RESUMO
Angiopoietins 1 and 2 (Ang1 and Ang2) regulate angiogenesis through their similar F-domains by activating Tie2 receptors on endothelial cells. Despite the similarity in the underlying receptor-binding interaction, the two angiopoietins have opposite effects: Ang1 induces phosphorylation of AKT, strengthens cell-cell junctions, and enhances endothelial cell survival while Ang2 can antagonize these effects, depending on cellular context. To investigate the molecular basis for the opposing effects, we examined the phenotypes of a series of computationally designed protein scaffolds presenting the Ang1 F-domain in a wide range of valencies and geometries. We find two broad phenotypic classes distinguished by the number of presented F-domains: Scaffolds presenting 3 or 4 F-domains have Ang2-like activity, upregulating pFAK and pERK but not pAKT, while scaffolds presenting 6, 8, 12, 30, or 60 F-domains have Ang1-like activity, upregulating pAKT and inducing migration and vascular stability. The scaffolds with 6 or more F-domains display super-agonist activity, producing stronger phenotypes at lower concentrations than Ang1. Tie2 super-agonist nanoparticles reduced blood extravasation and improved blood-brain barrier integrity four days after a controlled cortical impact injury.
Assuntos
Angiopoietinas , Células Endoteliais , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Transdução de SinaisRESUMO
Pluripotent cells from the early stages of embryonic development have the unlimited capacity to self-renew and undergo differentiation into all of the cell types of the adult organism. These properties are regulated by tightly controlled networks of gene expression, which in turn are governed by the availability of transcription factors and their interaction with the underlying epigenetic landscape. Recent data suggest that, perhaps unexpectedly, some key epigenetic marks, and thereby gene expression, are regulated by the levels of specific metabolites. Hence, cellular metabolism plays a vital role beyond simply the production of energy, and may be involved in the regulation of cell fate. In this Review, we discuss the metabolic changes that occur during the transitions between different pluripotent states both in vitro and in vivo, including during reprogramming to pluripotency and the onset of differentiation, and we discuss the extent to which distinct metabolites might regulate these transitions.
Assuntos
Diferenciação Celular/genética , Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Blastocisto/metabolismo , Carbono/metabolismo , Ciclo do Ácido Cítrico , Células-Tronco Embrionárias/citologia , Epigênese Genética , Humanos , Camundongos , Mitocôndrias/metabolismo , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Zigoto/metabolismoRESUMO
The polycomb repressive complex 2 (PRC2) histone methyltransferase plays a central role in epigenetic regulation in development and in cancer, and hence to interrogate its role in a specific developmental transition, methods are needed for disrupting function of the complex with high temporal and spatial precision. The catalytic and substrate recognition functions of PRC2 are coupled by binding of the N-terminal helix of the Ezh2 methylase to an extended groove on the EED trimethyl lysine binding subunit. Disrupting PRC2 function can in principle be achieved by blocking this single interaction, but there are few approaches for blocking specific protein-protein interactions in living cells and organisms. Here, we describe the computational design of proteins that bind to the EZH2 interaction site on EED with subnanomolar affinity in vitro and form tight and specific complexes with EED in living cells. Induction of the EED binding proteins abolishes H3K27 methylation in human embryonic stem cells (hESCs) and at all but the earliest stage blocks self-renewal, pinpointing the first critical repressive H3K27me3 marks in development.
Assuntos
Simulação por Computador , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Histonas/química , Células-Tronco Embrionárias Humanas/citologia , Humanos , Metilação , Complexo Repressor Polycomb 2/químicaRESUMO
In both mice and humans, pluripotent stem cells (PSCs) exist in at least two distinct states of pluripotency, known as the naïve and primed states. Our understanding of the intrinsic and extrinsic factors that enable PSCs to self-renew and to transition between different pluripotent states is important for understanding early development. In mouse embryonic stem cells (mESCs), Wnt proteins stimulate mESC self-renewal and support the naïve state. In human embryonic stem cells (hESCs), Wnt/ß-catenin signaling is active in naïve-state hESCs and is reduced or absent in primed-state hESCs. However, the role of Wnt/ß-catenin signaling in naïve hESCs remains largely unknown. Here, we demonstrate that inhibition of the secretion of Wnts or inhibition of the stabilization of ß-catenin in naïve hESCs reduces cell proliferation and colony formation. Moreover, we show that addition of recombinant Wnt3a partially rescues cell proliferation in naïve hESCs caused by inhibition of Wnt secretion. Notably, inhibition of Wnt/ß-catenin signaling in naïve hESCs did not cause differentiation. Instead, it induced primed hESC-like proteomic and metabolic profiles. Thus, our results suggest that naïve hESCs secrete Wnts that activate autocrine or paracrine Wnt/ß-catenin signaling to promote efficient self-renewal and inhibit the transition to the primed state.
Assuntos
Diferenciação Celular , Autorrenovação Celular , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Via de Sinalização Wnt , Apoptose , Benzotiazóis/farmacologia , Biomarcadores , Ciclo Celular/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Compostos Heterocíclicos com 3 Anéis/farmacologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Modelos Biológicos , Proteômica/métodos , RNA Interferente Pequeno/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
microRNAs are ~22bp nucleotide non-coding RNAs that play important roles in the post-transcriptional regulation of gene expression. Many studies have established that microRNAs are important for cell fate choices, including the naïve to primed pluripotency state transitions, and their intermediate state, the developmentally suspended diapause state in early development. However, the full extent of microRNAs associated with these stage transitions in human and mouse remain under-explored. By meta-analysis of microRNA-seq, RNA-seq, and metabolomics datasets from human and mouse, we found a set of microRNAs, and importantly, their experimentally validated target genes that show consistent changes in naïve to primed transitions (microRNA up, target genes down, or vice versa). The targets of these microRNAs regulate developmental pathways (e.g., the Hedgehog-pathway), primary cilium, and remodeling of metabolic processes (oxidative phosphorylation, fatty acid metabolism, and amino acid transport) during the transition. Importantly, we identified 115 microRNAs that significantly change in the same direction in naïve to primed transitions in both human and mouse, many of which are novel candidate regulators of pluripotency. Furthermore, we identified 38 microRNAs and 274 target genes that may be involved in diapause, where embryonic development is temporarily suspended prior to implantation to uterus. The upregulated target genes suggest that microRNAs activate stress response in the diapause stage. In conclusion, we provide a comprehensive resource of microRNAs and their target genes involved in naïve to primed transition and in the paused intermediate, the embryonic diapause stage.
Assuntos
Bases de Dados Genéticas , Células-Tronco Embrionárias Humanas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Células-Tronco Embrionárias Humanas/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , MicroRNAs/biossíntese , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/citologiaRESUMO
Aberrations in metabolism contribute to a large number of diseases, such as diabetes, obesity, cancer, and cardiovascular diseases, that have a substantial impact on the mortality rates and quality of life worldwide. However, the mechanisms leading to these changes in metabolic state--and whether they are conserved between diseases--is not well understood. Changes in metabolism similar to those seen in pathological conditions are observed during normal development in a number of different cell types. This provides hope that understanding the mechanism of these metabolic switches in normal development may provide useful insight in correcting them in pathological cases. Here, we focus on the metabolic remodeling observed both in early stage embryonic stem cells and during the maturation of cardiomyocytes.
Assuntos
Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Animais , Diferenciação Celular/fisiologia , HumanosRESUMO
Adult stem cells reside in hypoxic niches, and embryonic stem cells (ESCs) are derived from a low oxygen environment. However, it is not clear whether hypoxia is critical for stem cell fate since for example human ESCs (hESCs) are able to self-renew in atmospheric oxygen concentrations as well. We now show that hypoxia can govern cell fate decisions since hypoxia alone can revert hESC- or iPSC-derived differentiated cells back to a stem cell-like state, as evidenced by re-activation of an Oct4-promoter reporter. Hypoxia-induced "de-differentiated" cells also mimic hESCs in their morphology, long-term self-renewal capacity, genome-wide mRNA and miRNA profiles, Oct4 promoter methylation state, cell surface markers TRA1-60 and SSEA4 expression, and capacity to form teratomas. These data demonstrate that hypoxia can influence cell fate decisions and could elucidate hypoxic niche function.
Assuntos
Linhagem da Célula , Células-Tronco Pluripotentes/citologia , Adulto , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Desdiferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Histona Desacetilases/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Oxigênio/farmacologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismoRESUMO
Recent advances in patient-derived induced Pluripotent Stem Cell (iPSC) generation, improvement of cardiomyocyte-directed differentiation protocols, and the availability of new genome editing techniques have opened up new avenues for disease modeling of cardiomyopathies. Patients with cardiomyopathies often harbor a single-base substitution believed to be linked to the disease phenotype. Somatic cells derived from patients can be efficiently reprogrammed into iPSCs and subsequently engineered. The targeting of a precise mutation can be achieved by the introduction of double stranded breaks with CRISPR-Cas9 and by homology-directed repair when using a DNA donor template. This allows for the correction of a mutation in a patient iPSC line to generate an isogenic control. In addition, key mutations associated with cardiomyopathies can be introduced in an iPSC line derived from a healthy individual using the same techniques. In this chapter, we describe in detail how to engineer pluripotent stem cells to model cardiomyopathy in a dish using CRISPR-Cas9 technology.
Assuntos
Cardiomiopatias , Células-Tronco Pluripotentes Induzidas , Humanos , Miócitos Cardíacos , Mutação , Cardiomiopatias/genética , Genômica , Sistemas CRISPR-CasRESUMO
Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for investigating tooth development and regeneration. Here, we present a protocol for generating three-dimensional induced early ameloblasts (ieAMs) utilizing serum-free media and growth factors. We describe steps for directing hiPSCs toward oral epithelium and then toward ameloblast fate. These cells can form suspended early ameloblast organoids. This approach is critical for understanding, treating, and promoting regeneration in diseases like amelogenesis imperfecta. For complete details on the use and execution of this protocol, please refer to Alghadeer et al.1.
Assuntos
Ameloblastos , Técnicas de Cultura de Células , Células-Tronco Pluripotentes Induzidas , Ameloblastos/citologia , Ameloblastos/metabolismo , Humanos , Meios de Cultura Livres de Soro , Células-Tronco Pluripotentes Induzidas/citologia , Técnicas de Cultura de Células/métodos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Diferenciação Celular/fisiologia , Células CultivadasRESUMO
Tissue repair is significantly compromised in the aging human body resulting in critical disease conditions (such as myocardial infarction or Alzheimer's disease) and imposing a tremendous burden on global health. Reprogramming approaches (partial or direct reprogramming) are considered fruitful in addressing this unmet medical need. However, the efficacy, cellular maturity and specific targeting are still major challenges of direct reprogramming. Here we describe novel approaches in direct reprogramming that address these challenges. Extracellular signaling pathways (Receptor tyrosine kinases, RTK and Receptor Serine/Theronine Kinase, RSTK) and epigenetic marks remain central in rewiring the cellular program to determine the cell fate. We propose that modern protein design technologies (AI-designed minibinders regulating RTKs/RSTK, epigenetic enzymes, or pioneer factors) have potential to solve the aforementioned challenges. An efficient transdifferentiation/direct reprogramming may in the future provide molecular strategies to collectively reduce aging, fibrosis, and degenerative diseases.
RESUMO
In Finland the frequency of isolated cleft palate (CP) is higher than that of isolated cleft lip with or without cleft palate (CL/P). This trend contrasts to that in other European countries but its genetic underpinnings are unknown. We performed a genome-wide association study for orofacial clefts, which include CL/P and CP, in the Finnish population. We identified rs570516915, a single nucleotide polymorphism that is highly enriched in Finns and Estonians, as being strongly associated with CP ( P = 5.25 × 10 -34 , OR = 8.65, 95% CI 6.11-12.25), but not with CL/P ( P = 7.2 × 10 -5 ), with genome-wide significance. The risk allele frequency of rs570516915 parallels the regional variation of CP prevalence in Finland, and the association was replicated in independent cohorts of CP cases from Finland ( P = 8.82 × 10 -28 ) and Estonia ( P = 1.25 × 10 -5 ). The risk allele of rs570516915 disrupts a conserved binding site for the transcription factor IRF6 within a previously characterized enhancer upstream of the IRF6 gene. Through reporter assay experiments we found that the risk allele of rs570516915 diminishes the enhancer activity. Oral epithelial cells derived from CRISPR-Cas9 edited induced pluripotent stem cells demonstrate that the CP-associated allele of rs570516915 concomitantly decreases the binding of IRF6 and the expression level of IRF6 , suggesting impaired IRF6 autoregulation as a molecular mechanism underlying the risk for CP.
RESUMO
miRNAs are small non-coding RNAs that have emerged as crucial post-transcriptional regulators of gene expression. They are key players in various critical cellular processes such as proliferation, cell cycle progression, apoptosis and differentiation. Self-renewal capacity and differentiation potential are hallmarks of stem cells. The switch between self-renewal and differentiation requires rapid widespread changes in gene expression. Since miRNAs can repress the translation of many mRNA targets, they are good candidates to regulate cell fates. In the past few years, miRNAs have appeared as important new actors in stem cell development by regulating differentiation and maintenance of stem cells. In this chapter we will focus on the role of miRNAs in various stem cell populations. After an introduction on microRNA biogenesis, we will review the recent knowledge on miRNA expression and function in pluripotent cells and during the acquisition of stem cell fate. We will then briefly examine the role of miRNAs in adult and cancer stem cells.
Assuntos
Células-Tronco Adultas/metabolismo , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Células-Tronco Adultas/citologia , Animais , Ciclo Celular , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/citologia , Humanos , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Pluripotentes/citologia , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The cavovarus foot is a complex deformity that can be treated using multiple surgical procedures, ranging from soft tissue surgery to triple arthrodesis. Among these options, anterior midfoot tarsectomy is a three-dimensional closed-wedge osteotomy, traditionally performed slowly and progressively in a blind fashion, and remaining a challenge for unexperimented surgeons with variable outcomes. As such, we investigated and discussed the use of patient-specific cutting guides (PSCGs) in computer-assisted anterior midfoot tarsectomy in terms of accuracy, reproducibility, and safety.
Assuntos
Artrodese , Pé , Humanos , Reprodutibilidade dos Testes , Artrodese/métodos , Osteotomia/métodosRESUMO
Just like time and tide, embryonic development waits for no man but progresses forcefully to its completion, with just one exception. Diapause is an enigmatic, reversible, dormant halt that protects the developing embryo. Cancer cells have evolved to hijack many useful stem cell capabilities, and diapause is no exception. Recent work has revealed a diapause-like cancer cell state, prompting the quest for its key molecular regulators useful for cancer therapies. The present paper by Sun et al.1 addresses this knowledge gap by revealing a key player in regulating the diapause-like cancer cell stage, the condensin protein SMC4.
Assuntos
Embrião de Mamíferos , Desenvolvimento Embrionário , Feminino , Gravidez , Humanos , Células-TroncoRESUMO
Using embryonic stem cells (ESCs) in regenerative medicine or in disease modeling requires a complete understanding of these cells. Two main distinct developmental states of ESCs have been stabilized in vitro, a naïve pre-implantation stage and a primed post-implantation stage. Based on two recently published CRISPR-Cas9 knockout functional screens, we show here that the exit of the naïve state is impaired upon heme biosynthesis pathway blockade, linked in mESCs to the incapacity to activate MAPK- and TGFß-dependent signaling pathways after succinate accumulation. In addition, heme synthesis inhibition promotes the acquisition of 2 cell-like cells in a heme-independent manner caused by a mitochondrial succinate accumulation and leakage out of the cell. We further demonstrate that extracellular succinate acts as a paracrine/autocrine signal, able to trigger the 2C-like reprogramming through the activation of its plasma membrane receptor, SUCNR1. Overall, this study unveils a new mechanism underlying the maintenance of pluripotency under the control of heme synthesis.
Assuntos
Células-Tronco Embrionárias , Ácido Succínico , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias Murinas , Ácido Succínico/metabolismo , Animais , CamundongosRESUMO
Embryonic development is a continuum in vivo. Transcriptional analysis can separate established human embryonic stem cells (hESC) into at least four distinct developmental pluripotent stages, two naïve and two primed, early and late relative to the intact epiblast. In this study we primarily show that exposure of frozen human blastocysts to an inhibitor of checkpoint kinase 1 (CHK1) upon thaw greatly enhances establishment of karyotypically normal late naïve hESC cultures. These late naïve cells are plastic and can be toggled back to early naïve and forward to early primed pluripotent stages. The early primed cells are transcriptionally equivalent to the post inner cell mass intermediate (PICMI) stage seen one day following transfer of human blastocysts into in vitro culture and are stable at an earlier stage than conventional primed hESC.
Assuntos
Técnicas de Cultura de Células , Quinase 1 do Ponto de Checagem , Células-Tronco Embrionárias Humanas , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Blastocisto/citologia , Células-Tronco Pluripotentes/citologiaRESUMO
Following acute genotoxic stress, both normal and tumorous stem cells can undergo cell-cycle arrest to avoid apoptosis and later re-enter the cell cycle to regenerate daughter cells. However, the mechanism of protective, reversible proliferative arrest, "quiescence," remains unresolved. Here, we show that mitophagy is a prerequisite for reversible quiescence in both irradiated Drosophila germline stem cells (GSCs) and human induced pluripotent stem cells (hiPSCs). In GSCs, mitofission (Drp1) or mitophagy (Pink1/Parkin) genes are essential to enter quiescence, whereas mitochondrial biogenesis (PGC1α) or fusion (Mfn2) genes are crucial for exiting quiescence. Furthermore, mitophagy-dependent quiescence lies downstream of mTOR- and PRC2-mediated repression and relies on the mitochondrial pool of cyclin E. Mitophagy-dependent reduction of cyclin E in GSCs and in hiPSCs during mTOR inhibition prevents the usual G1/S transition, pushing the cells toward reversible quiescence (G0). This alternative method of G1/S control may present new opportunities for therapeutic purposes.
Assuntos
Proteínas de Drosophila , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Mitofagia/genética , Ciclina E/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Drosophila/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Serina-Treonina Quinases TOR , Células Germinativas/metabolismo , Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinases , Proteínas de Drosophila/genéticaRESUMO
Transplanted human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improve ventricular performance when delivered acutely post-myocardial infarction but are ineffective in chronic myocardial infarction/heart failure. 2'-deoxy-ATP (dATP) activates cardiac myosin and potently increases contractility. Here we engineered hPSC-CMs to overexpress ribonucleotide reductase, the enzyme controlling dATP production. In vivo, dATP-producing CMs formed new myocardium that transferred dATP to host cardiomyocytes via gap junctions, increasing their dATP levels. Strikingly, when transplanted into chronically infarcted hearts, dATP-producing grafts increased left ventricular function, whereas heart failure worsened with wild-type grafts or vehicle injections. dATP-donor cells recipients had greater voluntary exercise, improved cardiac metabolism, reduced pulmonary congestion and pathological cardiac hypertrophy, and improved survival. This combination of remuscularization plus enhanced host contractility offers a novel approach to treating the chronically failing heart.
RESUMO
Over 90% of the U.S. adult population suffers from tooth structure loss due to caries. Most of the mineralized tooth structure is composed of dentin, a material produced and mineralized by ectomesenchyme derived cells known as odontoblasts. Clinicians, scientists, and the general public share the desire to regenerate this missing tooth structure. To bioengineer missing dentin, increased understanding of human tooth development is required. Here we interrogate at the single cell level the signaling interactions that guide human odontoblast and ameloblast development and which determine incisor or molar tooth germ type identity. During human odontoblast development, computational analysis predicts that early FGF and BMP activation followed by later HH signaling is crucial. Application of this sci-RNA-seq analysis generates a differentiation protocol to produce mature hiPSC derived odontoblasts in vitro (iOB). Further, we elucidate the critical role of FGF signaling in odontoblast maturation and its biomineralization capacity using the de novo designed FGFR1/2c isoform specific minibinder scaffolded as a C6 oligomer that acts as a pathway agonist. We find that FGFR1c is upregulated in functional odontoblasts and specifically plays a crucial role in driving odontoblast maturity. Using computational tools, we show on a molecular level how human molar development is delayed compared to incisors. We reveal that enamel knot development is guided by FGF and WNT in incisors and BMP and ROBO in the molars, and that incisor and molar ameloblast development is guided by FGF, EGF and BMP signaling, with tooth type specific intensity of signaling interactions. Dental ectomesenchyme derived cells are the primary source of signaling ligands responsible for both enamel knot and ameloblast development.
RESUMO
Tooth enamel secreted by ameloblasts (AMs) is the hardest material in the human body, acting as a shield to protect the teeth. However, the enamel is gradually damaged or partially lost in over 90% of adults and cannot be regenerated due to a lack of ameloblasts in erupted teeth. Here, we use single-cell combinatorial indexing RNA sequencing (sci-RNA-seq) to establish a spatiotemporal single-cell census for the developing human tooth and identify regulatory mechanisms controlling the differentiation process of human ameloblasts. We identify key signaling pathways involved between the support cells and ameloblasts during fetal development and recapitulate those findings in human ameloblast in vitro differentiation from induced pluripotent stem cells (iPSCs). We furthermore develop a disease model of amelogenesis imperfecta in a three-dimensional (3D) organoid system and show AM maturation to mineralized structure in vivo. These studies pave the way for future regenerative dentistry.