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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
4.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360992

RESUMO

Several protocols exist for generating megakaryocytes (MKs) and platelets from human induced pluripotent stem cells (hiPSCs) with limited efficiency. We observed previously that mesoderm induction improved endothelial and stromal differentiation. We, therefore, hypothesized that a protocol modification prior to hemogenic endothelial cell (HEC) differentiation will improve MK progenitor (MKP) production and increase platelet output. We further asked if basic media composition affects MK maturation. In an iterative process, we first compared two HEC induction protocols. We found significantly more HECs using the modified protocol including activin A and CHIR99021, resulting in significantly increased MKs. MKs released comparable platelet amounts irrespective of media conditions. In a final validation phase, we obtained five-fold more platelets per hiPSC with the modified protocol (235 ± 84) compared to standard conditions (51 ± 15; p < 0.0001). The regenerative potency of hiPSC-derived platelets was compared to adult donor-derived platelets by profiling angiogenesis-related protein expression. Nineteen of 24 angiogenesis-related proteins were expressed equally, lower or higher in hiPSC-derived compared to adult platelets. The hiPSC-platelet's coagulation hyporeactivity compared to adult platelets was confirmed by thromboelastometry. Further stepwise improvement of hiPSC-platelet production will, thus, permit better identification of platelet-mediated regenerative mechanisms and facilitate manufacture of sufficient amounts of functional platelets for clinical application.


Assuntos
Plaquetas/citologia , Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Megacariócitos/citologia , Células Cultivadas , Meios de Cultura/química , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
5.
Methods Mol Biol ; 2341: 25-30, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34264457

RESUMO

Many strains of Staphylococcus aureus produce a variety of cytolysins that target many different cell types to both fight the immune system and acquire nutrients. This includes hemolysins which destroy erythrocytes and are well studied virulence factors. Traditionally, hemolysin activity is measured on blood agar plates due to the simplicity of the assay. While this is telling, it cannot encapsulate the full story because S. aureus is known to behave differently in broth and on agar. Furthermore, plate-based assays are primarily semiquantitative and often a more accurate determination of hemolytic potential is needed to discern differences between strains. Here, we describe a method to quantify hemolysin activity from broth or similarly grown cells.


Assuntos
Eritrócitos/fisiologia , Proteínas Hemolisinas/análise , Staphylococcus aureus/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Proteínas Hemolisinas/metabolismo , Hemólise , Humanos , Staphylococcus aureus/metabolismo , Staphylococcus aureus/patogenicidade , Fatores de Virulência/análise , Fatores de Virulência/metabolismo
6.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209789

RESUMO

Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT.


Assuntos
Materiais Biocompatíveis/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Meios de Cultura/farmacologia , Adulto , Materiais Biocompatíveis/química , Plaquetas/citologia , Meios de Cultura/química , Endotélio/citologia , Feminino , Humanos , Masculino , Teste de Materiais , Pessoa de Meia-Idade , Ativação Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Polímeros/farmacologia , Tecidos Suporte/química
7.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207579

RESUMO

Biomanufacturing processes may be optimized by storing cell culture media at room temperature, but this is currently limited by their instability and change in color upon long-term storage. This study demonstrates that one of the critical contributing factors toward media browning is tryptophan. LC-MS technology was utilized to identify tryptophan degradation products, which are likely formed primarily from oxidation reactions. Several of the identified compounds were shown to contribute significantly to color in solutions but also to exhibit toxicity against CHO cells. A cell-culture-compatible antioxidant, a-ketoglutaric acid, was found to be an efficient cell culture media additive for stabilizing components against degradation, inhibiting the browning of media formulations, and decreasing ammonia production, thus providing a viable method for developing room-temperature stable cell culture media.


Assuntos
Meios de Cultura/química , Triptofano/metabolismo , Animais , Células CHO , Cricetulus , Oxirredução , Triptofano/análise
8.
Methods Mol Biol ; 2319: 51-60, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331242

RESUMO

Cardiovascular disease is a worldwide health issue that affects millions of lives every year, and thus, researchers are in need of high-throughput model systems with which to investigate mechanisms of disease and to develop and test potential therapies. The use of human-derived induced pluripotent stem cells (iPSCs) differentiated into cardiomyocytes aims to address this need. While cardiac differentiation protocols have been established previously in iPSCs, optimization of cardiac differentiation remains crucial to obtaining high quality cardiomyocytes for future experimental analyses. Important factors to consider include cell density and rate of proliferation, temporal regulation of media changes throughout the differentiation process, and the concentration of the chemicals utilized. In this chapter, we present a detailed protocol to outline the process of differentiating cardiomyocytes from human iPSCs via modulation of Wnt signaling, characterization of cardiomyocytes by immunofluorescence, as well as guidelines for troubleshooting and optimizing these techniques.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultura/química , Células-Tronco Pluripotentes Induzidas/citologia , Desenvolvimento Muscular , Miócitos Cardíacos/citologia , Via de Sinalização Wnt , Imunofluorescência , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo
9.
Methods Mol Biol ; 2319: 69-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331244

RESUMO

There is increasing interest in the study of the mammalian lymphatic system, including the lymphatic endothelial cells (LECs) that make up lymphatic vessels. The ability to isolate primary LECs from tissue of normal and genetically modified mice permits detailed analysis of this unique cell type. Here, we describe a robust protocol for the isolation and in vitro expansion of LECs from mouse lung by antibody-based magnetic separation.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Células Endoteliais/citologia , Separação Imunomagnética/métodos , Pulmão/citologia , Animais , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Meios de Cultura/química , Células Endoteliais/metabolismo , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos
10.
Methods Mol Biol ; 2319: 93-104, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331247

RESUMO

Lightsheet microscopy is a form of fluorescence microscopy that can be used to visualize specimen with high resolution, a large depth-of-field, and minimal photodamage and photobleaching as compared to traditional confocal microscopy. As this technology becomes much more readily available, it will be useful in revealing new findings in the cardiovascular development field that may be hidden or difficult to image. In this manuscript, we describe an approach for mounting and culturing postimplantation mouse embryos to visualize blood vessel development with a lightsheet microscope.


Assuntos
Angiografia/métodos , Vasos Sanguíneos/diagnóstico por imagem , Técnicas de Cultura/métodos , Embrião de Mamíferos/diagnóstico por imagem , Desenvolvimento Embrionário , Microscopia de Fluorescência/métodos , Neovascularização Fisiológica , Animais , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Meios de Cultura/química , Dissecação/métodos , Embrião de Mamíferos/irrigação sanguínea , Camundongos , Camundongos Transgênicos , Microscopia Confocal
11.
Methods Mol Biol ; 2288: 91-102, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270006

RESUMO

We describe the production of doubled haploids through anther culture in caraway. Induction conditions for the cultivation of donor plants, anther collection, composition of culture media, and physical induction conditions for embryogenesis have been described. As a result, responsive lines with numerous haploid embryo production were obtained, which after colchicine treatment became fertile. From a practical point of view, two doubled haploid populations are tested under field conditions.


Assuntos
Carum/crescimento & desenvolvimento , Carum/genética , Melhoramento Vegetal/métodos , Carum/fisiologia , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Meios de Cultura/química , Diploide , Esterases/análise , Flores/genética , Flores/crescimento & desenvolvimento , Haploidia , Homozigoto , Isoenzimas/análise , Biologia Molecular/métodos , Pólen/genética , Pólen/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos
12.
Methods Mol Biol ; 2288: 129-144, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270009

RESUMO

Rapeseed (Brassica napus) is one of the most important oilseed crops worldwide. It is also a model system to study the process of microspore embryogenesis, due to the high response of some B. napus lines, and to the refinements of the protocols. This chapter presents a protocol for the induction of haploid and DH embryos in B. napus through isolated microspore culture in two specific backgrounds widely used in DH research, the high response DH4079 line and the low response DH12075 line. We also present methods to identify the best phenological window to identify buds with microspores/pollen at the right developmental stage to induce this process. Methods to determine microspore/pollen viability and to check the ploidy by flow cytometry are also described.


Assuntos
Brassica napus/crescimento & desenvolvimento , Brassica napus/genética , Melhoramento Vegetal/métodos , Aclimatação/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Meios de Cultura/química , Diploide , Citometria de Fluxo , Genótipo , Germinação/genética , Haploidia , Homozigoto , Biologia Molecular/métodos , Ploidias , Pólen/genética , Pólen/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos
13.
Methods Mol Biol ; 2288: 113-126, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270008

RESUMO

Carrot is a vegetable of increasing economic importance. New hybrid cultivars are constantly required to meet the changing market needs. The application of anther culture significantly shortens the difficult and long-lasting breeding of carrot. We examined all the stages of the process of generating androgenic plants: induction of embryos in anther cultures, regeneration and acclimatization of produced plants, their evaluation, ploidy and homozygosity, and many other factors affecting their effectiveness. Every factor has been optimized by experimentally selecting the optimal level. As a result, a full protocol of producing homozygous plants using anther cultures was developed, which is presented in this chapter.


Assuntos
Daucus carota/crescimento & desenvolvimento , Daucus carota/genética , Melhoramento Vegetal/métodos , Aclimatação/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/fisiologia , Meios de Cultura/química , Daucus carota/fisiologia , Flores/genética , Flores/crescimento & desenvolvimento , Heterozigoto , Homozigoto , Isoenzimas/análise , Biologia Molecular/métodos , Regeneração/genética , Técnicas de Cultura de Tecidos
14.
Methods Mol Biol ; 2288: 103-111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270007

RESUMO

Doubled haploidy technology is a powerful tool to accelerate the breeding of new crop varieties. Protocols are not universal, as even species within the same family require a specific process. Here we describe methods for developing doubled haploids for fennel and dill, both Apiaceae species which are used for food, flavorings, and medicine.


Assuntos
Anethum graveolens/crescimento & desenvolvimento , Anethum graveolens/genética , Foeniculum/crescimento & desenvolvimento , Foeniculum/genética , Melhoramento Vegetal/métodos , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Meios de Cultura/química , Diploide , Haploidia , Homozigoto , Biologia Molecular/métodos , Pólen/genética , Pólen/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos
15.
Methods Mol Biol ; 2288: 145-162, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270010

RESUMO

Culture of isolated microspores is a widely used method to obtain haploid and doubled haploid plants for many crop species. This protocol describes the steps necessary to obtain a large number of microspore derived embryos for pakchoi (Brassica rapa L. ssp. chinensis) and zicaitai (Brassica rapa L. ssp. сhinensis Hanelt var. purpuraria Kitam).


Assuntos
Brassica rapa/crescimento & desenvolvimento , Brassica rapa/genética , Melhoramento Vegetal/métodos , Brassica rapa/ultraestrutura , Cloroplastos/ultraestrutura , Cromossomos de Plantas/ultraestrutura , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Meios de Cultura/química , Diploide , Germinação/genética , Haploidia , Homozigoto , Microscopia de Fluorescência , Biologia Molecular/métodos , Ploidias , Pólen/genética , Pólen/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos
16.
Methods Mol Biol ; 2288: 163-180, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270011

RESUMO

Brassica carinata, also known as Ethiopian or Abyssinian mustard, is a drought- and heat-tolerant oilseed with great potential as a dedicated industrial feedstock crop for use in biofuel and other bio-based applications. Doubled haploid technology, a system that allows for the rapid development of doubled haploid, completely homozygous plants through microspore embryogenesis, has been applied routinely in both B. carinata breeding and basic research. Here, we present a comprehensive isolated microspore culture protocol detailing the various steps involved in doubled haploid plant production for this species, from growing donor plants over harvesting flower buds and isolating, culturing and inducing microspores to regenerating doubled haploid embryos and plantlets.


Assuntos
Mostardeira/crescimento & desenvolvimento , Mostardeira/genética , Melhoramento Vegetal/métodos , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Meios de Cultura/química , Diploide , Haploidia , Homozigoto , Biologia Molecular/métodos , Ploidias , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/ultraestrutura , Técnicas de Cultura de Tecidos
17.
Methods Mol Biol ; 2288: 181-199, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270012

RESUMO

The production of haploid and doubled haploid plants is a biotechnological tool that shortens the breeding process of new cultivars in many species. Doubled haploid plants are homozygous at every locus and they can be utilized as parents to produce F1 hybrids. In this chapter, we describe a protocol for the production of doubled haploid plants in Brassica rapa L. subsp. pekinensis using androgenesis induced by isolated microspore cultures.


Assuntos
Brassica rapa/crescimento & desenvolvimento , Brassica rapa/genética , Melhoramento Vegetal/métodos , Aclimatação/genética , Brassica rapa/fisiologia , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/fisiologia , Meios de Cultura/química , DNA de Plantas/genética , Diploide , Glucose-6-Fosfato Isomerase/genética , Haploidia , Homozigoto , Biologia Molecular/métodos , Pólen/genética , Pólen/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Regeneração/genética , Técnicas de Cultura de Tecidos
18.
Methods Mol Biol ; 2288: 201-216, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270013

RESUMO

Broccoli (Brassica olearecea var. italica) is a cole crop grown for its floral heads and stalks. It is rich in bioactive chemicals good for human health. Broccoli has been consumed as a vegetable since Roman times, but its production and consumption have increased significantly over the past few decades. Breeders try to develop new broccoli varieties with high yield, improved quality, and resistance to biotic and abiotic stresses. Almost all new broccoli varieties are F1 hybrids. Development of inbred broccoli lines that can be used as parents in hybrid production is a time-consuming and difficult process. Haploidization techniques can be utilized as a valuable support in broccoli breeding programs to speed up the production of genetically pure genotypes. Haploid plants of broccoli can be produced from immature male gametophytes via anther and microspore cultures with similar success rates. The most important parameters affecting the success of haploidization in broccoli are the genetic background (genotype) and the developmental stage of the microspores. Broccoli genotypes differ in their responses to androgenesis induction. The highest androgenesis response could be induced from microspores in late uninucleate and early binucleate stages. Recovery of diploid broccoli plants from haploids is possible via spontaneous and induced doubling. Doubled haploid (DH) broccoli lines are considered to be fully homozygous. Therefore, the production of DH lines is an alternative way to obtain pure inbred lines that can be utilized as parents in the development of new F1 hybrid varieties showing high levels of heterosis, high-quality heads, and uniform harvestable crop. We are using an anther culture-based haploid plant production system to develop DH broccoli lines in our broccoli breeding program. DH broccoli lines are produced from different genetic backgrounds within a year and handed to broccoli breeders.


Assuntos
Brassica/crescimento & desenvolvimento , Brassica/genética , Melhoramento Vegetal/métodos , Aclimatação/genética , Brassica/fisiologia , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Meios de Cultura/química , Diploide , Citometria de Fluxo , Flores/genética , Flores/crescimento & desenvolvimento , Haploidia , Homozigoto , Vigor Híbrido/genética , Biologia Molecular/métodos , Ploidias , Pólen/genética , Pólen/crescimento & desenvolvimento , Regeneração/genética , Técnicas de Cultura de Tecidos
19.
Methods Mol Biol ; 2288: 217-232, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270014

RESUMO

Here, we describe the first protocol of European radish (Raphanus sativus L. subsp. sativus convar. radicula) for obtaining doubled haploid plants through in vitro microspore culture, in which the full cycle of doubled haploid formation was successfully achieved. Using this protocol, a yield of up to eight embryoids per Petri dish can be obtained. Effectiveness of this protocol was confirmed for several genotypes of European radish.


Assuntos
Melhoramento Vegetal/métodos , Raphanus/crescimento & desenvolvimento , Raphanus/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/fisiologia , Meios de Cultura/química , Diploide , Corantes Fluorescentes , Genótipo , Haploidia , Homozigoto , Indóis , Biologia Molecular/métodos , Ploidias , Pólen/genética , Pólen/crescimento & desenvolvimento , Raphanus/fisiologia , Regeneração/genética , Coloração e Rotulagem , Técnicas de Cultura de Tecidos
20.
Methods Mol Biol ; 2288: 235-250, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270015

RESUMO

Eggplant is one of the five important, worldwide-distributed solanaceous crops. The use of anther culture technology to produce pure, 100% homozygous doubled haploid lines for hybrid seed production is possible since 1982, where the first protocol of wide application to different eggplant materials was published. From then on, different improvements and adaptations to different materials have been made. In parallel, protocols to implement isolated microspore culture technology in eggplant have been developed principally in the last decade, which opens the door for a more efficient DH production in this species. In this chapter, two protocols, one for anther and other for isolated microspore culture in eggplant, are described. Some steps and materials are common to both approaches. A detailed description of each step from is provided.


Assuntos
Melhoramento Vegetal/métodos , Solanum melongena/crescimento & desenvolvimento , Solanum melongena/genética , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/fisiologia , Meios de Cultura/química , Diploide , Flores/genética , Flores/crescimento & desenvolvimento , Corantes Fluorescentes , Haploidia , Homozigoto , Indóis , Biologia Molecular/métodos , Ploidias , Pólen/genética , Pólen/crescimento & desenvolvimento , Regeneração/genética , Solanum melongena/fisiologia , Coloração e Rotulagem , Técnicas de Cultura de Tecidos
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