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1.
Nat Biomed Eng ; 5(8): 926-940, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373601

RESUMO

Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.


Assuntos
Proteínas de Homeodomínio/genética , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Condrócitos/citologia , Condrócitos/metabolismo , Condrócitos/transplante , Condrogênese , Doenças do Colágeno/terapia , Meios de Cultura/química , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Pluripotentes/metabolismo , Polímeros/química , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Antígenos Thy-1/metabolismo , Engenharia Tecidual
2.
Sci Rep ; 11(1): 15811, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34349154

RESUMO

Although multiple studies have investigated the mesenchymal stem and progenitor cells (MSCs) that give rise to mature bone marrow, high heterogeneity in their morphologies and properties causes difficulties in molecular separation of their distinct populations. In this study, by taking advantage of the resolution of the single cell transcriptome, we analyzed Sca-1 and PDGFR-α fraction in the mouse bone marrow tissue. The single cell transcriptome enabled us to further classify the population into seven populations according to their gene expression profiles. We then separately obtained the seven populations based on candidate marker genes, and specified their gene expression properties and epigenetic landscape by ATAC-seq. Our findings will enable to elucidate the stem cell niche signal in the bone marrow microenvironment, reconstitute bone marrow in vitro, and shed light on the potentially important role of identified subpopulation in various clinical applications to the treatment of bone- and bone marrow-related diseases.

3.
Int J Mol Sci ; 22(3)2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573345

RESUMO

Skeletal disorders, such as osteoarthritis and bone fractures, are among the major conditions that can compromise the quality of daily life of elderly individuals. To treat them, regenerative therapies using skeletal cells have been an attractive choice for patients with unmet clinical needs. Currently, there are two major strategies to prepare the cell sources. The first is to use induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), which can recapitulate the skeletal developmental process and differentiate into various skeletal cells. Skeletal tissues are derived from three distinct origins: the neural crest, paraxial mesoderm, and lateral plate mesoderm. Thus, various protocols have been proposed to recapitulate the sequential process of skeletal development. The second strategy is to extract stem cells from skeletal tissues. In addition to mesenchymal stem/stromal cells (MSCs), multiple cell types have been identified as alternative cell sources. These cells have distinct multipotent properties allowing them to differentiate into skeletal cells and various potential applications for skeletal regeneration. In this review, we summarize state-of-the-art research in stem cell differentiation based on the understanding of embryogenic skeletal development and stem cells existing in skeletal tissues. We then discuss the potential applications of these cell types for regenerative medicine.


Assuntos
Desenvolvimento Ósseo/fisiologia , Osso e Ossos/fisiologia , Fraturas Ósseas/terapia , Osteoartrite/terapia , Medicina Regenerativa/métodos , Animais , Osso e Ossos/embriologia , Osso e Ossos/lesões , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário/fisiologia , Células-Tronco Embrionárias/fisiologia , Fraturas Ósseas/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Mesenquimais/fisiologia , Mesoderma/embriologia , Crista Neural/embriologia , Osteoartrite/fisiopatologia , Osteoblastos/fisiologia , Osteoblastos/transplante , Medicina Regenerativa/tendências , Transplante de Células-Tronco/métodos , Transplante de Células-Tronco/tendências
4.
Methods Mol Biol ; 2245: 167-178, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33315202

RESUMO

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a powerful tool to identify binding profiles of transcriptional regulators and chromatin regulators as well as histone modification patterns in a genome-wide manner. ChIP-seq consists of five major steps: (1) preparation of cells and chromatin, (2) ChIP, (3) ChIP-seq library construction, (4) sequencing of ChIP DNA with a next-generation sequencer (NGS), and (5) computational analysis of sequence data. Recent ChIP-seq studies in skeletal tissues enable us to understand the modes of action of key skeletal regulators, functional interaction among the enhancers bound by the regulators, the complex nature of regulatory inputs, and thereby the gene regulatory landscape in skeletal development. Here we describe a ChIP-seq protocol that we have employed in our studies, with particular focus on chromatin preparation and subsequent ChIP in skeletal cells, including chondrocytes.


Assuntos
Cartilagem/metabolismo , Condrócitos/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Animais , Animais Recém-Nascidos , Separação Celular/métodos , Cromatina/genética , Análise de Dados , Biblioteca Gênica , Humanos , Separação Imunomagnética , Mamíferos
5.
Exp Mol Med ; 52(8): 1166-1177, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32788657

RESUMO

Pluripotent stem cells (PSCs) are attractive regenerative therapy tools for skeletal tissues. However, a deep understanding of skeletal development is required in order to model this development with PSCs, and for the application of PSCs in clinical settings. Skeletal tissues originate from three types of cell populations: the paraxial mesoderm, lateral plate mesoderm, and neural crest. The paraxial mesoderm gives rise to the sclerotome mainly through somitogenesis. In this process, key developmental processes, including initiation of the segmentation clock, formation of the determination front, and the mesenchymal-epithelial transition, are sequentially coordinated. The sclerotome further forms vertebral columns and contributes to various other tissues, such as tendons, vessels (including the dorsal aorta), and even meninges. To understand the molecular mechanisms underlying these developmental processes, extensive studies have been conducted. These studies have demonstrated that a gradient of activities involving multiple signaling pathways specify the embryonic axis and induce cell-type-specific master transcription factors in a spatiotemporal manner. Moreover, applying the knowledge of mesoderm development, researchers have attempted to recapitulate the in vivo development processes in in vitro settings, using mouse and human PSCs. In this review, we summarize the state-of-the-art understanding of mesoderm development and in vitro modeling of mesoderm development using PSCs. We also discuss future perspectives on the use of PSCs to generate skeletal tissues for basic research and clinical applications.

6.
Bone ; 137: 115458, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32474244

RESUMO

The development of osteoblasts, a bone-forming cell population, occurs in conjunction with development of the skeleton, which creates our physical framework and shapes the body. In the past two decades, genetic studies have uncovered the molecular framework of this process-namely, transcriptional regulators and signaling pathways coordinate the cell fate determination and differentiation of osteoblasts in a spatial and temporal manner. Recently emerging genome-wide studies provide additional layers of understanding of the gene regulatory landscape during osteoblast differentiation, allowing us to gain novel insight into the modes of action of the key regulators, functional interaction among the regulator-bound enhancers, epigenetic regulations, and the complex nature of regulatory inputs. In this review, we summarize current understanding of the transcriptional regulation in osteoblasts, in terms of the gene regulatory landscape.


Assuntos
Regulação da Expressão Gênica , Osteogênese , Diferenciação Celular/genética , Osteoblastos , Transdução de Sinais
7.
Stem Cell Reports ; 15(1): 125-139, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32531191

RESUMO

Two genetic diseases, Gorlin syndrome and McCune-Albright syndrome (MAS), show completely opposite symptoms in terms of bone mineral density and hedgehog (Hh) activity. In this study, we utilized human induced pluripotent stem cell (iPSC)-based models of the two diseases to understand the roles of Hh signaling in osteogenesis. Gorlin syndrome-derived iPSCs showed increased osteoblastogenesis and mineralization with Hh signaling activation and upregulation of a set of transcription factors in an osteogenic culture, compared with the isogenic control. MAS-specific iPSCs showed poor mineralization with low Hh signaling activity in the osteogenic culture; impaired osteoblastogenesis was restored to the normal level by treatment with an Hh signaling-activating small molecule. These data suggest that Hh signaling is a key controller for differentiation of osteoblasts from precursors. This study may pave a path to new drug therapies for genetic abnormalities in calcification caused by dysregulation of Hh signaling.


Assuntos
Proteínas Hedgehog/metabolismo , Osteoblastos/metabolismo , Osteogênese , Síndrome do Nevo Basocelular/patologia , Técnicas de Cultura de Células , Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Perfilação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcriptoma/genética
8.
Int J Mol Sci ; 21(7)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244499

RESUMO

Antxr1/Tem8 is highly expressed in tumor endothelial cells and is a receptor for anthrax toxin. Mutation of Antxr1 causes GAPO syndrome, which is characterized by growth retardation, alopecia, pseudo-anodontia, and optic atrophy. However, the mechanism underlying the growth retardation remains to be clarified. Runx2 is essential for osteoblast differentiation and chondrocyte maturation and regulates chondrocyte proliferation through Ihh induction. In the search of Runx2 target genes in chondrocytes, we found that Antxr1 expression is upregulated by Runx2. Antxr1 was highly expressed in cartilaginous tissues and was directly regulated by Runx2. In skeletal development, the process of endochondral ossification proceeded similarly in wild-type and Antxr1-/- mice. However, the limbs of Antxr1-/- mice were shorter than those of wild-type mice from embryonic day 16.5 due to the reduced chondrocyte proliferation. Chondrocyte-specific Antxr1 transgenic mice exhibited shortened limbs, although the process of endochondral ossification proceeded as in wild-type mice. BrdU-uptake and apoptosis were both increased in chondrocytes, and the apoptosis-high regions were mineralized. These findings indicated that Antxr1, of which the expression is regulated by Runx2, plays an important role in chondrocyte proliferation and that overexpression of Antxr1 causes chondrocyte apoptosis accompanied by matrix mineralization.


Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Cartilagem , Diferenciação Celular/fisiologia , Condrócitos/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Células Endoteliais , Feminino , Fêmur/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Mutação , Osteogênese/fisiologia , Receptores de Superfície Celular/genética , Esqueleto/embriologia , Esqueleto/patologia , Tíbia/patologia , Transcriptoma , Regulação para Cima
9.
Regen Ther ; 14: 19-31, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31988991

RESUMO

Clinically relevant human induced pluripotent stem cell (hiPSC) derivatives require efficient protocols to differentiate hiPSCs into specific lineages. Here we developed a fully defined xeno-free strategy to direct hiPSCs toward osteoblasts within 21 days. The strategy successfully achieved the osteogenic induction of four independently derived hiPSC lines by a sequential use of combinations of small-molecule inducers. The induction first generated mesodermal cells, which subsequently recapitulated the developmental expression pattern of major osteoblast genes and proteins. Importantly, Col2.3-Cherry hiPSCs subjected to this strategy strongly expressed the cherry fluorescence that has been observed in bone-forming osteoblasts in vivo. Moreover, the protocol combined with a three-dimensional (3D) scaffold was suitable for the generation of a xeno-free 3D osteogenic system. Thus, our strategy offers a platform with significant advantages for bone biology studies and it will also contribute to clinical applications of hiPSCs to skeletal regenerative medicine.

10.
Int J Mol Sci ; 20(24)2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31847446

RESUMO

Chondrogenesis is a key developmental process that molds the framework of our body and generates the skeletal tissues by coupling with osteogenesis. The developmental processes are well-coordinated by spatiotemporal gene expressions, which are hardwired with gene regulatory elements. Those elements exist as thousands of modules of DNA sequences on the genome. Transcription factors function as key regulatory proteins by binding to regulatory elements and recruiting cofactors. Over the past 30 years, extensive attempts have been made to identify gene regulatory mechanisms in chondrogenesis, mainly through biochemical approaches and genetics. More recently, newly developed next-generation sequencers (NGS) have identified thousands of gene regulatory elements on a genome scale, and provided novel insights into the multiple layers of gene regulatory mechanisms, including the modes of actions of transcription factors, post-translational histone modifications, chromatin accessibility, the concept of pioneer factors, and three-dimensional chromatin architecture. In this review, we summarize the studies that have improved our understanding of the gene regulatory mechanisms in chondrogenesis, from the historical studies to the more recent works using NGS. Finally, we consider the future perspectives, including efforts to improve our understanding of the gene regulatory landscape in chondrogenesis and potential applications to the treatment of chondrocyte-related diseases.


Assuntos
Condrócitos/fisiologia , Redes Reguladoras de Genes/genética , Animais , Condrogênese/genética , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Fatores de Transcrição/genética
11.
Arthritis Res Ther ; 21(1): 247, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31771658

RESUMO

BACKGROUND: Both loss- and gain-of-function of Wnt/ß-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/ß-catenin signaling in the superficial zone (SFZ) of articular cartilage. METHODS: Wnt/ß-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice for loss- and gain-of-function, respectively, of Wnt/ß-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/ß-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/ß-catenin signaling in SFZ cells. RESULTS: Wnt/ß-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific ß-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific ß-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by ß-catenin knockout, while it was upregulated by ß-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/ß-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4. CONCLUSIONS: We demonstrated that Wnt/ß-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.


Assuntos
Cartilagem Articular/metabolismo , Homeostase/genética , Osteoartrite/genética , Proteoglicanas/genética , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/metabolismo , Regulação da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteoglicanas/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo
12.
Stem Cell Reports ; 13(3): 530-544, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31402337

RESUMO

A simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5-9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and ß-catenin association with DNA showed that retinoic acid and Wnt/ß-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation.


Assuntos
Benzoatos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Condrócitos/metabolismo , Células-Tronco Pluripotentes/citologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Retinoides/farmacologia , Animais , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/citologia , Condrócitos/transplante , Condrogênese , Cromatina/metabolismo , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Células-Tronco Pluripotentes/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismo , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
13.
Regen Ther ; 9: 10-14, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30525070

RESUMO

The 17th Congress of the Japanese Society for Regenerative Medicine was held on March 21-23, 2018 at PACIFICO Yokohama (Kanagawa Prefecture) with 3860 participants. The theme of the congress was 'The Integration of Wisdom from All Sectors.' With this theme, this congress aimed to provide people from all sectors (including individuals from various industries, regulatory authorities, academia, and citizens) with opportunities for exchanging views on regenerative medicine under one roof. A broad spectrum of topics related to regenerative medicine was covered by one presidential lecture, one keynote lecture, one collaborative lecture by the Congress Chair and the Governor of Kanagawa Prefecture, three special lectures (six topics), four award lectures, 43 symposia (235 talks), 337 oral presentations (59 sessions), 358 poster presentations (43 sessions), 25 co-organized seminars (35 talks), two sessions for junior high school and high school students (basic and advanced), and the state-of-the-art technology showcase (158 organizations).

14.
Nat Commun ; 9(1): 4216, 2018 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-30310071

RESUMO

Organoids generated from pluripotent stem cells are used in the development of organ replacement regenerative therapy by recapitulating the process of organogenesis. These processes are strictly regulated by morphogen signalling and transcriptional networks. However, the precise transcription factors involved in the organogenesis of exocrine glands, including salivary glands, remain unknown. Here, we identify a specific combination of two transcription factors (Sox9 and Foxc1) responsible for the differentiation of mouse embryonic stem cell-derived oral ectoderm into the salivary gland rudiment in an organoid culture system. Following orthotopic transplantation into mice whose salivary glands had been removed, the induced salivary gland rudiment not only showed a similar morphology and gene expression profile to those of the embryonic salivary gland rudiment of normal mice but also exhibited characteristics of mature salivary glands, including saliva secretion. This study suggests that exocrine glands can be induced from pluripotent stem cells for organ replacement regenerative therapy.


Assuntos
Células-Tronco Embrionárias Murinas/citologia , Glândulas Salivares/crescimento & desenvolvimento , Animais , Células Cultivadas , Ectoderma/metabolismo , Feminino , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Mucosa Bucal/embriologia , Mucosa Bucal/metabolismo , Glândulas Salivares/citologia , Glândulas Salivares/transplante , Glândulas Salivares/ultraestrutura , Fatores de Transcrição/metabolismo
15.
Stem Cell Res Ther ; 9(1): 24, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29391049

RESUMO

BACKGROUND: Human dental pulp stem cells (DPSCs), which have the ability to differentiate into multiple lineages, were recently identified. DPSCs can be collected readily from extracted teeth and are now considered to be a type of mesenchymal stem cell with higher clonogenic and proliferative potential than bone marrow stem cells (BMSCs). Meanwhile, the treatment of severe bone defects, such as fractures, cancers, and congenital abnormalities, remains a great challenge, and novel bone regenerative techniques are highly anticipated. Several studies have previously shown that 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH), a helioxanthin derivative, induces osteogenic differentiation of preosteoblastic and mesenchymal cells. However, the osteogenic differentiation activities of TH have only been confirmed in some mouse cell lines. Therefore, in this study, toward the clinical use of TH in humans, we analyzed the effect of TH on the osteogenic differentiation of DPSCs, and the in-vivo osteogenesis ability of TH-induced DPSCs, taking advantage of the simple transplantation system using cell-sheet technology. METHODS: DPSCs were obtained from dental pulp of the wisdom teeth of five healthy patients (18-22 years old) and cultured in regular medium and osteogenic medium with or without TH. To evaluate osteogenesis of TH-induced DPSCs in vivo, we transplanted DPSC sheets into mouse calvaria defects. RESULTS: We demonstrated that osteogenic conditions with TH induce the osteogenic differentiation of DPSCs more efficiently than those without TH and those with bone morphogenetic protein-2. However, regular medium with TH did not induce the osteogenic differentiation of DPSCs. TH induced osteogenesis in both DPSCs and BMSCs, although the gene expression pattern in DPSCs differed from that in BMSCs up to 14 days after induction with TH. Furthermore, we succeeded in bone regeneration in vivo using DPSC sheets with TH treatment, without using any scaffolds or growth factors. CONCLUSIONS: Our results demonstrate that TH-induced DPSCs are a useful cell source for bone regenerative medicine, and the transplantation of DPSC sheets treated with TH is a convenient scaffold-free method of bone healing.


Assuntos
Regeneração Óssea , Células Imobilizadas , Polpa Dentária , Lignanas/química , Transplante de Células-Tronco , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Células Imobilizadas/metabolismo , Células Imobilizadas/transplante , Polpa Dentária/metabolismo , Polpa Dentária/transplante , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Osteogênese
16.
Sci Adv ; 3(5): e1602875, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28508073

RESUMO

The development of in vitro models for the maintenance and differentiation of pluripotent stem cells (PSCs) is an active area of stem cell research. The strategies used so far are based mainly on two-dimensional (2D) cultures, in which cellular phenotypes are regulated by soluble factors. We show that a 3D culture system with atelocollagen porous scaffolds can significantly improve the outcome of the current platforms intended for the maintenance and lineage specification of mouse PSCs (mPSCs). Unlike 2D conditions, the 3D conditions maintained the undifferentiated state of mouse embryonic stem cells (mESCs) without exogenous stimulation and also supported endoderm, mesoderm, and ectoderm differentiation of mESCs under serum-free conditions. Moreover, 3D mPSC-derived mesodermal cells showed accelerated osteogenic differentiation, giving rise to functional osteoblast-osteocyte populations within calcified structures. The present strategy offers a 3D platform suitable for the formation of organoids that mimic in vivo organs containing various cell types, and it may be adaptable to the generation of ectoderm-, mesoderm-, and endoderm-derived tissues when combined with appropriate differentiation treatments.


Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular , Osteócitos/metabolismo , Osteogênese , Células-Tronco Pluripotentes/metabolismo , Tecidos Suporte/química , Animais , Camundongos , Osteócitos/citologia , Células-Tronco Pluripotentes/citologia
17.
J Artif Organs ; 20(1): 91-98, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-27485094

RESUMO

Repair of large segmental defects of long bones are a tremendous challenge that calls for a novel approach to supporting immediate weight bearing and bone regeneration. This study investigated the functional and biological characteristics of a combination of a tailor-made titanium mesh cage with a plate (tTMCP) with tetrapod-shaped alpha tricalcium phosphate granules (TB) and basic fibroblast growth factor (bFGF)-binding ion complex gel (f-IC gel) to repair 20-mm segmental radial defects in dogs. The defects were created surgically in 18 adult beagle dogs and treated by implantation of tTMCPs with TB with (TB-gel group) or without (TB group) f-IC gel. Each tTMCP fitted the defect well, and all dogs could bear weight on the affected limb immediately after surgery. Dogs were euthanized 4, 8 and 24 weeks after implantation. Histomorphometry showed greater infiltration of new vessels and higher bone union rate in the TB-gel group than in the TB group. The lamellar bone volume and mineral apposition rate did not differ significantly between the groups, indicating that neovascularization may be the primary effect of f-IC gel on bone regeneration. This combination method which is tTMCP combined with TB and f-IC gel, would be useful for the treatment of segmental long bone defects.


Assuntos
Placas Ósseas , Regeneração Óssea/fisiologia , Rádio (Anatomia)/cirurgia , Titânio , Cicatrização/fisiologia , Animais , Regeneração Óssea/efeitos dos fármacos , Fosfatos de Cálcio/uso terapêutico , Cães , Fator 2 de Crescimento de Fibroblastos/uso terapêutico , Suporte de Carga , Cicatrização/efeitos dos fármacos
18.
Regen Ther ; 6: 100-107, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30271844

RESUMO

A class of gene-regulatory elements called enhancers are the main mediators controlling quantitative, temporal and spatial gene expressions. In the course of evolution, the enhancer landscape of higher organisms such as mammals has become quite complex, exerting biological functions precisely and coordinately. In mammalian skeletal development, the master transcription factors Sox9, Runx2 and Sp7/Osterix function primarily through enhancers on the genome to achieve specification and differentiation of skeletal cells. Recently developed genome-scale analyses have shed light on multiple layers of gene regulations, uncovering not only the primary mode of actions of these transcription factors on skeletal enhancers, but also the relation of the epigenetic landscape to three-dimensional chromatin architecture. Here, we review findings on the emerging framework of gene-regulatory networks involved in skeletal development. We further discuss the power of genome-scale analyses to provide new insights into genetic diseases and regenerative medicine in skeletal tissues.

19.
Trends Genet ; 32(12): 774-787, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27814929

RESUMO

Skeletal development creates the physical framework that shapes our body and its actions. In the past two decades, genetic studies have provided important insights into the molecular processes at play, including the roles of signaling pathways and transcriptional effectors that coordinate an orderly, progressive emergence and expansion of distinct cartilage and bone cell fates in an invariant temporal and spatial pattern for any given skeletal element within that specific vertebrate species. Genome-scale studies have provided additional layers of understanding, moving from individual genes to the gene regulatory landscape, integrating regulatory information through cis-regulatory modules into cell type-specific gene regulatory programs. This review discusses our current understanding of the transcriptional control of mammalian skeletal development, focusing on recent genome-scale studies.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Mamíferos/genética , Organogênese/genética , Esqueleto/crescimento & desenvolvimento , Animais , Redes Reguladoras de Genes/genética , Genoma , Mamíferos/crescimento & desenvolvimento , Transdução de Sinais/genética
20.
Biochem Biophys Res Commun ; 479(4): 772-778, 2016 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-27693795

RESUMO

Bone fracture healing is processed through multiple biological stages including the transition from cartilaginous callus to bony callus formation. Because of its specific, temporal and indispensable functions demonstrated by mouse genetic studies, Hedgehog (Hh) signaling is one of the most potent signaling pathways involved in these processes, but the effect of Hh-signaling activation by small compounds on the repair process had not yet been addressed. Here we examined therapeutic effects of local and one shot-administration of the Hh agonist known as smoothened agonist (SAG) on bone fracture healing in a mouse model. A quantitative analysis with three-dimensional micro-computed tomography showed that SAG administration increased the size of both the cartilaginous callus and bony callus at 14 days after the surgery. A histological analysis showed that SAG administration increased the number of cells expressing a proliferation marker and a chondrocyte marker in cartilaginous callus as well as the cells expressing an osteoblast marker in bony callus. These results indicate that the SAG administration resulted in an enhancement of callus formation during bone fracture healing, which is at least in part mediated by an increase in chondrocyte proliferation in cartilaginous callus and the promotion of bone formation in bony callus. Therapeutic strategies with a SAG-mediated protocol may thus be useful for the treatment of bone fractures.


Assuntos
Cicloexilaminas/administração & dosagem , Consolidação da Fratura/efeitos dos fármacos , Proteínas Hedgehog/agonistas , Tiofenos/administração & dosagem , Animais , Densidade Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/metabolismo , Calo Ósseo/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Modelos Animais de Doenças , Consolidação da Fratura/fisiologia , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fraturas da Tíbia/diagnóstico por imagem , Fraturas da Tíbia/tratamento farmacológico , Fraturas da Tíbia/patologia , Microtomografia por Raio-X
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