RESUMO
Teeth are comprised of epithelial and mesenchymal cells, and regenerative teeth rely on the regeneration of both cell types. Transcription factors play a pivotal role in cell fate determination. In this study, we establish fluorescence models based on transcription factors to monitor and analyze dental epithelial cells. Using Pitx2-P2A-copGFP mice, we observe that Pitx2+ epithelial cells, when combined with E14.5 dental mesenchymal cells, are sufficient for the reconstitution of teeth. Induced-Pitx2+ cells, directly isolated from the embryoid body that employs the Pitx2-GFP embryonic stem cell line, exhibit the capacity to differentiate into ameloblasts and develop into teeth when combined with dental mesenchymal cells. The regenerated teeth exhibit a complete structure, including dental pulp, dentin, enamel, and periodontal ligaments. Subsequent exploration via RNA-seq reveals that induced-Pitx2+ cells exhibit enrichment in genes associated with FGF receptors and WNT ligands compared with induced-Pitx2- cells. Our results indicate that both primary Pitx2+ and induced Pitx2+ cells possess the capability to differentiate into enamel-secreting ameloblasts and grow into teeth when combined with dental mesenchymal cells.
RESUMO
Many human diseases share a developmental origin that manifests during childhood or maturity. Aneuploid syndromes are caused by supernumerary or reduced number of chromosomes and represent an extreme example of developmental disease, as they have devastating consequences before and after birth. Investigating how alterations in gene dosage drive these conditions is relevant because it might help treat some clinical aspects. It may also provide explanations as to how quantitative differences in gene expression determine phenotypic diversity and disease susceptibility among natural populations. Here, we aimed to produce induced pluripotent stem cell (iPSC) lines that can be used to improve our understanding of aneuploid syndromes. We have generated iPSCs from monosomy X [Turner syndrome (TS)], trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome) and partial trisomy 11;22 (Emanuel syndrome), using either skin fibroblasts from affected individuals or amniocytes from antenatal diagnostic tests. These cell lines stably maintain the karyotype of the donors and behave like embryonic stem cells in all tested assays. TS iPSCs were used for further studies including global gene expression analysis and tissue-specific directed differentiation. Multiple clones displayed lower levels of the pseudoautosomal genes ASMTL and PPP2R3B than the controls. Moreover, they could be transformed into neural-like, hepatocyte-like and heart-like cells, but displayed insufficient up-regulation of the pseudoautosomal placental gene CSF2RA during embryoid body formation. These data support that abnormal organogenesis and early lethality in TS are not caused by a tissue-specific differentiation blockade, but rather involves other abnormalities including impaired placentation.
Assuntos
Aneuploidia , Transtornos Cromossômicos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular , Células Cultivadas , Transtornos Cromossômicos/metabolismo , Transtornos Cromossômicos/fisiopatologia , Feminino , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Masculino , Modelos GenéticosRESUMO
Ecto-mesenchymal cells of mammalian tooth germ develops from cranial neural crest cells. These cells are recognised as a promising source for tooth development and regeneration. Despite the high heterogeneity of the neural crest, the cellular landscape of in vitro cultured cranial neural crest cells (CNCCs) for odontogenesis remains unclear. In this study, we used large-scale single-cell RNA sequencing to analyse the cellular landscape of in vitro cultured mouse CNCCs for odontogenesis. We revealed distinct cell trajectories from primary cells to passage 5 and identified a rare Alx3+/Barx1+ sub-population in primary CNCCs that differentiated into two odontogenic clusters characterised by the up-regulation of Pax9/Bmp3 and Lhx6/Dmp1. We successfully induced whole tooth-like structures containing enamel, dentin, and pulp under the mouse renal capsule using in vitro cultured cells from both cranial and trunk neural crests with induction rates of 26.7% and 22.1%, respectively. Importantly, we confirmed only cells sorted from odontogenic path can induce tooth-like structures. Cell cycle and DNA replication genes were concomitantly upregulated in the cultured NCCs of the tooth induction groups. Our data provide valuable insights into the cell heterogeneity of in vitro cultured CNCCs and their potential as a source for tooth regeneration.
Assuntos
Diferenciação Celular , Crista Neural , Odontogênese , RNA-Seq , Análise de Célula Única , Animais , Crista Neural/citologia , Crista Neural/metabolismo , Camundongos , Odontogênese/genética , Análise de Célula Única/métodos , Células Cultivadas , Germe de Dente/metabolismo , Germe de Dente/citologia , Análise da Expressão Gênica de Célula ÚnicaRESUMO
The spatiotemporal relationships in high-resolution during odontogenesis remain poorly understood. We report a cell lineage and atlas of developing mouse teeth. We performed a large-scale (92,688 cells) single cell RNA sequencing, tracing the cell trajectories during odontogenesis from embryonic days 10.5 to 16.5. Combined with an assay for transposase-accessible chromatin with high-throughput sequencing, our results suggest that mesenchymal cells show the specific transcriptome profiles to distinguish the tooth types. Subsequently, we identified key gene regulatory networks in teeth and bone formation and uncovered spatiotemporal patterns of odontogenic mesenchymal cells. CD24+ and Plac8+ cells from the mesenchyme at the bell stage were distributed in the upper half and preodontoblast layer of the dental papilla, respectively, which could individually induce nonodontogenic epithelia to form tooth-like structures. Specifically, the Plac8+ tissue we discovered is the smallest piece with the most homogenous cells that could induce tooth regeneration to date. Our work reveals previously unknown heterogeneity and spatiotemporal patterns of tooth germs that may lead to tooth regeneration for regenerative dentistry.
Assuntos
Células-Tronco Mesenquimais , Dente , Camundongos , Animais , Odontogênese/genética , Germe de Dente , EpitélioRESUMO
The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover, these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology, positive staining for alkaline phosphatase, normal karyotype, and expression of hESC-like markers including Nanog, Rex1, Oct4, TRA-1-60, TRA-1-80, SSEA-3, and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers, and could as well be readily differentiated into functional motor neurons. Among other things, our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming, differentiation ability, stability of the resulting lineages, and the risk of associated abnormalities.
Assuntos
Âmnio/metabolismo , Técnicas de Cultura de Células/métodos , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Células-Tronco Pluripotentes/citologia , Cordão Umbilical/metabolismo , Animais , Linhagem Celular , Células Cultivadas/citologia , Humanos , Cariotipagem , Células-Tronco Mesenquimais/metabolismo , Camundongos , Modelos Biológicos , Neurônios Motores/metabolismo , Técnicas de Patch-Clamp , Células-Tronco Pluripotentes/metabolismo , Cordão Umbilical/citologiaRESUMO
We have previously demonstrated that tooth size is determined by dental mesenchymal factors. Exogenous bone morphogenetic protein (BMP)4, Noggin, fibroblast growth factor (FGF)3 and FGF10 have no effect on tooth size, despite the expressions of Bmp2, Bmp4, Fgf3, Fgf10 and Lef1 in the dental mesenchyme. Among the wingless (Wnt) genes that are differentially expressed during tooth development, only Wnt5a is expressed in the dental mesenchyme. The aims of the present study were to clarify the expression pattern of Wnt5a in developing tooth germs and the role of Wnt5a in the regulation of tooth size by treatment with exogenous WNT5A with/without an apoptosis inhibitor on in vitro tooth germs combined with transplantation into kidney capsules. Wnt5a was intensely expressed in both the dental epithelium and mesenchyme during embryonic days 14-17, overlapping partly with the expressions of both Shh and Bmp4. Moreover, WNT5A retarded the development of tooth germs by markedly inducing cell death in the non-dental epithelium and mesenchyme but not widely in the dental region, where the epithelial-mesenchymal gene interactions among Wnt5a, Fgf10, Bmp4 and Shh might partly rescue the cells from death in the WNT5A-treated tooth germ. Together, these results indicate that WNT5A-induced cell death inhibited the overall development of the tooth germ, resulting in smaller teeth with blunter cusps after tooth-germ transplantation. Thus, it is suggested that Wnt5a is involved in regulating cell death in non-dental regions, while in the dental region it acts as a regulator of other genes that rescue tooth germs from cell death.
Assuntos
Dente/anatomia & histologia , Dente/embriologia , Proteínas Wnt/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Bioensaio , Padronização Corporal/efeitos dos fármacos , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Soluções Tampão , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Modelos Biológicos , Tamanho do Órgão/efeitos dos fármacos , Dente/citologia , Dente/efeitos dos fármacos , Germe de Dente/citologia , Germe de Dente/efeitos dos fármacos , Germe de Dente/embriologia , Germe de Dente/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/farmacologia , Proteína Wnt-5aRESUMO
A stable, rapid and effective neural differentiation method is essential for the clinical applications of human embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) in treating neurological disorders and diseases. Herein, we established a novel and robust monolayer differentiation method to produce functional neural progenitor cells (NPCs) from human ESC/iPSCs on Type I Collagen. The derived cells not only displayed the requisite markers, but also behaved similarly to classic NPCs both in vitro and in vivo. Upon transplantation into traumatic brain injury model, the derived NPCs facilitated recovery from injury. We also found that SMAD signaling stayed down throughout the differentiation process on Type I Collagen, and the pluripotent signals were rapidly downregulated along with raising up of neural early markers on the third day. Meanwhile, ATAC-seq data showed the related mediation of distinct transcriptome and global chromatin dynamics during NPC induction. Totally, our results thus provide a convenient way to generate NPCs from human ESC/iPSCs for neural diseases' treatment.
Assuntos
Diferenciação Celular , Células-Tronco Embrionárias Humanas/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Neurais , Lesões Encefálicas Traumáticas/terapia , Técnicas de Cultura de Células , Colágeno Tipo I , Humanos , Células-Tronco Neurais/transplante , Análise de Sequência de RNARESUMO
Teeth, an excellent model for studying organogenesis, develop from a series of epithelial-mesenchymal interactions that are mediated by a complex molecular network. Bcor (BCL-6 interacting corepressor) has recently been discovered, but little is known about its function in tooth development. Mutations in BCOR affect humans with oculofaciocardiodental syndrome, which is an X-linked dominant disorder with presumed male lethality and which comprises microphthalmia, congenital cataracts, radiculomegaly, and cardiac and digital abnormalities. In this study, the Bcor expression pattern has been intensively investigated during mouse molar development. Bcor is expressed in both dental epithelium and the mesenchyme at E11.5. To understand the function of Bcor, knockdown of Bcor has been examined by using lentivirus-mediated RNA interference. Silencing of Bcor expression in dental mesenchymal cells at E14.5 causes dentinogenesis defects and retardation of tooth root development. Thus, our results suggest that Bcor expressed in the mesenchyme plays crucial roles during early tooth development. The function of Bcor expressed in the epithelium remains to be elucidated.
Assuntos
Mesoderma/embriologia , Odontogênese , Proteínas Repressoras/metabolismo , Dente/embriologia , Animais , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lentivirus/genética , Masculino , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Odontogênese/genética , Interferência de RNA , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Dente/metabolismoRESUMO
Various cellular and molecular events underlie the elevation and fusion of the developing palate that occurs during embryonic development. This includes convergent extension, where the medial edge epithelium is intercalated into the midline epithelial seam. We examined the expression patterns of Wnt11 and Fgfr1b - which are believed to be key factors in convergent extension - in mouse palate development. Wnt-11 overexpression and beads soaked in SU5402 (an Fgfr1 inhibitor) were employed in in vitro organ cultures. The results suggested that interactions between Wnt11 and Fgfr1b are important in modulating cellular events such as cell proliferation for growth and apoptosis for fusion. Moreover, the Wnt11 siRNA results showed that Wnt11-induced apoptosis was necessary for palatal fusion. In summary, Fgfr1b induces cell proliferation in the developing palate mesenchyme so that the palate grows and contacts each palatal shelf, with negative feedback of Fgfs triggered by excessive cell proliferation then inhibiting the expression of Fgfr1b and activating the expression of Wnt11 to fuse each palate by activating apoptosis.
Assuntos
Palato/citologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Proteínas Wnt/fisiologia , Envelhecimento/fisiologia , Animais , Eletroporação , Imuno-Histoquímica , Hibridização In Situ , Maxila/citologia , Maxila/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos ICR , Técnicas de Cultura de Órgãos , Palato/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/genética , Receptor Cross-Talk/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Proteínas Wnt/genéticaRESUMO
Rodent incisors are continuously growing teeth that include all stages of amelogenesis. Understanding amelogenesis requires investigations of the genes and their gene products control the ameloblast phenotype. One of the mechanisms related to tooth differentiation is mitogen-activated protein kinase (MAPK) signaling. The extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK) cascade is associated with mechanisms that control the cell cycle and cell survival. However, the roles of cascades in incisor development remain to be determined. In this study, we investigated incisor development and growth in the mouse based on MAPK signaling. Moreover, heat-shock protein (Hsp)-25 is well known to be a useful marker of odontoblast differentiation. We used anisomycin (a protein-synthesis inhibitor that activates MAPKs) and U0126 (a MAPK inhibitor that blocks ERK1/2 phosphorylation) to examine the role of MAPKs in Hsp25 signaling in the development of the mouse incisor. We performed immunohistochemistry and in vitro culture using incisor tooth germ, and found that phospho-ERK (pERK), pMEK, and Hsp25 localized in developing incisor ameloblasts and anisomycin failed to produce incisor development. In addition, Western blotting results showed that anisomycin stimulated the phosphorylation of ERK, MEK, and Hsp25, and that some of these proteins were blocked by the U0126. These findings suggest that MAPK signals play important roles in incisor formation, differentiation, and development by mediating Hsp25 signaling.
Assuntos
Ameloblastos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico/metabolismo , Incisivo/crescimento & desenvolvimento , MAP Quinase Quinase Quinases/metabolismo , Proteínas de Neoplasias/metabolismo , Ameloblastos/citologia , Ameloblastos/efeitos dos fármacos , Animais , Anisomicina/farmacologia , Butadienos/farmacologia , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Proteínas de Choque Térmico/efeitos dos fármacos , Incisivo/efeitos dos fármacos , Incisivo/metabolismo , Antígeno Ki-67/efeitos dos fármacos , Antígeno Ki-67/metabolismo , MAP Quinase Quinase Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Chaperonas Moleculares , Proteínas de Neoplasias/efeitos dos fármacos , Nitrilas/farmacologia , Técnicas de Cultura de Órgãos , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Teeth have been missing from Aves for almost 100 million years. However, it is believed that the avian oral epithelium retains the molecular signaling required to induce odontogenesis, and this has been widely examined using heterospecific recombinations with mouse dental mesenchyme. It has also been argued that teeth can form from the avian oral epithelium owing to contamination of the mouse mesenchyme with mouse dental epithelial cells. To investigate the possibility of tooth formation from chick oral epithelium and the characteristics of possible chick enamel, we applied LacZ transgenic mice during heterospecific recombination and examined the further tooth formation. Transmission electron microscopy was used to identify the two tissues during development after heterospecific recombination. No mixing was detected between chick oral epithelium and mouse dental mesenchyme after 2 days, and secretory ameloblasts with Tomes' processes were observed after 1 week. Teeth were formed after 3 weeks with a single cusp pattern, possibly determined by epithelial factors, which is similar to that of the avian tooth in the late Jurassic period. These recombinant teeth were smaller than mouse molars, whereas perfect structures of both ameloblasts and enamel showed histological characteristics similar to those of mice. Together these observations consistent with previous report that odontogenesis is initially directed by species-specific mesenchymal signals interplaying with common epithelial signals.
Assuntos
Embrião de Galinha/fisiologia , Mucosa Bucal/fisiologia , Animais , Galinhas , Primers do DNA , Células Epiteliais/fisiologia , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Dente Molar/embriologia , Boca/embriologia , Mucosa Bucal/citologia , Mucosa Bucal/embriologia , Mucosa Bucal/ultraestrutura , Odontogênese/genética , Recombinação Genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta-Galactosidase/genéticaRESUMO
The tooth is one of the ectodermal organs that develop from epithelial-mesenchymal interactions during embryonic development. An understanding of the underlying molecular mechanisms would improve our knowledge of the growth factors that regulate cell proliferation and differentiation. One of the related aspects is mitogen-activated protein kinase (MAPK) signaling in tooth differentiation. The extracellular-signal regulated kinase (ERK)/mitogen-activated protein kinase kinase (MEK) cascade plays a pivotal role in many of the essential cellular processes underlying embryonic development, including responses to major developmental changes. However, the role of the ERK pathway in molar development is unclear. This study investigated epithelial patterning and tooth growth in the mouse embryo by monitoring ERK and fibroblast growth factor (FGF) signaling. ERK, MEK, and phosphatase and tensin homolog (PTEN) were activated at different levels and locations in the developing tooth at E13.5 to E16.5 and PN2. ERK was activated in the inner dental epithelium and cervical loop, while PTEN was activated in the outer dental epithelium. In addition, only ERK was activated in secretory ameloblast at PN2. To further define the pathways involving FGF and ERK, tooth germs were cultured in the presence of compounds to inhibit MAPK/ERK-mediated signaling. Western blot analysis indicated that pERK2 was strongly activated in the tooth germ. Moreover, the activation level of pERK1 was dramatically increased by exogenous FGF10 alone and by combined treatment with FGF10 and U0126. The reported results will improve our understanding of the unique developmental processes of the dental epithelium and tooth growth, and will help to elucidate the fundamental mechanisms of ERK signaling underlying tooth development.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 10 de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Dente/crescimento & desenvolvimento , Animais , Western Blotting , Ativação Enzimática , Imuno-Histoquímica , Camundongos , Camundongos Nus , Microscopia Eletrônica de TransmissãoRESUMO
Dental pulp elaborates both bone and dentin under pathological conditions such as tooth replantation/transplantation. This study aims to clarify the capability of dental pulp to elaborate bone tissue in addition to dentin by allogenic tooth transplantation using immunohistochemistry and histochemistry. After extraction of the molars of 3-week-old mice, the roots and pulp floor were resected and immediately allografted into the sublingual region in a littermate. In addition, we studied the contribution of donor and host cells to the regenerated pulp tissue using a combination of allogenic tooth transplantation and lacZ transgenic ROSA26 mice. On Days 5-7, tubular dentin formation started next to the preexisting dentin at the pulp horn where nestin-positive odontoblast-like cells were arranged. Until Day 14, bone-like tissue formation occurred in the pulp chamber, where intense tartrate-resistant acid phosphatase-positive cells appeared. Furthermore, allogenic transplantation using ROSA26 mice clearly showed that both donor and host cells differentiated into osteoblast-like cells with the assistance of osteoclast-lineage cells, whereas newly differentiated odontoblasts were exclusively derived from donor cells. These results suggest that the odontoblast and osteoblast lineage cells reside in the dental pulp and that both donor and host cells contribute to bone-like tissue formation in the regenerated pulp tissue.
Assuntos
Polpa Dentária/citologia , Dente Molar/citologia , Fosfatase Ácida/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Polpa Dentária/metabolismo , Histocitoquímica , Proteínas de Filamentos Intermediários/metabolismo , Isoenzimas/metabolismo , Óperon Lac , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Dente Molar/metabolismo , Dente Molar/transplante , Soalho Bucal , Proteínas do Tecido Nervoso/metabolismo , Nestina , Osteopontina/metabolismo , Fosfatase Ácida Resistente a Tartarato , Transplante HomólogoRESUMO
The development of induced pluripotent stem cells (iPS cells) has raised the prospect of patient-specific treatments for various diseases. Theoretically, iPS cell technology avoids the limitations of human embryonic stem cells (ES cells), including poor establishment, ethical issues, and immune rejection of allogeneic transplantation. However, the immunogenicity of iPS cells has attracted the attention of researchers, and it remains unclear whether iPS cells and their derivatives will be recognized as a patient's own cells. Even though iPS-derived functional cells have been used in the treatment of some diseases, the process of somatic cell reprogramming and iPS cell differentiation is time-consuming, making it difficult to use iPS cells in acute illness or injury. In recent years, it has been suggested that iPS cells may be used as allografts by establishing an iPS cell bank and HLA matching, providing a novel strategy for the clinical application of iPS cells. This article provides a concise overview of iPS cell immunogenicity, and summarizes published data regarding the application of iPS cells in both autologous and allogeneic transplantation in order to help develop more reliable biotechnical strategies utilizing iPS cells.
Assuntos
Diferenciação Celular , Doença , Células-Tronco Pluripotentes Induzidas/citologia , Transplante de Células-Tronco , Animais , Humanos , Transplante Autólogo , Transplante HomólogoRESUMO
Glucosamine (GA) is an important cartilage matrix precursor for the glycosaminoglycan biochemical synthesis, and has positive effects on cartilage regeneration, particularly in osteoarthritis therapy. However, it has not been used as a bioactive group in scaffolds for cartilage repair widely. In this study, we synthesized modified polyethylene glycol (PEG) hydrogel with glucosamine and then encapsulated human bone mesenchymal stem cells (hBMSCs) in the hydrogel to induce the differentiation of hBMSCs into chondrocytes in three-dimensional culture. The GA-modified PEG hydrogels promoted the chondrogenesis of hBMSCs, particularly in the concentration of 5mM and 10mM. The subcutaneous transplantation of 10mM GA-modified hydrogels with hBMSCs formed cartilage-like blocks in vivo for 8weeks. Importantly, with glucosamine increase, the modified hydrogels down-regulated the fibrosis and hypertrophic cartilage markers in protein level. Therefore, glucosamine modified PEG hydrogels facilitated the chondrogenesis of hBMSCs, which might represent a new method for cartilage repair using a tissue-engineering approach.
Assuntos
Diferenciação Celular , Condrogênese , Glucosamina , Humanos , Hidrogéis , Células-Tronco Mesenquimais , PolietilenoglicóisRESUMO
BACKGROUND: A number of studies have shown that tooth-like structures can be regenerated using induced pluripotent stem cells and mouse embryonic stem (mES) cells. However, few studies have reported the regeneration of tooth-periodontium complex structures, which are more suitable for clinical tooth transplantation. We established an optimized approach to induce high-odontogenic potential dental epithelium derived from mES cells by temporally controlling bone morphogenic protein 4 (BMP4) function and regenerated tooth-periodontium complex structures in vivo. METHODS: First, immunofluorescence and quantitative reverse transcription-polymerase chain reaction were used to identify the watershed of skin and the oral ectoderm. LDN193189 was then used to inhibit the BMP4 receptor around the watershed, followed by the addition of exogenous BMP4 to promote BMP4 function. The generated dental epithelium was confirmed by western blot analysis and immunofluorescence. The generated epithelium was ultimately combined with embryonic day 14.5 mouse mesenchyme and transplanted into the renal capsules of nude mice. After 4 weeks, the tooth-periodontium complex structure was examined by micro-computed tomography (CT) and hematoxylin and eosin (H&E) staining. RESULTS: Our study found that the turning point of oral ectoderm differentiation occurred around day 3 after the embryoid body was transferred to a common culture plate. Ameloblastin-positive dental epithelial cells were detected following the temporal regulation of BMP4. Tooth-periodontium complex structures, which included teeth, a periodontal membrane, and alveolar bone, were formed when this epithelium was combined with mouse dental mesenchyme and transplanted into the renal capsules of nude mice. Micro-CT and H&E staining revealed that the generated tooth-periodontium complex structures shared a similar histological structure with normal mouse teeth. CONCLUSIONS: An optimized induction method was established to promote the differentiation of mES cells into dental epithelium by temporally controlling the function of BMP4. A novel tooth-periodontium complex structure was generated using the epithelium.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Embrionárias Murinas , Odontogênese/genética , Periodonto/citologia , Animais , Diferenciação Celular/genética , Epitélio/crescimento & desenvolvimento , Células-Tronco Pluripotentes Induzidas/classificação , Mesoderma/citologia , Camundongos , Regeneração/genética , Dente/citologiaRESUMO
AIMS: The aim of this study was to improve a method that induce cartilage differentiation of human embryoid stem cells (hESCs) in vitro, and test the effect of in vivo environments on the further maturation of hESCs derived cells. MAIN METHODS: Embryoid bodies (EBs) formed from hESCs, with serum-free KSR-based medium and mesodermal specification related factors, CHIR, and Noggin for first 8days. Then cells were digested and cultured as micropellets in serum-free KSR-based chondrogenic medium that was supplemented with PDGF-BB, TGF ß3, BMP4 in sequence for 24days. The morphology, FACS, histological staining as well as the expression of chondrogenic specific genes were detected in each stage, and further in vivo experiments, cell injections and tissue transplantations, further verified the formation of chondrocytes. KEY FINDINGS: We were able to obtain chondrocyte/cartilage from hESCs using serum-free KSR-based conditioned medium. qPCR analysis showed that expression of the chondroprogenitor genes and the chondrocyte/cartilage matrix genes. Morphology analysis demonstrated we got PG+COL2+COL1-particles. It indicated we obtained hyaline cartilage-like particles. 32-Day differential cells were injected subcutaneous. Staining results showed grafts developed further mature in vivo. But when transplanted in subrenal capsule, their effect was not good as in subcutaneous. Microenvironment might affect the cartilage formation. SIGNIFICANCE: The results of this study provide an absolute serum-free and efficient approach for generation of hESC-derived chondrocytes, and cells will become further maturation in vivo. It provides evidence and technology for the hypothesis that hESCs may be a promising therapy for the treatment of cartilage disease.
Assuntos
Cartilagem/fisiologia , Técnicas de Cultura de Células , Meios de Cultura Livres de Soro/farmacologia , Células-Tronco Embrionárias Humanas/citologia , Regeneração/efeitos dos fármacos , Indutores da Angiogênese/farmacologia , Animais , Becaplermina , Proteína Morfogenética Óssea 4/farmacologia , Cartilagem/citologia , Proteína de Matriz Oligomérica de Cartilagem/genética , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro/química , Citometria de Fluxo , Células-Tronco Embrionárias Humanas/transplante , Humanos , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-sis/farmacologia , Fator de Crescimento Transformador beta3/farmacologiaRESUMO
During embryonic organogenesis, the odontogenic potential resides in dental mesenchyme from the bud stage until birth. Mouse dental mesenchymal cells (mDMCs) isolated from the inductive dental mesenchyme of developing molars are frequently used in the context of tooth development and regeneration. We wondered if and how the odontogenic potential could be retained when mDMCs were cultured in vitro. In the present study, we undertook to test the odontogenic potential of cultured mDMCs and attempted to maintain the potential during culturing. We found that cultured mDMCs could retain the odontogenic potential for 24 h with a ratio of 60% for tooth formation, but mDMCs were incapable of supporting tooth formation after more than 24 h in culture. This loss of odontogenic potential was accompanied by widespread transcriptomic alteration and, specifically, the downregulation of some dental mesenchyme-specific genes, such as Pax9, Msx1, and Pdgfrα. To prolong the odontogenic potential of mDMCs in vitro, we then cultured mDMCs in a serum-free medium with Knockout Serum Replacement (KSR) and growth factors (fibroblastic growth factor 2 and epidermal growth factor). In this new micromilieu, mDMCs could maintain the odontogenic potential for 48 h with tooth formation ratio of 50%. Moreover, mDMCs cultured in KSR-supplemented medium gave rise to tooth-like structures when recombined with non-dental second-arch epithelium. Among the supplements, KSR is essential for the survival and adhesion of mDMCs, and both Egf and Fgf2 induced the expression of certain dental mesenchyme-related genes. Taken together, our results demonstrated that the transcriptomic changes responded to the alteration of odontogenic potential in cultured mDMCs and a new micromilieu partly retained this potential in vitro, providing insight into the long-term maintenance of odontogenic potential in mDMCs.
Assuntos
Dente/crescimento & desenvolvimento , Animais , Proliferação de Células , Células Cultivadas , Meios de Cultura Livres de Soro , Camundongos , Regeneração , Dente/citologia , Dente/fisiologia , TranscriptomaRESUMO
Background. Mouse dental mesenchymal cells (mDMCs) from tooth germs of cap or later stages are frequently used in the context of developmental biology or whole-tooth regeneration due to their odontogenic potential. In vitro-expanded mDMCs serve as an alternative cell source considering the difficulty in obtaining primary mDMCs; however, cultured mDMCs fail to support tooth development as a result of functional failures of specific genes or pathways. The goal of this study was to identify the genes that maintain the odontogenic potential of mDMCs in culture. Methods. We examined the odontogenic potential of freshly isolated versus cultured mDMCs from the lower first molars of embryonic day 14.5 mice. The transcriptome of mDMCs was detected using RNA sequencing and the data were validated by qRT-PCR. Differential expression analysis and pathway analysis were conducted to identify the genes that contribute to the loss of odontogenic potential. Results. Cultured mDMCs failed to develop into well-structured tooth when they were recombined with dental epithelium. Compared with freshly isolated mDMCs, we found that 1,004 genes were upregulated and 948 were downregulated in cultured mDMCs. The differentially expressed genes were clustered in the biological processes and signaling pathways associated with tooth development. Following in vitro culture, genes encoding a wide array of components of MAPK, TGF-ß/BMP, and Wnt pathways were significantly downregulated. Moreover, the activities of Bdnf, Vegfα, Bmp2, and Bmp7 were significantly inhibited in cultured mDMCs. Supplementation of VEGFα, BMP2, and BMP7 restored the expression of a subset of downregulated genes and induced mDMCs to form dentin-like structures in vivo. Conclusions. Vegfα, Bmp2, and Bmp7 play a role in the maintenance of odontogenic potential in mDMCs.
RESUMO
As a key factor for cell pluripotent and self-renewing phenotypes, SOX2 has attracted scientists' attention gradually in recent years. However, its exact effects in dental pulp stem cells (DPSCs) are still unclear. In this study, we mainly investigated whether SOX2 could affect some biological functions of DPSCs. DPSCs were isolated from the dental pulp of human impacted third molar. SOX2 overexpressing DPSCs (DPSCs-SOX2) were established through retroviral infection. The effect of SOX2 on cell proliferation, migration and adhesion ability was evaluated with CCK-8, trans-well system and fibronectin-induced cell attachment experiment respectively. Whole genome expression of DPSCs-SOX2 was analyzed with RNA microarray. Furthermore, a rescue experiment was performed with SOX2-siRNA in DPSC-SOX2 to confirm the effect of SOX2 overexpression in DPSCs. We found that SOX2 overexpression could result in the enhancement of cell proliferation, migration, and adhesion in DPSCs obviously. RNA microarray analysis indicated that some key genes in the signal pathways associated with cell cycle, migration and adhesion were upregulated in different degree, and the results were further confirmed with qPCR and western-blot. Finally, DPSC-SOX2 transfected with SOX2-siRNA showed a decrease of cell proliferation, migration and adhesion ability, which further confirmed the biological effect of SOX2 in human DPSCs. This study indicated that SOX2 could improve the cell proliferation, migration and adhesion ability of DPSCs through regulating gene expression about cell cycle, migration and adhesion, and provided a novel strategy to develop seed cells with strong proliferation, migration and adhesion ability for tissue engineering.