Neural crest E-cadherin loss drives cleft lip/palate by epigenetic modulation via pro-inflammatory gene-environment interaction

Gene-environment interactions are believed to play a role in multifactorial phenotypes, although poorly described mechanistically. Cleft lip/palate (CLP), the most common craniofacial malformation, has been associated with both genetic and environmental factors, with little gene-environment interaction experimentally demonstrated. Here, we study CLP families harbouring CDH1/E-Cadherin variants with incomplete penetrance and we explore the association of pro-inflammatory conditions to CLP. By studying neural crest (NC) from mouse, Xenopus and humans, we show that CLP can be explained by a 2-hit model, where NC migration is impaired by a combination of genetic (CDH1 loss-of-function) and environmental (pro-inflammatory activation) factors, leading to CLP. Finally, using in vivo targeted methylation assays, we demonstrate that CDH1 hypermethylation is the major target of the pro-inflammatory response, and a direct regulator of E-cadherin levels and NC migration. These results unveil a gene-environment interaction during craniofacial development and provide a 2-hit mechanism to explain cleft lip/palate aetiology

Human Stem Cell Cultures from Cleft Lip/Palate Patients Show Enrichment of Transcripts Involved in Extracellular Matrix Modeling By Comparison to Controls

Nonsyndromic cleft lip and palate (NSCL/P) is a complex disease resulting from failure of fusion of facial primordia, a complex developmental process that includes the epithelial-mesenchymal transition (EMT). Detection of differential gene transcription between NSCL/P patients and control individuals offers an interesting alternative for investigating pathways involved in disease manifestation. Here we compared the transcriptome of 6 dental pulp stem cell (DPSC) cultures from NSCL/P patients and 6 controls. Eighty-seven differentially expressed genes (DEGs) were identified. The most significant putative gene network comprised 13 out of 87 DEGs of which 8 encode extracellular proteins: ACAN, COL4A1, COL4A2, GDF15, IGF2, MMP1, MMP3 and PDGFa. Through clustering analyses we also observed that MMP3, ACAN, COL4A1 and COL4A2 exhibit co-regulated expression. Interestingly, it is known that MMP3 cleavages a wide range of extracellular proteins, including the collagens IV, V, IX, X, proteoglycans, fibronectin and laminin. It is also capable of activating other MMPs. Moreover, MMP3 had previously been associated with NSCL/P. The same general pattern was observed in a further sample, confirming involvement of synchronized gene expression patterns which differed between NSCL/P patients and controls. These results show the robustness of our methodology for the detection of differentially expressed genes using the RankProd method. In conclusion, DPSCs from NSCL/P patients exhibit gene expression signatures involving genes associated with mechanisms of extracellular matrix modeling and palate EMT processes which differ from those observed in controls. This comparative approach should lead to a more rapid identification of gene networks predisposing to this complex malformation syndrome than conventional gene mapping technologies

Investigating craniofacial malformations with the use of neural crest-derived cell models

As malformações craniofaciais (MCFs) compreendem uma vasta e heterogênea gama de doenças que envolvem o acometimento de tecidos do crânio e da face, sendo que indivíduos afetados enfrentam morbidade e deficiências funcionais relevantes. O entendimento da etiologia das MCFs é de extrema importância, pois poderá levar ao desenvolvimento ou melhoria de estratégias preventivas e terapêuticas. As MCFs são oriundas principalmente de distúrbios no desenvolvimento da crista neural cranial e seus derivados mesenquimais. Neste contexto, modelos baseados em células derivadas de crista neural são de grande potencial para o entendimento das MCFs, já que estudos funcionais podem ser realizados nestas células para averiguar fenótipos diretamente relacionados às doenças. Neste trabalho, nós empregamos esta estratégia na investigação de três tipos de MCFs: fissuras labiopalatinas não sindrômicas (FLP NS), síndrome de Richieri-Costa-Pereira (SRCP) e síndrome de Treacher Collins (STC). As FLP NS foram investigadas por meio de ensaios transcriptômicos e funcionais em células-tronco de polpa de dente decíduo, que são células mesenquimais adultas derivadas de crista neural cranial. Identificamos uma assinatura de expressão gênica específica às FLP NS, com desregulação de uma rede gênica responsável pelo reparo de quebras duplas no DNA, resultando no acúmulo deste tipo de lesão em células de indivíduos afetados pela doença. Estes achados revelam um novo mecanismo patogênico para as FLP NS e corroboram observações prévias que sugeriam sobreposição de etiologias entre esta doença e o câncer. A SRCP e a STC foram investigadas com o uso de uma nova metodologia para a geração de células de crista neural a partir de células-tronco pluripotentes induzidas (induced pluripotent stem cells; iPSCs) para recapitular o desenvolvimento craniofacial. Realizamos triagem de fenótipos celulares e identificamos desregulação de diferenciação osteogênica em células mesenquimais derivadas de crista neural de pacientes com SRCP, o que foi corroborado por ensaios de RNA de interferência. Além disso, mostramos que células mesenquimais de crista neural de pacientes com STC apresentam apoptose elevada aliada a alterações durante diferenciação osteogênica e condrogênica. Estes resultados revelam que células mesenquimais de crista neural estão alteradas na SRCP e STC, colaborando para o esclarecimento dos mecanismos patogênicos responsáveis por estas síndromes. Assim sendo, nós evidenciamos a aplicabilidade da modelagem de MCFs por meio de células oriundas da crista neural, e esses achados inéditos contribuirão para um melhor entendimento da etiologia das MCFs, e servem de base para futuras estratégias de pesquisa na área de doenças craniofaciaisCraniofacial malformations (CFMs) comprise a large and heterogeneous group of disorders in which tissues of the skull and face are affected. Affected subjects suffer from significant functional impairment and morbidity, and understanding the aetiology of these disorders is of great importance, as it may lead to the development or improvement of preventive and therapeutic strategies in the future. CFMs are largely considered to arise from developmental disturbances in the cranial neural crest and its cranioskeletal and cartilaginous mesenchymal derivatives. Neural crest-derived cell models have the potential to provide invaluable insight into the pathogenesis of CFMs, as functional studies can be to assess phenotypes in disease-relevant cell lineages. In this work, we applied this strategy to investigate three craniofacial disorders: non-syndromic cleft lip/palate (NSCL/P), Richieri-Costa-Pereira syndrome (RCPS), and Treacher Collins syndrome (TCS). NSCL/P was investigated through transcriptomic and functional assays on stem cells from human exfoliated deciduous teeth, which are neural crest-derived, adult mesenchymal cells. We identified a NSCL/P-specific dysregulated transcriptional signature involving a gene network responsible for DNA double-strand break repair that results in accumulation of DNA damage in patients\' cells. These findings revealed a novel pathogenetic mechanism for NSCL/P and support previous observations pointing towards an aetiological overlap between this disease and cancer. RCPS and TCS were investigated with the use of a novel approach to generate neural crest cells from patient-specific induced pluripotent stem cells (iPSCs) as a means to recapitulate craniofacial development. We demonstrated that RCPS and TCS somatic cells can be successfully used to generate iPSCs and iPSC-derived neural crest cells and their mesenchymal derivatives. Phenotype screening showed that RCPS neural crest-derived mesenchymal cells display dysregulation of osteogenic differentiation, which was supported by confirmatory knockdown assays. Further, we report elevated apoptosis in TCS neural crest-derived mesenchymal cells, which was allied to alterations in chondrogenic and osteogenic differentiation. These results will aid in clarifying the pathogenic mechanism determining RCPS and TCS, revealing that neural crest mesenchymal cells are altered in these syndromes. In conclusion, we attested the applicability of NC-derived cell types to provide clues regarding the pathogenetic mechanisms leading to CFMs, and these novel findings will aid in dissecting the aetiology of CFMs by providing grounds to direct future efforts in craniofacial researc

Transcriptome analysis of mesenchymal stem cells to investigate the aetiology of non-syndromic cleft lip and palate

A fissura lábio-palatina não-sindrômica (FLP NS) é uma doença multifatorial, determinada pela interação entre fatores genéticos e ambientais e sua incidência é estimada entre 0,34 e 2,29 a cada 1000 nascimentos. Trata-se de uma embriopatia causada por erros durante a morfogênese orofacial, a qual depende de uma fina regulação de mecanismos como proliferação celular, remodelagem de matriz extracelular, e transição epitélio-mesenquimal. Apesar de intensos esforços para se determinar fatores genéticos e ambientais de susceptibilidade, a etiologia desta malformação permanece pouco compreendida. Vários loci associados às FLP NS vêm sendo identificados por meio de estudos de mapeamento gênico convencionais, entretanto, a grande maioria dos resultados não se replica em diferentes estudos, e não há clareza acerca do efeito funcional das variantes detectadas. Neste contexto, uma abordagem interessante para investigar a etiologia da doença é a análise de expressão gênica, que pode ser utilizada para identificar alterações de vias biológicas que convergem na manifestação do quadro clínico. Em vista disso, neste trabalho nós utilizamos a análise do transcriptoma de células-tronco de polpa dental de pacientes portadores de FLP NS, com o intuito de identificar padrões de expressão relacionados a mecanismos biológicos relevantes para a embriopatogênese da doença. Obtivemos padrões de expressão que sugerem desregulação de mecanismos associados à remodelagem de matriz extracelular e à transição epitélio-mesenquimal. Além disso, ao utilizarmos diferentes condições de cultura celular, verificamos em uma nova amostra de pacientes a desregulação de vias biológicas relacionadas ao reparo de DNA e checkpoint do ciclo celular. Nossos dados revelam a aplicabilidade das células-tronco de polpa dental para este tipo de abordagem, e indicam que tais perfis de expressão podem levar ao acometimento da morfogênese lábio-palatina. Além disso, mostramos pela primeira vez uma conexão entre desregulação de expressão gênica e a documentada maior incidência de formas esporádicas de câncer em famílias segregando a FLP NS. Nossos resultados abrem novas possibilidades para a investigação da etiologia das FLP NS, e ajudarão na compreensão dos eventos embrionários que predispõem a essa malformação.Non-syndromic cleft lip and palate (NSCL/P) is a multifactorial disease determined by the interplay between genetic and environmental factors, with a variable incidence of 0.34-2.29:1000 births. This malformation arises from errors during lip and palate morphogenesis, which requires tight regulation of biological mechanisms such as cellular proliferation, extracellular matrix remodelling, and epithelial-mesenchymal transition. Albeit much effort has been put into determining the genetic and environmental factors underlying disease susceptibility, the aetiology of NSCL/P remains obscure. Many candidate loci have been identified through conventional gene mapping strategies, however, there is a general lack of reproducibility across studies, and there is no consensus with regard to the functional implications of the identified genetic variants. In this context, an alternative approach resides in assessing differential expression patterns to identify alterations in biological networks that could lead to phenotype manifestation. Here, we analysed the transcriptome of dental pulp stem cells from NSCL/P patients in order to pinpoint dysregulated pathways involved in the embryopathogenesis of the disease. We encountered expression patterns related to dysregulation of extracellular matrix remodelling and epithelial mesenchymal transition. Moreover, by subjecting a novel NSCL/P sample to differential cell culture conditions, we observed abnormal transcription of genes partaking in DNA repair and cell cycle checkpoint pathways. Our results show the applicability of dental pulp stem cells to this strategy and suggest that the observed expression patterns could lead to impairment of lip and palate morphogenesis. Moreover, we described for the first time a connection between abnormal gene expression in these individuals and the elevated occurrence of sporadic cancer types in NSCL/P families. Our results open new possibilities to investigate the aetiology of NSCL/P and provide further insight into the ontogenetic events underlying disease predisposition

An experimental model for the study of craniofacial deformities Modelo experimental para o estudo de deformidades craniofaciais

PURPOSE: To develop an experimental surgical model in rats for the study of craniofacial abnormalities. METHODS: Full thickness calvarial defects with 10x10-mm and 5x8-mm dimensions were created in 40 male NIS Wistar rats, body weight ranging from 320 to 420 g. The animals were equally divided into two groups. The periosteum was removed and dura mater was left intact. Animals were killed at 8 and 16 weeks postoperatively and cranial tissue samples were taken from the defects for histological analysis. RESULTS: Cranial defects remained open even after 16 weeks postoperatively. CONCLUSION: The experimental model with 5x8-mm defects in the parietal region with the removal of the periosteum and maintenance of the integrity of the dura mater are critical and might be used for the study of cranial bone defects in craniofacial abnormalities.<br>OBJETIVO: Desenvolver um modelo experimental em ratos para o estudo de deformidades craniofaciais. MÉTODOS: Foram realizados defeitos ósseos de espessura total com diâmetro de 5 x 8 mm e 10 x 10mm na calota craniana em 40 ratos, machos, NIS Wistar, com peso de 320 a 420 g divididos igualmente em dois grupos. O periósteo foi retirado e a dura-máter mantida intacta. Os animais foram sacrificados na 8ª e 16ª semana de pós-operatório e amostras de tecido ósseo foram extraídas para realização da análise histológica. RESULTADOS: Os defeitos cranianos permaneceram abertos mesmo após 16 semanas após a cirurgia. CONCLUSÃO: O modelo experimental com defeitos de 5x8mm na região parietal, com remoção do periósteo e manutenção da integridade da dura-máter são considerados críticos, e poderá ser utilizado para o estudo dos defeitos ósseos cranianos nas anomalias craniofaciais

mir152 hypomethylation as a mechanism for non-syndromic cleft lip and palate

Non-syndromic cleft lip with or without cleft palate (NSCLP), the most common human craniofacial malformation, is a complex disorder given its genetic heterogeneity and multifactorial component revealed by genetic, epidemiological, and epigenetic findings. Epigenetic variations associated with NSCLP have been identified; however, functional investigation has been limited. Here, we combined a reanalysis of NSCLP methylome data with genetic analysis and used both in vitro and in vivo approaches to dissect the functional effects of epigenetic changes. We found a region in mir152 that is frequently hypomethylated in NSCLP cohorts (21–26%), leading to mir152 overexpression. mir152 overexpression in human neural crest cells led to downregulation of spliceosomal, ribosomal, and adherens junction genes. In vivo analysis using zebrafish embryos revealed that mir152 upregulation leads to craniofacial cartilage impairment. Also, we suggest that zebrafish embryonic hypoxia leads to mir152 upregulation combined with mir152 hypomethylation and also analogous palatal alterations. We therefore propose that mir152 hypomethylation, potentially induced by hypoxia in early development, is a novel and frequent predisposing factor to NSCLP

Improvement of In Vitro Osteogenic Potential through Differentiation of Induced Pluripotent Stem Cells from Human Exfoliated Dental Tissue towards Mesenchymal-Like Stem Cells

Constraints for the application of MSCs for bone reconstruction include restricted self-renewal and limited cell amounts. iPSC technology presents advantages over MSCs, providing homogeneous cellular populations with prolonged self-renewal and higher plasticity. However, it is unknown if the osteogenic potential of iPSCs differs from that of MSCs and if it depends on the iPSCs originating cellular source. Here, we compared the in vitro osteogenesis between stem cells from human deciduous teeth (SHED) and MSC-like cells from iPSCs from SHED (iPS-SHED) and from human dermal fibroblasts (iPS-FIB). MSC-like cells from iPS-SHED and iPS-FIB displayed fibroblast-like morphology, downregulation of pluripotency markers and upregulation of mesenchymal markers. Comparative in vitro osteogenesis analysis showed higher osteogenic potential in MSC-like cells from iPS-SHED followed by MSC-like cells from iPS-FIB and SHED. CD105 expression, reported to be inversely correlated with osteogenic potential in MSCs, did not display this pattern, considering that SHED presented lower CD105 expression. Higher osteogenic potential of MSC-like cells from iPS-SHED may be due to cellular homogeneity and/or to donor tissue epigenetic memory. Our findings strengthen the rationale for the use of iPSCs in bone bioengineering. Unveiling the molecular basis behind these differences is important for a thorough use of iPSCs in clinical scenarios