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1.  Craniofacial and Dental Development in Costello Syndrome 
Costello syndrome (CS) is a RASopathy characterized by a wide range of cardiac, musculoskeletal, dermatological, and developmental abnormalities. The RASopathies are defined as a group of syndromes caused by activated Ras/mitogen-activated protein kinase (MAPK) signaling. Specifically, CS is caused by activating mutations in HRAS. Although receptor tyrosine kinase (RTK) signaling, which is upstream of Ras/MAPK, is known to play a critical role in craniofacial and dental development, the craniofacial and dental features of CS have not been systematically defined in a large group of individuals. In order to address this gap in our understanding and fully characterize the CS phenotype, we evaluated the craniofacial and dental phenotype in a large cohort (n=41) of CS individuals. We confirmed that the craniofacial features common in CS include macrocephaly, bitemporal narrowing, convex facial profile, full cheeks, and large mouth. Additionally, CS patients have a characteristic dental phenotype that includes malocclusion with anterior open bite and posterior crossbite, enamel hypo-mineralization, delayed tooth development and eruption, gingival hyperplasia, thickening of the alveolar ridge, and high palate. Comparison of the craniofacial and dental phenotype in CS with other RASopathies, such as cardio-facio-cutaneous syndrome (CFC), provides insight into the complexities of Ras/MAPK signaling in human craniofacial and dental development.
doi:10.1002/ajmg.a.36475
PMCID: PMC4115793  PMID: 24668879
Costello syndrome; CS; craniofacial development; malocclusion; MAPK pathway; occlusion; Ras; RASopathy; receptor tyrosine kinase; signal transduction; tooth development; gingival hyperplasia; enamel
2.  Craniofacial morphometric analysis of individuals with X-linked hypohidrotic ectodermal dysplasia 
Hypohidrotic ectodermal dysplasia (HED) is the most prevalent type of ectodermal dysplasia (ED). ED is an umbrella term for a group of syndromes characterized by missing or malformed ectodermal structures, including skin, hair, sweat glands, and teeth. The X-linked recessive (XL), autosomal recessive (AR), and autosomal dominant (AD) types of HED are caused by mutations in the genes encoding ectodysplasin (EDA1), EDA receptor (EDAR), or EDAR-associated death domain (EDARADD). Patients with HED have a distinctive facial appearance, yet a quantitative analysis of the HED craniofacial phenotype using advanced three-dimensional (3D) technologies has not been reported. In this study, we characterized craniofacial morphology in subjects with X-linked hypohidrotic ectodermal dysplasia (XLHED) by use of 3D imaging and geometric morphometrics (GM), a technique that uses defined landmarks to quantify size and shape in complex craniofacial morphologies. We found that the XLHED craniofacial phenotype differed significantly from controls. Patients had a smaller and shorter face with a proportionally longer chin and midface, prominent midfacial hypoplasia, a more protrusive chin and mandible, a narrower and more pointed nose, shorter philtrum, a narrower mouth, and a fuller and more rounded lower lip. Our findings refine the phenotype of XLHED and may be useful both for clinical diagnosis of XLHED and to extend understanding of the role of EDA in craniofacial development.
doi:10.1002/mgg3.84
PMCID: PMC4190877  PMID: 25333067
3D imaging; craniofacial development; ectodysplasin; geometric morphometrics; X-linked hypohidrotic ectodermal dysplasia
3.  Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor 
Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal's life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.
doi:10.3791/51266
PMCID: PMC4141628  PMID: 24834972
Stem Cell Biology; Issue 87; Epithelial Stem Cells; Adult Stem Cells; Incisor; Cervical Loop; Cell Culture
4.  Characterization of Dental Epithelial Stem Cells from the Mouse Incisor with Two-Dimensional and Three-Dimensional Platforms 
Dental epithelial stem cells (DESCs) drive continuous growth in the adult mouse incisors. To date, a robust system for the primary culture of these cells has not been reported, and little is known about the basic molecular architecture of these cells or the minimal extracellular scaffolding that is necessary to maintain the epithelial stem cell population in an undifferentiated state. We report a method of isolating DESCs from the cervical loop of the mouse mandibular incisor. Cells were viable in a two-dimensional culture system and did not demonstrate preferential proliferation when grown on top of various substrates. Characterization of these cells indicated that E-cadherin, integrin alpha-6, and integrin beta-4 mark the DESCs both in vivo and in vitro. We also grew these cells in a three-dimensional microenvironment and obtained spheres with an epithelial morphology and expression patterns. Insights into the mechanisms of stem cell maintenance in vitro will help lay the groundwork for the successful generation of bioengineered teeth from adult DESCs.
doi:10.1089/ten.tec.2012.0232
PMCID: PMC3522131  PMID: 22742471
5.  Revitalization of a Diastemal Tooth Primordium in Spry2 Null Mice Results From Increased Proliferation and Decreased Apoptosis 
An understanding of the factors that promote or inhibit tooth development is essential for designing biological tooth replacements. The embryonic mouse dentition provides an ideal system for studying such factors because it consists of two types of tooth primordia. One type of primordium will go on to form a functional tooth, whereas the other initiates development but arrests at or before the bud stage. This developmental arrest contributes to the formation of the toothless mouse diastema. It is accompanied by the apoptosis of the rudimentary diastemal buds, which presumably results from the insufficient activity of anti-apoptotic signals such as fibroblast growth factors (FGFs). We have previously shown that the arrest of a rudimentary tooth bud can be rescued by inactivating Spry2, an antagonist of FGF signaling. Here, we studied the role of the epithelial cell death and proliferation in this process by comparing the development of a rudimentary diastemal tooth bud (R2) and the first molar in the mandibles of Spry2−/− and wild-type (WT) embryos using histological sections, image analysis and 3D reconstructions. In the WT R2 at embryonic day 13.5, significantly increased apoptosis and decreased proliferation were found compared with the first molar. In contrast, increased levels of FGF signaling in Spry2−/− embryos led to significantly decreased apoptosis and increased proliferation in the R2 bud. Consequently, the R2 was involved in the formation of a supernumerary tooth primordium. Studies of the revitalization of rudimentary tooth primordia in mutant mice can help to lay the foundation for tooth regeneration by enhancing our knowledge of mechanisms that regulate tooth formation.
doi:10.1002/jez.b.21266
PMCID: PMC2880865  PMID: 19127536
6.  Temporal Analysis of Ectopic Enamel Production in Incisors From Sprouty Mutant Mice 
The mouse incisor has two unusual features: it grows continuously and it is covered by enamel exclusively on the labial side. The continuous growth is driven in part by epithelial stem cells in the cervical loop region that can both self-renew and give rise to ameloblasts. We have previously reported that ectopic enamel is found on the lingual side of the incisor in mice with loss-of-function of sprouty (spry) genes. Spry2+/−; Spry4−/− mice, in which three sprouty alleles have been inactivated, have ectopic enamel as a result of upregulation of epithelial-mesenchymal FGF signaling in the lingual part of the cervical loop. Interestingly, lingual enamel is also present in the early postnatal period in Spry4−/− mice, in which only two sprouty alleles have been inactivated, but ectopic enamel is not found in adults of this genotype. To explore the mechanisms underlying the disappearance of lingual enamel in Spry4−/− adults, we studied the fate of the lingual enamel in Spry4−/− mice by comparing the morphology and growth of their lower incisors with wild type and Spry2+/−; Spry4−/− mice at several timepoints between the perinatal period and adulthood. Ameloblasts and enamel were detected on the lingual side in postnatal Spry2+/−; Spry4−/+ incisors. By contrast, new ectopic ameloblasts ceased to differentiate after postnatal day 3 in Spry4−/− incisors, which was followed by a progressive loss of lingual enamel. Both the posterior extent of lingual enamel and the time of its last deposition were variable early postnatally in Spry4−/− incisors, but in all Spry4−/− adult incisors the lingual enamel was ultimately lost through continuous growth and abrasion of the incisor.
doi:10.1002/jez.b.21254
PMCID: PMC2837846  PMID: 19101957
7.  Tooth, hair and claw: comparing epithelial stem cell niches of ectodermal appendages 
Experimental cell research  2014;325(2):96-103.
The vertebrate ectoderm gives rise to organs that produce mineralized or keratinized substances, including teeth, hair, and claws. Most of these ectodermal derivatives grow continuously throughout the animal’s life and have active pools of adult stem cells that generate all the necessary cell types. These organs provide powerful systems for understanding the mechanisms that enable stem cells to regenerate or renew ectodermally derived tissues, and remarkable progress in our understanding of these systems has been made in recent years using mouse models. We briefly compare what is known about stem cells and their niches in incisors, hair follicles, and claws, and we examine expression of Gli1 as a potential example of a shared stem cell marker. We summarize some of the features, structures, and functions of the stem cell niches in these ectodermal derivatives; definition of the basic elements of the stem cell niches in these organs will provide guiding principles for identification and characterization of the niche in similar systems.
doi:10.1016/j.yexcr.2014.02.003
PMCID: PMC4072742  PMID: 24530577
stem cells; niche; tooth; hair; nail; claw; ectodermal derivatives; Gli1
8.  Phenotypic and evolutionary implications of modulating the ERK-MAPK cascade using the dentition as a model 
Scientific Reports  2015;5:11658.
The question of phenotypic convergence across a signalling pathway has important implications for both developmental and evolutionary biology. The ERK-MAPK cascade is known to play a central role in dental development, but the relative roles of its components remain unknown. Here we investigate the diversity of dental phenotypes in Spry2−/−, Spry4−/−, and Rsk2−/Y mice, including the incidence of extra teeth, which were lost in the mouse lineage 45 million years ago (Ma). In addition, Sprouty-specific anomalies mimic a phenotype that is absent in extant mice but present in mouse ancestors prior to 9 Ma. Although the mutant lines studied display convergent phenotypes, each gene has a specific role in tooth number determination and crown patterning. The similarities found between teeth in fossils and mutants highlight the pivotal role of the ERK-MAPK cascade during the evolution of the dentition in rodents.
doi:10.1038/srep11658
PMCID: PMC4485067  PMID: 26123406
9.  Transcriptome-wide Analysis Reveals Hallmarks of Human Intestine Development and Maturation In Vitro and In Vivo 
Stem Cell Reports  2015;4(6):1140-1155.
Summary
Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation.
Highlights
•HIOs derived from hPSCs remain fetal in vitro•HIOs become adult-like when transplanted into mice•Transcriptional profiling across time reveals hallmarks of human gut maturation•The intestinal stem cell protein OLFM4 is a marker of human crypt maturation
Human pluripotent stem cell-derived intestinal organoids (HIOs) are an in vitro model of the small intestine. Spence and colleagues used transcriptional profiling to demonstrate that HIOs remain fetal in vitro and show that they undergo maturation into adult-like tissue when transplanted in vivo. Their results demonstrate that HIOs are a valuable in vitro model to study the fetal intestine.
doi:10.1016/j.stemcr.2015.04.010
PMCID: PMC4471827  PMID: 26050928
10.  Characterization of X-linked Hypohidrotic Ectodermal Dysplasia (XL-HED) Hair and Sweat Gland Phenotypes Using Phototrichogram Analysis and Live Confocal Imaging 
Hypohidrotic ectodermal dysplasia (HED) is the most common type of ectodermal dysplasia (ED), which encompasses a large group of syndromes that share several phenotypic features such as missing or malformed ectodermal structures, including skin, hair, sweat glands, and teeth. X-linked hypohidrotic ectodermal dysplasia (XL-HED) is associated with mutations in ectodysplasin (EDA1). Hypohidrosis due to hypoplastic sweat glands and thin, sparse hair are phenotypic features that significantly affect the daily lives of XL-HED individuals and therefore require systematic analysis. We sought to determine the quality of life of individuals with XL-HED and to quantify sweat duct and hair phenotypes using confocal imaging, pilocarpine iontophoresis, and phototrichogram analysis. Using these highly sensitive and non-invasive techniques, we demonstrated that 11/12 XL-HED individuals presented with a complete absence of sweat ducts and that none produced sweat. We determined that the thin hair phenotype observed in XL-HED was due to multiple factors, such as fewer terminal hairs with decreased thickness and slower growth rate, as well as fewer follicular units and fewer hairs per unit. The precise characterization of XL-HED phenotypes using sensitive and non-invasive techniques presented in our study will improve upon larger genotype-phenotype studies and in the assessment of future therapies in XL-HED.
doi:10.1002/ajmg.a.35959
PMCID: PMC4414120  PMID: 23687000
X-Linked hypohidrotic ectodermal dysplasia; ectodysplasin; hair; sweat gland; terminal hair; confocal imaging; pilocarpine iontophoresis; phototrichogram
11.  Hyperplasia of Interstitial Cells of Cajal in Sprouty Homolog 4 Deficient Mice 
PLoS ONE  2015;10(4):e0124861.
Gastrointestinal stromal tumors, which are thought to derive from interstitial cells of Cajal or their precursors, often harbor an oncogenic mutation of the KIT receptor tyrosine kinase. Sprouty homolog 4, a known negative regulator of ERK pathway, has been identified in the interstitial cells of Cajal in the KitK641E murine model of gastrointestinal stromal tumors. Sprouty homolog 4 was upregulated both at the mRNA and protein level in these cells, suggesting that Sprouty homolog 4 is downstream of oncogenic KIT activation and potentially engaged in the negative feedback loop of ERK activation in this model. Here, we used KitK641E heterozygous and Sprouty homolog 4 knock out animals to quantify interstitial cells of Cajal in situ, using quantitative immunofluorescence for the receptor tyrosine kinase Kit and for phosphodiesterase 3a (PDE3A). In the antrum of Sprouty homolog 4 knock out mice, hyperplasia of interstitial cells of Cajal was reminiscent of the KitK641E heterozygous mice antrum. Additionally, the density of interstitial cells of Cajal was higher in the colon of adult Sprouty homolog 4 knock out mice than in WT littermates, although hyperplasia seemed more severe in KitK641E heterozygous mice. Functional transit studies also show similarities between Sprouty homolog 4 knock out and KitK641E heterozygous mice, as the total transit time in 9 month old animals was significantly increased in both genotypes compared to WT littermates. We concluded that the lack of Sprouty homolog 4 expression leads to hyperplasia of the interstitial cells of Cajal and is functionally associated with a delayed transit time.
doi:10.1371/journal.pone.0124861
PMCID: PMC4414615  PMID: 25923139
12.  Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis 
Cell reports  2015;11(1):33-42.
Summary
Proper organ homeostasis requires tight control of adult stem cells and differentiation through integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a de-repression of the Wnt signaling pathway, leading to mis-expression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology.
doi:10.1016/j.celrep.2015.03.007
PMCID: PMC4394041  PMID: 25818302
13.  In vitro generation of human pluripotent stem cell derived lung organoids 
eLife  null;4:e05098.
Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease.
DOI: http://dx.doi.org/10.7554/eLife.05098.001
eLife digest
Cell behavior has traditionally been studied in the lab in two-dimensional situations, where cells are grown in thin layers on cell-culture dishes. However, most cells in the body exist in a three-dimensional environment as part of complex tissues and organs, and so researchers have been attempting to re-create these environments in the lab. To date, several such ‘organoids’ have been successfully generated, including models of the human intestine, stomach, brain and liver. These organoids can mimic the responses of real tissues and can be used to investigate how organs form, change with disease, and how they might respond to potential therapies.
Here, Dye et al. developed a new three-dimensional model of the human lung by coaxing human stem cells to become specific types of cells that then formed complex tissues in a petri dish. To make these lung organoids, Dye et al. manipulated several of the signaling pathways that control the formation of organs during the development of animal embryos. First, the stem cells were instructed to form a type of tissue called endoderm, which is found in early embryos and gives rise to the lung, liver and other several other internal organs.
Then, Dye et al. activated two important developmental pathways that are known to make endoderm form three-dimensional intestinal tissue. However, by inhibiting two other key developmental pathways at the same time, the endoderm became tissue that resembles the early lung found in embryos instead.
This early lung-like tissue formed three-dimensional spherical structures as it developed. The next challenge was to make these structures develop into lung tissue. Dye et al. worked out a method to do this, which involved exposing the cells to additional proteins that are involved in lung development. The resulting lung organoids survived in laboratory cultures for over 100 days and developed into well-organized structures that contain many of the types of cells found in the lung.
Further analysis revealed the gene activity in the lung organoids resembles that of the lung of a developing human fetus, suggesting that lung organoids grown in the dish are not fully mature. Dye et al.'s findings provide a new approach for creating human lung organoids in culture that may open up new avenues for investigating lung development and diseases.
DOI: http://dx.doi.org/10.7554/eLife.05098.002
doi:10.7554/eLife.05098
PMCID: PMC4370217  PMID: 25803487
pluripotent stem cells; organoids; endoderm; lung; foregut; spheroid; human
14.  On the cutting edge of organ renewal: identification, regulation and evolution of incisor stem cells 
The rodent incisor is one of a number of organs that grow continuously throughout the life of an animal. Continuous growth of the incisor arose as an evolutionary adaptation to compensate for abrasion at the distal end of the tooth. The sustained turnover of cells that deposit the mineralized dental tissues is made possible by epithelial and mesenchymal stem cells residing at the proximal end of the incisor. A complex network of signaling pathways and transcription factors regulates the formation, maintenance, and differentiation of these stem cells during development and throughout adulthood. Research over the past 15 years has led to significant progress in our understanding of this network, which includes FGF, BMP, Notch, and Hh signaling, as well as cell adhesion molecules and microRNAs. This review surveys key historical experiments that laid the foundation of the field and discusses more recent findings that definitively identified the stem cell population, elucidated the regulatory network, and demonstrated possible genetic mechanisms for the evolution of continuously growing teeth.
doi:10.1002/dvg.22732
PMCID: PMC4252016  PMID: 24307456
hypselodont; tissue regeneration; tooth; dental; renewal
15.  A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable 
Nature  2011;478(7368):255-259.
The small intestine epithelium renews every 2 to 5 days, making it one of the most regenerative mammalian tissues. Genetic inducible fate mapping studies have identified two principal epithelial stem cell pools in this tissue. One pool consists of columnar Lgr5-expressing cells that cycle rapidly and are present predominantly at the crypt base1. The other pool consists of Bmi1-expressing cells that largely reside above the crypt base2. However, the relative functions of these two pools and their interrelationship are not understood. Here, we specifically ablated Lgr5-expressing cells using a diphtheria toxin receptor (DTR) gene knocked into the Lgr5 locus. We found that complete loss of the Lgr5-expressing cells did not perturb homeostasis of the epithelium, indicating that other cell types can compensate for elimination of this population. After ablation of Lgr5-expressing cells, progeny production by Bmi1-expressing cells increased, suggesting that Bmi1-expressing stem cells compensate for the loss of Lgr5-expressing cells. Indeed, lineage tracing showed that Bmi1-expressing cells gave rise to Lgr5-expressing cells, pointing to a hierarchy of stem cells in the intestinal epithelium. Our results demonstrate that Lgr5-expressing cells are dispensable for normal intestinal homeostasis. In the absence of these cells, the Bmi1-expressing cells can serve as an alternative stem cell pool, providing the first experimental evidence for the interrelationship between these populations. The Bmi1-expressing stem cells may represent both a reserve stem cell pool in case of injury to the small intestine epithelium and a source for replenishment of the Lgr5-expressing cells under non-pathological conditions.
doi:10.1038/nature10408
PMCID: PMC4251967  PMID: 21927002
16.  Developmental disorders of the dentition: an update 
Dental anomalies are common congenital malformations that can occur either as isolated findings or as part of a syndrome. This review focuses on genetic causes of abnormal tooth development and the implications of these abnormalities for clinical care. As an introduction, we describe general insights into the genetics of tooth development obtained from mouse and zebrafish models. This is followed by a discussion of isolated as well as syndromic tooth agenesis, including Van der Woude syndrome, ectodermal dysplasias, oral-facial-digital syndrome type I, Rieger syndrome, holoprosencephaly, and tooth anomalies associated with cleft lip and palate. Next, we review delayed formation and eruption of teeth, as well as abnormalities in tooth size, shape and form. Finally, isolated and syndromic causes of supernumerary teeth are considered, including cleidocranial dysplasia and Gardner syndrome.
doi:10.1002/ajmg.c.31382
PMCID: PMC3844689  PMID: 24124058
mouse; zebrafish; teeth; hypodontia; supernumerary teeth; craniofacial; syndrome; tooth
17.  Craniofacial and Dental Development in Cardio-facio-cutaneous Syndrome: The Importance of Ras Signaling Homeostasis 
Clinical genetics  2012;83(6):539-544.
Cardio-facio-cutaneous syndrome (CFC) is a RASopathy that is characterized by craniofacial, dermatologic, gastrointestinal, ocular, cardiac, and neurologic anomalies. CFC is caused by activating mutations in the Ras/mitogen-activated protein kinase (MAPK) signaling pathway that lies downstream of receptor tyrosine kinase (RTK) signaling. RTK signaling is known to play a central role in craniofacial and dental development, but to date, no studies have systematically examined individuals with CFC to define key craniofacial and dental features. To fill this critical gap in our knowledge, we evaluated the craniofacial and dental phenotype of a large cohort (n=32) of CFC individuals who attended the 2009 and 2011 CFC International Family Conferences. We determined that the craniofacial features common in CFC include macrocephaly, bitemporal narrowing, convex facial profile, and hypoplastic supraorbital ridges. In addition, there is a characteristic dental phenotype in CFC syndrome that includes malocclusion with open bite, posterior crossbite, dental crowding, and a high-arched palate. This thorough evaluation of the craniofacial and dental phenotype in CFC individuals provides a step forward in our understanding of the role of RTK/MAPK signaling in human craniofacial development and will aid clinicians who treat patients with CFC.
doi:10.1111/cge.12005
PMCID: PMC4115672  PMID: 22946697
Cardio-facio-cutaneous syndrome; CFC; craniofacial development; malocclusion; MAPK pathway; occlusion; Ras; RASopathy; receptor tyrosine kinase; signal transduction; tooth development
18.  Injectable Bone Tissue Engineering Using Expanded Mesenchymal Stem Cells 
Stem cells (Dayton, Ohio)  2013;31(3):572-580.
Patients suffering from bone defects are often treated with autologous bone transplants, but this therapy can cause many complications. New approaches are therefore needed to improve treatment for bone defects, and stem cell therapy presents an exciting alternative approach. Although extensive evidence from basic studies using stem cells has been reported, very few clinical applications using stem cells for bone tissue engineering have been developed. We investigated whether injectable tissue-engineered bone composed of mesenchymal stem cells (MSCs) and platelet rich plasma was able to regenerate functional bone in alveolar deficiencies. We performed these studies in animals and subsequently carried out pilot trial cases in patients with long-term follow up; these showed good bone formation using minimally invasive MSC transplantation. All patients exhibited significantly improved bone volume with no side effects. Newly formed bone areas at 3 months was significantly higher than the pre-operation baseline (P <0.001) and reached levels equivalent to that of native bone. No significant bone resorption occurred during long term follow-up. Injectable tissue-engineered bone restored masticatory function in patients. This novel clinical approach represents an effective therapeutic utilization of bone tissue engineering.
doi:10.1002/stem.1300
PMCID: PMC4043220  PMID: 23225744
tissue engineering; regenerative medicine; bone; cell transplantation; clinical application
19.  From molecules to mastication: the development and evolution of teeth 
Teeth are unique to vertebrates and have played a central role in their evolution. The molecular pathways and morphogenetic processes involved in tooth development have been the focus of intense investigation over the past few decades, and the tooth is an important model system for many areas of research. Developmental biologists have exploited the clear distinction between the epithelium and the underlying mesenchyme during tooth development to elucidate reciprocal epithelial/mesenchymal interactions during organogenesis. The preservation of teeth in the fossil record makes these small organs essential for the work of paleontologists, anthropologists, and evolutionary biologists. In addition, with the recent identification and characterization of dental stem cells, teeth have become of interest to the field of regenerative medicine. Here, we review the major research areas and studies in the development and evolution of teeth, including morphogenesis, genetics and signaling, evolution of tooth development, and dental stem cells. Brief discussions of microRNAs and human disease as they apply to teeth are also included.
doi:10.1002/wdev.63
PMCID: PMC3632217  PMID: 24009032
20.  Bmi1 represses Ink4a/Arf and Hox genes to regulate stem cells in the rodent incisor 
Nature cell biology  2013;15(7):846-852.
The polycomb group gene Bmi1 is required for maintenance of adult stem cells in many organs1, 2. Inactivation of Bmi1 leads to impaired stem cell self-renewal due to deregulated gene expression. One critical target of BMI1 is Ink4a/Arf, which encodes the cell cycle inhibitors p16ink4a and p19Arf3. However, deletion of Ink4a/Arf only partially rescues Bmi1 null phenotypes4, indicating that other important targets of BMI1 exist. Here, using the continuously-growing mouse incisor as a model system, we report that Bmi1 is expressed by incisor stem cells and that deletion of Bmi1 resulted in fewer stem cells, perturbed gene expression, and defective enamel production. Transcriptional profiling revealed that Hox expression is normally repressed by BMI1 in the adult, and functional assays demonstrated that BMI1-mediated repression of Hox genes preserves the undifferentiated state of stem cells. As Hox gene upregulation has also been reported in other systems when Bmi1 is inactivated1, 2, 5–7, our findings point to a general mechanism whereby BMI1-mediated repression of Hox genes is required for the maintenance of adult stem cells and for prevention of inappropriate differentiation.
doi:10.1038/ncb2766
PMCID: PMC3735916  PMID: 23728424
21.  Oral epithelial stem cells in tissue maintenance and disease: the first steps in a long journey 
The identification and characterization of stem cells is a major focus of developmental biology and regenerative medicine. The advent of genetic inducible fate mapping techniques has made it possible to precisely label specific cell populations and to follow their progeny over time. When combined with advanced mathematical and statistical methods, stem cell division dynamics can be studied in new and exciting ways. Despite advances in a number of tissues, relatively little attention has been paid to stem cells in the oral epithelium. This review will focus on current knowledge about adult oral epithelial stem cells, paradigms in other epithelial stem cell systems that could facilitate new discoveries in this area and the potential roles of epithelial stem cells in oral disease.
doi:10.1038/ijos.2013.46
PMCID: PMC3967329  PMID: 23887128
cancer stem cell; invariant asymmetry; neutral drift; oral epithelial stem cell; population asymmetry
22.  Sox2+ Stem Cells Contribute to All Epithelial Lineages of the Tooth via Sfrp5+ Progenitors 
Developmental cell  2012;23(2):317-328.
SUMMARY
The continuously growing mouse incisor serves as a valuable model to study stem cell regulation during organ renewal. Epithelial stem cells are localized in the proximal end of the incisor in the labial cervical loop. Here, we show that the transcription factor Sox2 is a specific marker for these stem cells. Sox2+ cells became restricted to the labial cervical loop during tooth morphogenesis, and they contributed to the renewal of enamel-producing ameloblasts as well as all other epithelial cell lineages of the tooth. The early progeny of Sox2-positive stem cells transiently expressed the Wnt inhibitor Sfrp5. Sox2 expression was regulated by the tooth initiation marker FGF8 and specific miRNAs, suggesting a fine-tuning to maintain homeostasis of the dental epithelium. The identification of Sox2 as a marker for the dental epithelial stem cells will facilitate further studies on their lineage segregation and differentiation during tooth renewal.
doi:10.1016/j.devcel.2012.05.012
PMCID: PMC3690347  PMID: 22819339
23.  A genome-wide association study identifies susceptibility loci for non-syndromic sagittal craniosynostosis near BMP2 and within BBS9 
Nature genetics  2012;44(12):1360-1364.
Sagittal craniosynostosis is the most common form of craniosynostosis, affecting approximately one of 5,000 newborns. We conducted the first genome-wide association study (GWAS) for non-syndromic sagittal craniosynostosis (sNSC) using 130 non-Hispanic white (NHW) case-parent trios. Robust associations were observed in a 120 kb region downstream of BMP2, flanked by rs1884302 (P = 1.13 × 10−14; odds ratio [OR] = 4.58) and rs6140226 (P = 3.40 × 10−11; OR = 0.24) and within a 167 kb region of BBS9 between rs10262453 (P = 1.61 × 10−10; OR=0.19) and rs17724206 (P = 1.50 × 10−8; OR = 0.22). We replicated the associations to both loci [rs1884302 (P = 4.39 × 10−31); rs10262453 (P = 3.50 × 10−14)] in an independent NHW population of 172 unrelated sNSC probands and 548 controls. Both BMP2 and BBS9 are genes with a role in skeletal development warranting functional studies to further understand the etiology of sNSC.
doi:10.1038/ng.2463
PMCID: PMC3736322  PMID: 23160099
genome-wide association study; non-syndromic sagittal craniosynostosis; BMP2; BBS9; meta-analysis; nonsyndromic
24.  Sprouty genes regulate proliferation and survival of human embryonic stem cells 
Scientific Reports  2013;3:2277.
Sprouty (Spry) genes encode negative regulators of receptor tyrosine kinase (RTK) signaling, which plays important roles in human embryonic stem cells (hESCs). SPRY2 and SPRY4 are the two most highly expressed Sprouty family members in hESCs, suggesting that they may influence self-renewal. To test this hypothesis, we performed siRNA-mediated knock down (KD) studies. SPRY2 KD resulted in increased cell death and decreased proliferation, whereas SPRY4 KD enhanced survival. In both cases, after KD the cells were able to differentiate into cells of the three germ layers, although after SPRY2 KD there was a tendency toward increased ectodermal differentiation. SPRY2 KD cells displayed impaired mitochondrial fusion and cell membrane damage, explaining in part the increased cell death. These data indicate that Sprouty genes regulate pathways involved in proliferation and cell death in hESCs.
doi:10.1038/srep02277
PMCID: PMC3721083  PMID: 23880645
25.  Stem Cell and Biomaterials Research in Dental Tissue Engineering and Regeneration 
Dental clinics of North America  2012;56(3):495-520.
doi:10.1016/j.cden.2012.05.009
PMCID: PMC3494412  PMID: 22835534
Tissue engineering; Regenerative medicine; Dental tissues; Scaffold

Results 1-25 (35)