2006). Mineralization of enamel and dentin, in compar- marrow, each as heterogeneous cell populations, appear ison to the unmineralized dental pulp, affords a unique opportu- to undergo more rapid proliferation ex vivo than bone nity for studying the contrasting fate of a single origin of stem marrow MSCs. Rapid proliferation does not necessarily cells, dental papilla in this case, that differentiate into mineralized guarantee that orofacial CTS cells can be propagated in dentin, and unmineralized dental pulp. Whereas the hair follicle greater numbers for therapeutic purposes. bulge and the intestinal crypt are subjects of robust investiga- tions toward understanding of stem cell behavior, relatively Orofacial CTS Cells: Future Directions less is known about lineage commitment, migration, and differ- Despite a well justified motivation to harness the presumed ther- entiation of dental epithelium and mesenchymal stem cells of apeutic potential of orofacial CTS cells, fundamental biology the developing tooth organ. Few studies exist on putative stem studies must be pursued and will fuel translational effort toward cells in oral epithelium and salivary gland epithelium. A notable orofacial regeneration. Virtually untapped are the mechanisms exception is a recent report of an epithelial stem cell axis in the by which orofacial CTS cells may cells contribute to the patho- salivary gland, showing that acetylcholine signaling increased genesis of congenital and acquired diseases. epithelial morphogenesis and proliferation of the keratin 5-posi- d In vivo lineage tracing studies that tag and track various or- tive progenitor cells, whereas parasympathetic innervation ofacial CTS cells using transgenic and/or interventional maintains the stemness of the epithelial progenitor cell popula- models. In vitro multilineage differentiation of heteroge- tion (Knox et al., 2010). neous orofacial CTS cell populations is of little value. During tooth development, DiI labeling and BrdU pulse chase/ Clonal differentiation is valuable but in itself still does not label retention shows that dental epithelial stem cells undergo fully establish stemness. continuous self renewal (Harada et al., 1999; Kawano et al., d Focus on the understanding of how stem cells give 2004). Dental epithelial stem cells further undergo asymmetric rise to specialized orofacial cells that are not found else- division, with some daughters retaining their stemness, while where in the body including odontoblasts (and how they others depart from the niche, migrate and differentiate into differ from osteoblasts), ameloblasts or enamel-forming ameloblasts, or enamel-forming cells that synthesize enamel cells (e.g., what equips them with outstanding mineraliza- matrix (Smith, 1980; Harada et al., 1999, 2002; Wang et al., tion), cementoblasts, salivary gland cells and oral mucosa 2007). Continuous self-renewal and asymmetric division of den- cells. tal epithelial stem cells are directly responsible not only for the d Benchmark studies that compare orofacial CTS cells with replenishment of functional ameloblasts, but also continuing appendicular marrow MSCs in humans and other species, eruption of rodent incisors (Harada et al., 1999, 2002; Wang including the use of donor-matched samples. et al., 2007). d Immunoepitope panels and molecular assays that serve as The action of dental epithelium stem cells is only a part of hallmarks for each of the orofacial CTS cell populations at the story in tooth organogenesis. Dental mesenchymal stem critical stages of differentiation and self-renewal. cells surround dental epithelium stem cells in the cervical loop d Develop and validate heterotopic and orthotopic animal (Figure 1B) (Rothova´ et al., 2011). During epithelium-orches- models that reproducibly test the behavior of transplanted trated amelogenesis, dental mesenchymal stem cells line up and tagged orofacial CTS cells in vivo. opposite the row of enameling-forming ameloblasts initially d Signaling pathways that regulate stemness, differentiation with nothing but a basement membrane in between (Harada and trophic effects of orofacial CTS cells have received et al., 1999, 2002; Thesleff, 2006; Wang et al., 2007). Amelo- little attention and need to be better understood. blasts, while laying down enamel matrix, generate an indispens- d Study how orofacial CTS cells may be involved in the path- able induction signal for mesenchymally derived odontoblasts ogenesis of congenital anomalies and acquired diseases, to lay down dentin matrix (Kawano et al., 2004; Yoshida et al., exemplified as birth defects and periodontal disease or 2008; Fujimori et al., 2010). By the time the developing tooth jaw joint disorders. organ reaches the bud stage, dental mesenchyme takes over d A critical question that needs to be answered is whether as signal generator for the developing ameloblasts to undergo orofacial cells, including stem/progenitor cells, offer higher maturation (Kollar and Fisher, 1980; Tucker and Sharpe, 2004; efficiency and safety for reprogramming, including direct Thesleff, 2006). This mutual induction of dental epithelium and transformation into cells that safely propagate into suffi- mesenchyme has contributed a great deal to the understanding cient numbers and regenerate orofacial or nonorofacial of epithelial-mesenchymal interactions, along with observations tissues. in other organ systems such as the skin and hair follicle (Moore

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and Lemischka, 2006; Hsu et al., 2011). However, little is known for tooth formation. When embryonic dental epithelium is recon- about what governs the differentiation of dental mesenchyme stituted with either dental or nondental mesenchyme, odonto- stem cells not only into mineralized dentin and cementum, but genesis genes are upregulated and multiple tooth organs are also unmineralized dental pulp. formed upon transplantation in the adult renal capsule or jaw An additional striking feature of dental epithelium stem cells in bone (Ohazama et al., 2004; Modino and Sharpe, 2005). rodent incisors is that enamel is only formed on the labial surface, Similarly, E14.5 oral epithelium and dental mesenchyme can but not the lingual surface (Figure 1B), providing a rare model for be reconstituted in collagen gel and, when cultured ex vivo, studying the polarity of stem cell distribution and function (Har- yield multiple dental tissue analogs (Nakao et al., 2007). ada et al., 1999, 2002; Thesleff, 2006; Wang et al., 2007). In the When similarly reconstituted mouse E14.5 tooth germ cells cervical loop, epithelial stem cells proliferate and migrate along were transplanted into tooth extraction sockets of 5-week-old the labial surface, differentiating into enamel-forming amelo- mice, a complete tooth organ was formed with both the blasts (Figure 1B) (Wang et al., 2007). In contrast, the lingual crown and root, followed by eruption into the oral cavity (Ikeda cervical loop has few proliferating epithelial stem cells or amelo- et al., 2009). Recently, reconstituted E14.5 mouse tooth germ blasts, and hence is devoid of enamel (Figure 1B) (Thesleff et al., cells further yielded complex tooth organ structures with 2007). mechanical stiffness approaching that of native tooth structures Considerable insight on signaling in tooth development has with a putative periodontal ligament after eruption (Oshima enriched our understanding of epithelial and mesenchymal et al., 2011). These studies underscore the capacity of embry- stem cells. TGFb, Wnt, FGF, Lrp4, and Hedgehog are among onic dental epithelium and mesenchyme cells, even following some of the highly conserved signaling pathways that regulate disassociation and reconstitution, to form a complete tooth many aspects of dental stem cells in development (Thesleff, organ. 2003; Ja¨ rvinen et al., 2006; Yokohama-Tamaki et al., 2006; Klein The developing tooth germ continues to grow in postnatal life, et al., 2008; Lin et al., 2009). FGF signaling in dental mesen- including human wisdom teeth that are frequently extracted chyme regulates Notch signaling in dental epithelium (Harada to alleviate or prevent peridental infections. However, whether et al., 1999; Kawano et al., 2004; Mitsiadis et al., 2010). Notch these postnatal stem/progenitor cells, without reprogramming, signaling, in turn, is required for regulating the survival of are able to regenerate an entire tooth organ is fully understood epithelial stem cells in the continuously growing mouse incisor at this time. Disassociated cells of postnatal porcine or rat (Felszeghy et al., 2010). Sonic hedgehog produced by the dif- tooth buds, when seeded in biomaterials and implanted in the ferentiating progeny of rodent incisor stem cells, though not abdominal cavity, yielded multiple dentin and enamel organs necessary for survival, is essential for ameloblastic differentia- (Young et al., 2002; Duailibi et al., 2004). Transplantation of post- tion (Seidel et al., 2010). Activin signaling regulates the prolifera- natal autologous tooth germ cells from unerupted molar tooth tion and differentiation of dental epithelial stem cells (Wang et al., germ yielded dentin/pulp-like tissues with odontoblast-like cells 2004). Stimulation of Wnt or Wnt/BMP pathways in dental epi- and cementum-like structures (Kuo et al., 2008). Multipotent thelium in transgenic mice not only mediates continuous growth cells of the tooth apical papilla, a transient structural derivative of mouse incisors, but also leads to multiple newly formed teeth of dental papilla, generated mineralized tissues with a putative (Ja¨ rvinen et al., 2006; Wang et al., 2009; O’Connell et al., 2012). periodontal ligament when transplanted in porous tricalcium However, signaling pathways in tooth development are only phosphate in the extraction socket of an incisor in a miniature partially understood, and are virtually not studied at all in the pig (Sonoyama et al., 2006). Seeding dental follicle cells from context of tooth regeneration. surgically extracted wisdom teeth in dentin matrix sheets acti- vates expression of multiple odontogenesis/osteogenesis genes Regeneration of Orofacial Tissues (Yang et al., 2012). In contrast to mouse E14.5 tooth germ cells, The face, including the oral cavity and the teeth, is of tremen- reconstituted postnatal tooth germ cells have only generated dous therapeutic interest for tissue regeneration (Mao et al., fragmented dental structures and/or miniature tooth organs 2006). In addition to functional reconstruction, patients who upon in vivo transplantation, rather than an anatomically correct suffer from tooth loss, cleft lip or facial trauma have a strong sized tooth organ. desire for restoring esthetics. Mammalian teeth do not sponta- Given the presence of stem/progenitor cells in many dental neously regenerate upon trauma or pathological insult. Sharks tissues, the idea of promoting tooth regeneration through manip- and certain lizards, however, continuously generate new sets ulating endogenous stem/progenitor cells is a clinically translat- of teeth, albeit rootless, throughout life in ways that are only able but nonetheless under-explored possibility. A first attempt peripherally understood (Boyne, 1970; Samuel et al., 1983; has recently been made to deliver two growth factors, SDF1 Handrigan et al., 2010). This section uses tooth regeneration and BMP7, in the microchannels of anatomically correct bioma- as a model to exemplify challenges and strategies for orofacial terial tooth scaffolds that were implanted orthotopically in tooth regeneration. extraction sockets in vivo (Kim et al., 2010a). Nine weeks The classic experiment of Kollar and Fisher (1980) shows that following implantation, codelivery of SDF1 and BMP7 induced grafting of 5-day chick epithelium from the first/second pharyn- the regeneration of mineralized tissue in biomaterial root scaf- geal arch combined with E16–18 mouse molar mesenchyme folds with de novo formation of a putative periodontal ligament produced tooth crowns with enamel and dentin in the ocular and newly formed alveolar bone by the recruitment of endoge- chamber, suggesting that (1) inductive signals for tooth organo- nous host cells (Kim et al., 2010a; Yildirim et al., 2011). Whether genesis may derive from nondental epithelium such as the other factors, including other members of bone morphogenetic toothless chick epithelium, and (2) the oral cavity is not privileged proteins, contribute to tooth regeneration warrants additional

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investigations (Nakashima and Reddi, 2003). However, amelo- tulous (CDC). Can adult stem/progenitor cells, regardless of genesis was not observed (Kim et al., 2010a; Yildirim sources, regenerate a complete, anatomically correct tooth? et al., 2011), similar to the lack of enamel formation upon trans- The short answer for now is no, as ameloblasts or enamel-form- plantation of postnatal tooth germ cells or apical papilla cells ing cells are no longer present following crown formation and (Sonoyama et al., 2006; Kuo et al., 2008). Tooth regeneration tooth eruption. However, the paucity of tissue progenitor cells by recruitment of host endogenous stem/progenitor cells is for enamel regeneration is hardly a unique problem for tooth consistent with tissue regeneration by cell homing in several regeneration, as this challenge exists for regeneration of other other structures such cartilage, skeletal muscle and pancreatic tissues. tissues (Lee et al., 2006; Karp and Leng Teo, 2009; Lee et al., Projected Strategies for Tooth Regeneration 2010b), and appears to offer an advantage towards clinical Tooth regeneration needs to have multiple milestones with the translation. eventual endpoint as regeneration of entire tooth organs in General difficulties associated with cell therapy also apply to patients. First, translational approaches are called for to regen- cell sources that could potentially be used in tooth regeneration, erate singular or multiple dental tissues such as dental pulp including teratoma formation and inappropriate lineage differen- and/or dentin, enamel and cementum (e.g., Cordeiro et al., tiation for embryonic stem cells (ESCs) or induced pluripotent 2008; Kim et al., 2010b; Iohara et al., 2011; Galler et al., 2012). stem cells (iPSCs). Regardless of cell source, cell transplantation In parallel, it is meritorious to produce scalable enamel and for tooth regeneration encounters additional translational dentin crystals that serve as native replacement fillers (Du barriers including excessive costs associated with ex vivo cell et al., 2005; Huang et al., 2010; Aida et al., 2012). Furthermore, culture and manipulation, potential contamination, complexities there is a clinical need to regenerate a biological tooth root of sterilization, shipping, storage and handling, and potential that is connected to the supporting alveolar bone with a peri- oncogenic mutation associated with ex vivo cell manipulation. odontal ligament. A prosthetic tooth crown can readily be Tumorigenecity becomes a real concern upon prolonged ex vivo attached to a biologically regenerated tooth root and may serve culture or immortalization. Cell sources and biomaterial selec- as a first generation regenerative tooth therapy. The ultimate tions for tooth regeneration are topics of intense interest (for goal is to regenerate anatomically correct, entire tooth organs reviews see Yelick and Vacanti, 2006; Thesleff and Tummers, with the enamel, dentin, cementum, and dental pulp, as well as 2008; Volponi et al., 2010; Yildirim et al., 2011; Keller et al., the periodontal ligament, using clinically compatible cell types 2011; Yuan et al., 2011). and approaches. Cell Sources for Tooth Regeneration Life ends in numerous wild life species upon complete tooth Developmentally, the tooth originates from the epithelium that loss, suggesting that spontaneous tooth regeneration is not forms the enamel, and the mesenchyme that differentiates into phylogenically embedded in postnatal orofacial stem/progenitor the dentin, cementum and dental pulp. Indeed, epithelium cells. However, cellular reprogramming prompts the imagina- stem cells and mesenchyme stem cells from the embryonic tion of whether bioengineered embryonic-like cells, or reprog- tooth germ have formed tooth organs that erupt into the oral rammed tooth germ cells, can regenerate an entire tooth organ. cavity in a rat model. However, embryonic tooth germ cells are After all, inductive signals that trigger dental mesenchyme for difficult, if not impossible, to be applied clinically, tooth organogenesis can originate from toothless species, or conversely, dental epithelium can direct nondental epithelium d Autologous human embryonic tooth germ cells are inac- toward tooth formation. Thus, it is perhaps not too farfetched cessible for tissue regeneration in the adult. Allogeneic to conceptualize that inductive signals with the same potency human embryonic tooth germ cells are ethically unaccept- as embryonic dental epithelium and mesenchyme may be able, and also may cause immunorejection and pathogen teased out by high-throughput screening approaches. Novel transmission. bioengineering and imaging tools are necessary for advancing d Xenogenic, nonhuman embryonic tooth germ cells suffer our understanding of fundamental biology and translation from immune rejection and tooth dysmorphogenesis toward the development of therapeutics. resulting from genetically patterned crown and root shape, and altered shape and dimensions of nonhuman, Concluding Remarks donor species. Diversity of the face not only among humans, but also among d Postnatal autologous tooth germ cells (e.g., third molars) or myriad vertebrate species, inspires numerous investigations autologous dental stem/progenitor cells are of limited about the amazing ability of stem cells of the embryonic epithe- availability, and appear to lack the potency to regenerate lium, mesoderm, and neural crest derived mesenchyme in an anatomically correct complete tooth organ. patterning highly individualized structures. Additionally, there is d Clinical trials embedded with intrinsic risks and high cost clearly a need for viable pathways to develop regenerative may be justified for potentially life-threatening diseases therapies for patients with congenital anomalies and acquired that current medicine deems incurable, such as Parkin- orofacial defects. Translational studies may well take place son’s disease, diabetes or spinal cord injuries, but likely without the obligation to wait for full understanding of every not for tooth regeneration. thread of fundamental biology of orofacial stem/progenitor cells. However, basic understanding of the potency and limita- Tooth loss is the most common organ failure. By 2030, 30 tions of orofacial stem/progenitor cells will serve as an instructive million individuals in the United States, where dental care is cue for better translation. Despite recent exponential growth among the most advanced worldwide, will be completely eden- in the volume of studies on orofacial stem/progenitor cells,

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we only understand bits and pieces of their functions in develop- Duailibi, M.T., Duailibi, S.E., Young, C.S., Bartlett, J.D., Vacanti, J.P., and Ye- ment, pathogenesis, and regeneration. At a minimum, orofacial lick, P.C. (2004). Bioengineered teeth from cultured rat tooth bud cells. J. Dent. Res. 83, 523–528. structures including the tooth are among some of the powerful and under-explored models for studying how stem cells work Felszeghy, S., Suomalainen, M., and Thesleff, I. (2010). Notch signalling is required for the survival of epithelial stem cells in the continuously growing in development, wound healing, and genetic and acquired mouse incisor. Differentiation 80, 241–248. diseases. Is postnatal tissue regeneration a faithful recapitula- tion of embryonic development? Orofacial tissues appear to Feng, J., Mantesso, A., De Bari, C., Nishiyama, A., and Sharpe, P.T. (2011). Dual origin of mesenchymal stem cells contributing to organ growth and repair. be well poised to address questions such as this. A photo Proc. Natl. Acad. Sci. USA 108, 6503–6508. of a child with a cleft lip and palate stimulates unlimited imagina- Friedenstein, A.J., Chailakhyan, R.K., Latsinik, N.V., Panasyuk, A.F., and Kei- tion of how the human face can possibly be reconstructed liss-Borok, I.V. (1974). Stromal cells responsible for transferring the microenvi- by innovative therapies based on the knowledge of stem/ ronment of the hemopoietic tissues. Cloning in vitro and retransplantation progenitor cells. in vivo. Transplantation 17, 331–340. Fujimori, S., Novak, H., Weissenbo¨ ck, M., Jussila, M., Gonc¸ alves, A., Zeller, R., Galloway, J., Thesleff, I., and Hartmann, C. (2010). Wnt/b-catenin signaling in ACKNOWLEDGMENTS the dental mesenchyme regulates incisor development by regulating Bmp4. Dev. Biol. 348, 97–106. We thank Dr. N. Lumelsky and Dr. P.G. Robey from NIH/NIDCR for critical comments and Dr. M. Embree for interactions with medical graphics for illus- Galler, K.M., Hartgerink, J.D., Cavender, A.C., Schmalz, G., and D’Souza, R.N. tration. The work for composition of this manuscript is supported by NIH grants (2012). A customized self-assembling peptide hydrogel for dental pulp tissue engineering. Tissue Eng. Part A 18, 176–184. R01DE018248, R01EB009663, and RC2DE020767 (to J.J.M.); New York Stem Cell Foundation Grant (to J.J.M.); and NIH grants R01HL073755, Gandia, C., Armin˜ an, A., Garcı´a-Verdugo, J.M., Lledo´ , E., Ruiz, A., Min˜ ana, P01RR17447, and R21EY020962 (to D.J.P.). J.J.M. and D.J.P. conceived M.D., Sanchez-Torrijos, J., Paya´ , R., Mirabet, V., Carbonell-Uberos, F., et al. the general framework of the manuscript. J.J.M. composed the first draft (2008). Human dental pulp stem cells improve left ventricular function, induce and revised the manuscript. D.J.P. provided insightful and critical sugges- angiogenesis, and reduce infarct size in rats with acute myocardial infarction. tions and participated in the revision of the manuscript. Stem Cells 26, 638–645.

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