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


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.

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
Bioorg Med Chem Lett ; 25(20): 4464-71, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26364943


Phytohormone auxin is a master regulator in plant growth and development. Regulation of cellular auxin level plays a central role in plant development. Auxin polar transport system modulates an auxin gradient that determines plant developmental process in response to environmental conditions and developmental programs. Photolabile caged auxins allow optical control of artificial auxin gradients at cellular resolution. Especially, two-photon uncaging system achieves high spatiotemporal control of photolysis reaction at two-photon cross-section. However, the development of caged versions of auxin has been limited by the instability of the caged auxins to higher plant metabolic activities. Here, we describe the synthesis and application of highly stable caged auxins, 4-methoxy-7-nitroindolinyl (MNI)-caged auxins. Natural auxin, indole 3-acetic acid, and two synthetic auxins, 1-NAA and 2,4-D were caged by MNI caging group. MNI-caged auxins showed a high stability in planta and a rapid release the original auxin when photolyzed. We demonstrated that optical control of auxin-responsive gene expression and auxin-related physiological responses by using MNI-caged auxins. We anticipate that MNI-caged auxins will be an effective tool for high-resolution control of endogenous auxin level.

Arabidopsis/química , Ácidos Indolacéticos/química , Indóis/química , Reguladores de Crescimento de Plantas/química , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Fotólise , Reguladores de Crescimento de Plantas/metabolismo
Bioorg Med Chem Lett ; 22(17): 5663-7, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22832308


Cytokinins are phytohormones that regulate diverse developmental processes throughout the life of a plant. trans-Zeatin, kinetin, benzyladenine and dihydrozeatin are adenine-type cytokinins that are perceived by the AHK cytokinin receptors. Endogenous cytokinin levels are critical for regulating plant development. To manipulate intracellular cytokinin levels, caged cytokinins were designed on the basis of the crystal structure of the AHK4 cytokinin receptor. The caged cytokinin was photolyzed to release the cytokinin molecule inside the cells and induce cytokinin-responsive gene expression. The uncaging of intracellular caged cytokinins demonstrated that cytokinin-induced root growth inhibition can be manipulated with photo-irradiation. This caged cytokinin system could be a powerful tool for cytokinin biology.

Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Citocininas/química , Citocininas/metabolismo , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/metabolismo , Citocininas/síntese química , Modelos Moleculares , Fotólise , Reguladores de Crescimento de Plantas/síntese química , Raízes de Plantas/metabolismo
Chembiochem ; 10(13): 2195-202, 2009 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-19637145


Auxin, a plant hormone, is polar transported from its site of production. This auxin polar transport system establishes an auxin gradient in plant tissue that is necessary for proper plant development. Therefore, the spatial effect of the auxin gradient on plant development is highly important for the understanding of plant auxin responses. Herein we report the design, syntheses and biological properties of esterase-resistant caged auxins. The conventional caging group, 2-nitrobenzyl ester, was found to be enzymatically hydrolyzed in plant cells and released original auxin without photolysis. The esterase-resistant caging group, (2,5-dimethoxyphenyl)(2-nitrobenzyl) ester, (DMPNB) was designed to improve the stability of caged auxins. Three auxins, indole 3-acetic acid, naphthalene 1-acetic acid and 2,4-dichlorophenoxy acetic acid were caged with the DMPNB caging group. DMPNB-caged auxins were inactive within a plant cell until photolysis, but they release auxins with photoirradiation to activate auxin-responsive gene expression. We demonstrated spatial and temporal control of intracellular auxin levels with photoirradiation by using this caged auxin system and were able to photocontrol the physiological auxin response in Arabidopsis plants. Additionally, the photoirradiation of DMPNB-caged auxin within a single cell can manipulate the intracellular auxin level and triggers auxin response.

Esterases/metabolismo , Ácidos Indolacéticos/química , Raios Ultravioleta , Arabidopsis/química , Ácidos Indolacéticos/síntese química , Fotólise , Raízes de Plantas/crescimento & desenvolvimento
Bioorg Med Chem ; 16(9): 5331-44, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18343120


The auxins, plant hormones, regulate many aspects of the growth and development of plants. Terfestatin A (TrfA), a novel auxin signaling inhibitor, was identified in a screen of Streptomyces sp. F40 extracts for inhibition of the expression of an auxin-inducible gene. However, the mode of action of TrfA has not been elucidated. To identify the active core structure, 25 derivatives of TrfA were synthesized and their inhibitory activities against auxin-inducible gene expression were evaluated. The structure-activity relationships revealed the essential active core structure of TrfA, 3-butoxy-4-methylbiphenyl-2,6-diol, which will lead to the design of biotin-tagged active TrfA.

Glucosídeos/farmacologia , Ácidos Indolacéticos/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Compostos de Terfenil/farmacologia , Sítios de Ligação , Grupo dos Citocromos b/efeitos dos fármacos , Grupo dos Citocromos b/genética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Complexo IV da Cadeia de Transporte de Elétrons/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucosídeos/síntese química , Glucosídeos/química , Glucuronidase/efeitos dos fármacos , Glucuronidase/genética , Ácidos Indolacéticos/farmacologia , Modelos Moleculares , Estrutura Molecular , Estereoisomerismo , Streptomyces/química , Relação Estrutura-Atividade , Compostos de Terfenil/síntese química , Compostos de Terfenil/química
Bioorg Med Chem Lett ; 16(9): 2470-4, 2006 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-16480868