RESUMO
We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs), partially reprogrammed cells, somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over â¼10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free, enriched to 95%-99% purity with >80% survival, and had normal transcriptional profiles, differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells.
Assuntos
Adesão Celular , Células-Tronco Pluripotentes/citologia , Diferenciação Celular , Separação Celular , Humanos , CariotipagemRESUMO
The differentiation of pluripotent stem cells as embryoid bodies (EBs) remains a common method for inducing differentiation toward many lineages. However, differentiation via EBs typically yields a significant amount of heterogeneity in the cell population, as most cells differentiate simultaneously toward different lineages, while others remain undifferentiated. Moreover, physical parameters, such as the size of EBs, can modulate the heterogeneity of differentiated phenotypes due to the establishment of nutrient and oxygen gradients. One of the challenges in examining the cellular composition of EBs is the lack of analytical methods that are capable of determining the phenotype of all of the individual cells that comprise a single EB. Therefore, the objective of this work was to examine the ability of a microfluidic cell trapping array to analyze the heterogeneity of cells comprising EBs during the course of early differentiation. The heterogeneity of single cell phenotype on the basis of protein expression of the pluripotent transcription factor OCT-4 was examined for populations of EBs and single EBs of different sizes at distinct stages of differentiation. Results from the cell trap device were compared with flow cytometry and whole mount immunostaining. Additionally, single cells from dissociated pooled EBs or individual EBs were examined separately to discern potential differences in the value or variance of expression between the different methods of analysis. Overall, the analytical method described represents a novel approach for evaluating how heterogeneity is manifested in EB cultures and may be used in the future to assess the kinetics and patterns of differentiation in addition to the loss of pluripotency.
Assuntos
Corpos Embrioides/química , Microfluídica/métodos , Análise de Célula Única/métodos , Animais , Diferenciação Celular , Linhagem Celular , Corpos Embrioides/citologia , Imunofluorescência , Camundongos , Técnicas Analíticas Microfluídicas/instrumentação , Células-Tronco PluripotentesRESUMO
The field of tissue engineering and regenerative medicine will tremendously benefit from the development of three dimensional scaffolds with defined micro- and macro-architecture that replicate the geometry and chemical composition of native tissues. The current report describes a freeform fabrication technique that permits the development of nerve regeneration scaffolds with precisely engineered architecture that mimics that of native nerve, using the native extracellular matrix component hyaluronic acid (HA). To demonstrate the flexibility of the fabrication system, scaffolds exhibiting different geometries with varying pore shapes, sizes and controlled degradability were fabricated in a layer-by-layer fashion. To promote cell adhesion, scaffolds were covalently functionalized with laminin. This approach offers tremendous spatio-temporal flexibility to create architecturally complex structures such as scaffolds with branched tubes to mimic branched nerves at a plexus. We further demonstrate the ability to create bidirectional gradients within the microfabricated nerve conduits. We believe that combining the biological properties of HA with precise three dimensional micro-architecture could offer a useful platform for the development of a wide range of bioartificial organs.
Assuntos
Ácido Hialurônico/química , Microtecnologia/métodos , Regeneração Nervosa , Alicerces Teciduais , Materiais Biocompatíveis/química , Adesão Celular , Células Cultivadas , Compostos de Epóxi/química , Matriz Extracelular/química , Humanos , Imageamento Tridimensional/métodos , Metacrilatos/química , Tecido Nervoso/química , Tecido Nervoso/citologia , Engenharia Tecidual/métodosRESUMO
The development of biomedical scaffolds mimicking a heterogeneous cellular microenvironment for a specified regulation of cell-fates is very promising for tissue engineering. In this study, three-dimensional scaffolds with heterogeneous microstructure were developed using a DMD-PP apparatus. During the fabrication process, this apparatus can efficiently switch monomers to form microstructures with localized, different material properties; the resolution in the arrangement of material properties is comparable to the characteristic size of functional subunits in living organs, namely, a hundred microns. The effectiveness of this DMD-PP apparatus is demonstrated by a woodpile microstructure with heterogeneous fluorescence and also by a microporous cell-culturing scaffold with selected sites for protein adhesion. Cell-cultivation experiment was performed with the microporous scaffold, in which selective cell adhesion was observed.
Assuntos
Microtecnologia/instrumentação , Engenharia Tecidual , Alicerces Teciduais , Animais , Adesão Celular , Técnicas de Cultura de Células , Dispositivos Lab-On-A-Chip , Proteínas/metabolismo , Células de Schwann/citologiaRESUMO
Purpose The TAXYNERGY trial ( ClinicalTrials.gov identifier: NCT01718353) evaluated clinical benefit from early taxane switch and circulating tumor cell (CTC) biomarkers to interrogate mechanisms of sensitivity or resistance to taxanes in men with chemotherapy-naïve, metastatic, castration-resistant prostate cancer. Patients and Methods Patients were randomly assigned 2:1 to docetaxel or cabazitaxel. Men who did not achieve ≥ 30% prostate-specific antigen (PSA) decline by cycle 4 (C4) switched taxane. The primary clinical endpoint was confirmed ≥ 50% PSA decline versus historical control (TAX327). The primary biomarker endpoint was analysis of post-treatment CTCs to confirm the hypothesis that clinical response was associated with taxane drug-target engagement, evidenced by decreased percent androgen receptor nuclear localization (%ARNL) and increased microtubule bundling. Results Sixty-three patients were randomly assigned to docetaxel (n = 41) or cabazitaxel (n = 22); 44.4% received prior potent androgen receptor-targeted therapy. Overall, 35 patients (55.6%) had confirmed ≥ 50% PSA responses, exceeding the historical control rate of 45.4% (TAX327). Of 61 treated patients, 33 (54.1%) had ≥ 30% PSA declines by C4 and did not switch taxane, 15 patients (24.6%) who did not achieve ≥ 30% PSA declines by C4 switched taxane, and 13 patients (21.3%) discontinued therapy before or at C4. Of patients switching taxane, 46.7% subsequently achieved ≥ 50% PSA decrease. In 26 CTC-evaluable patients, taxane-induced decrease in %ARNL (cycle 1 day 1 v cycle 1 day 8) was associated with a higher rate of ≥ 50% PSA decrease at C4 ( P = .009). Median composite progression-free survival was 9.1 months (95% CI, 4.9 to 11.7 months); median overall survival was not reached at 14 months. Common grade 3 or 4 adverse events included fatigue (13.1%) and febrile neutropenia (11.5%). Conclusion The early taxane switch strategy was associated with improved PSA response rates versus TAX327. Taxane-induced shifts in %ARNL may serve as an early biomarker of clinical benefit in patients treated with taxanes.
Assuntos
Células Neoplásicas Circulantes/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Taxoides/administração & dosagem , Idoso , Biomarcadores Tumorais/sangue , Núcleo Celular/metabolismo , Intervalo Livre de Doença , Docetaxel , Esquema de Medicação , Humanos , Calicreínas/sangue , Masculino , Células Neoplásicas Circulantes/patologia , Antígeno Prostático Específico/sangue , Neoplasias de Próstata Resistentes à Castração/sangue , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores Androgênicos/metabolismo , Taxoides/efeitos adversosRESUMO
Implant-associated inflammation and bacterial infection severely limit the functional performance of medical devices and are a major cause of implant failure. Therefore, it is crucial to develop methodologies to monitor/image implant-associated aseptic inflammation and bacterial infection in a minimally invasive manner. Here, we exploited near-infrared fluorescence (NIRF) molecular probes injected locally at the implant site to perform minimally invasive, simultaneous imaging of inflammation, and infection associated with implanted polymer disks. The hydro-sulfo-Cy5 (H-s-Cy5) probe detected reactive oxygen species associated with inflammatory responses to both aseptic and biofilm-containing implants, whereas diaminocyanine sulfonate selectively detected nitric oxide associated with a biofilm on the biomaterial at acute time points (<4 days). This imaging modality also allows longitudinal monitoring because of high specificity and fast clearance rate of the fluorescent probes. Taken together, these NIRF molecular probes represent a useful tool to directly image inflammatory responses and infections associated with implanted devices for the diagnosis of device-associated inflammation and infection as well as the development of effective therapies.
Assuntos
Materiais Biocompatíveis/efeitos adversos , Inflamação/etiologia , Animais , Biofilmes , Contagem de Colônia Microbiana , Fluorescência , Corantes Fluorescentes , Inflamação/metabolismo , Inflamação/microbiologia , Camundongos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Biomaterial-associated infection is one of the most common complications related to the implantation of any biomedical device. Several in vivo imaging platforms have emerged as powerful diagnostic tools to longitudinally monitor biomaterial-associated infections in small animal models. In this study, we directly compared two imaging approaches: bacteria engineered to produce luciferase to generate bioluminescence and reactive oxygen species (ROS) imaging of the inflammatory response associated with the infected implant. We performed longitudinal imaging of bioluminescence associated with bacteria strains expressing plasmid-integrated luciferase driven by different promoters or a strain with the luciferase gene integrated into the chromosome. These luminescent strains provided an adequate signal for acute (0-4 days) monitoring of the infection, but the bioluminescence signal decreased over time and leveled off at 7 days post-implantation. This loss in the bioluminescence signal was attributed to changes in the metabolic activity of the bacteria. In contrast, near-infrared fluorescence imaging of ROS associated with inflammation to the implant provided sensitive and dose-dependent signals of biomaterial-associated bacteria. ROS imaging exhibited higher sensitivity than the bioluminescence imaging and was independent of the bacteria strain. Near-infrared fluorescence imaging of inflammatory responses represents a powerful alternative to bioluminescence imaging for monitoring biomaterial-associated bacterial infections.
Assuntos
Materiais Biocompatíveis/efeitos adversos , Infecções/etiologia , Staphylococcus aureus/metabolismo , Animais , Fluorescência , CamundongosRESUMO
Recently there has been an increased interest in the effects of paracrine signaling between groups of cells, particularly in the context of better understanding how stem cells contribute to tissue repair. Most current 3D co-culture methods lack the ability to effectively separate two cell populations after the culture period, which is important for simultaneously analyzing the reciprocal effects of each cell type on the other. Here, we detail the development of a 3D hydrogel co-culture system that allows us to culture different cell types for up to 7 days and subsequently separate and isolate the different cell populations using enzyme-sensitive glues. Separable 3D co-culture laminates were prepared by laminating PEG-based hydrogels with enzyme-degradable hydrogel adhesives. Encapsulated cell populations exhibited good segregation with well-defined interfaces. Furthermore, constructs can be separated on-demand upon addition of the appropriate enzyme, while cell viability remains high throughout the culture period, even after laminate separation. This platform offers great potential for a variety of basic cell signaling studies as the incorporation of an enzyme-sensitive adhesive interface allows the on-demand separation of individual cell populations for immediate analysis or further culture to examine persistence of co-culture effects and paracrine signaling on cell populations.
Assuntos
Técnicas de Cultura de Células/instrumentação , Separação Celular/métodos , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Análise de Variância , Técnicas de Cultura de Células/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura/instrumentação , Técnicas de Cocultura/métodos , Desenho de Equipamento , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Polietilenoglicóis/químicaRESUMO
In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments.
Assuntos
Células-Tronco Embrionárias/citologia , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Diferenciação Celular , Células Cultivadas , Corpos Embrioides/citologia , Corpos Embrioides/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mesoderma/citologia , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , TransfecçãoRESUMO
Various neural tissue engineering approaches that are under development for applications ranging from guidance conduits to cell-based therapies rely on the ability to encapsulate cells in three-dimensional (3D) scaffolds. Schwann cells play a key role in peripheral nerve regeneration by forming oriented paths for regrowing axons. We have engineered collagen and hyaluronic acid interpenetrating polymer network (IPN) hydrogels with and without laminin as a 3D culture system for Schwann cells in an attempt to devise novel neural regeneration therapies. Encapsulation of Schwann cells in 3D hydrogel constructs did not affect cell viability and cells were viable for 2 weeks in all hydrogel samples. Moreover, in hydrogels with high cell density, cells underwent spreading and proliferation, and the cell numbers increased by day 14 as assessed qualitatively using a Live/dead assay and scanning electron microscopy (SEM), and quantitatively using a CellTiter 96 AQueous non-radioactive cell proliferation assay. In some cases, the cells aligned parallel to each other and formed structures reminiscent of Bands of Büngner. Schwann cells in cell-hydrogel constructs with high cell density were not only viable but also actively secreting nerve growth factor and brain-derived neurotrophic factor. Of particular importance was the observation that addition of laminin in these hydrogels increased the overall production of nerve growth factor and brain-derived neurotrophic factor from the cells. Immunostaining revealed that S100 expression and cell spreading were differentially affected by cell density. Interestingly, in the co-culture of dissociated neurons with Schwann cells, neurons were able to extend neurites and some neurites were observed to follow Schwann cells. Therefore, we conclude that Schwann cells encapsulated in the 3D extracellular matrix-mimicking hydrogel may hold promise in nerve regeneration therapies and may form the basis for understanding the underlying mechanisms of Schwann cell interactions with neurons and various extracellular matrix components.
Assuntos
Colágeno/farmacologia , Ácido Hialurônico/farmacologia , Laminina/farmacologia , Sistema Nervoso/efeitos dos fármacos , Células de Schwann/metabolismo , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Apoptose/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Imageamento Tridimensional , Imuno-Histoquímica , Fator de Crescimento Neural/biossíntese , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas S100/metabolismo , Células de Schwann/citologia , Células de Schwann/efeitos dos fármacos , Células de Schwann/ultraestruturaRESUMO
To engineer complex tissues, it is necessary to create hybrid scaffolds with micropatterned structural and biomechanical properties, which can closely mimic the intricate body tissues. The current report describes the synthesis of a novel photocrosslinkable interpenetrating polymeric network (IPN) of collagen and hyaluronic acid (HA) with precisely controlled structural and biomechanical properties. Both collagen and HA are present in crosslinked form in IPNs, and the two networks are entangled with each other. IPNs were also compared with semi-IPNs (SIPN), in which only collagen was in network form and HA chains were entangled in the collagen network without being photocrosslinked. Scanning electron microscopy images revealed that IPNs are denser than SIPNs, which results in their molecular reinforcement. This was further confirmed by rheological experiments. Because of the presence of the HA crosslinked network, the storage modulus of IPNs was almost two orders of magnitude higher than SIPNs. The degradation of the collagen-HA IPNs was slower than the SIPNs because of the presence of the crosslinked HA network. Increasing concentration of HA further altered the properties among IPNs. Cytocompatibility of IPNs was confirmed by Schwann cell and dermal fibroblasts adhesion and proliferation studies. We also fabricated patterned scaffolds with regions of IPNs and SIPNs within a bulk hydrogel, resulting in zonal distribution of crosslinking densities, viscoelasticities, water content and pore sizes at the micro- and macro-scales. With the ability to fine-tune the scaffold properties by performing structural modifications and to create patterned scaffolds, these hydrogels can be employed as potential candidates for regenerative medicine applications.
Assuntos
Materiais Biocompatíveis/química , Colágeno/química , Compostos de Epóxi/química , Fibroblastos/fisiologia , Ácido Hialurônico/química , Hidrogéis/química , Metacrilatos/química , Células de Schwann/fisiologia , Adsorção , Materiais Biocompatíveis/efeitos da radiação , Adesão Celular , Técnicas de Cultura de Células/métodos , Proliferação de Células , Células Cultivadas , Colágeno/efeitos da radiação , Matriz Extracelular/química , Humanos , Ácido Hialurônico/efeitos da radiação , Hidrogéis/efeitos da radiação , Luz , Teste de Materiais , Tamanho da Partícula , Polímeros/química , Polímeros/efeitos da radiação , Porosidade , Propriedades de Superfície , Engenharia Tecidual/métodosRESUMO
Polymer-based, injectable systems that can simultaneously deliver multiple bioactive agents in a controlled manner could significantly enhance the efficacy of next generation therapeutics. For immunotherapies to be effective, both prophylactically or therapeutically, it is not only critical to drive the antigen (Ag)-specific immune response strongly towards either T helper type 1 (Th1) or Th2 phenotype, but also to promote recruitment of a high number of antigen-presenting cells (APCs) at the site of immunization. We have recently reported a microparticle-based system capable of simultaneously delivering siRNA and DNA to APCs. Here we present an in-situ crosslinkable, injectable formulation containing dendritic cell (DC)-chemo-attractants and dual-mode DNA-siRNA loaded microparticles to attract immature DCs and simultaneously deliver, to the migrated cells, immunomodulatory siRNA and plasmid DNA antigens. These low crosslink density hydrogels were designed to degrade within 2-7 days in vitro and released chemokines in a sustained manner. Chemokine carrying gels attracted 4-6 folds more DCs over a sustained period in vitro, compared to an equivalent bolus dose. Interestingly, migrated DCs were able to infiltrate the hydrogels and efficiently phagocytose the siRNA-DNA carrying microparticles. Hydrogel embedded microparticles co-delivering Interleukin-10 siRNA and plasmid DNA antigens exhibited efficient Interleukin-10 gene knockdown in migrated primary DCs in vitro.
Assuntos
Quimiocinas/administração & dosagem , DNA/administração & dosagem , Células Dendríticas/citologia , Portadores de Fármacos/química , Hidrogéis/química , RNA Interferente Pequeno/administração & dosagem , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células Cultivadas , Quimiocinas/imunologia , Reagentes de Ligações Cruzadas , DNA/imunologia , Células Dendríticas/imunologia , Feminino , Técnicas de Silenciamento de Genes , Interleucina-10/genética , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/administração & dosagem , Plasmídeos/imunologia , RNA Interferente Pequeno/imunologiaRESUMO
The purpose of a tissue engineered (TE) scaffold is to provide a support structure that can aid the regeneration of damaged tissue. Unlike native tissues, currently existing TE scaffolds are structurally simple, with homogeneous bulk properties that are unable to induce cells to regenerate architecturally complex healthy tissue. Thus, there is a need for methods that can create structural complexity within TE scaffolds to guide tissue regeneration. In this paper we have engineered novel dual-crosslinked hyaluronic acid hydrogel scaffolds with photopatterned anisotropic swelling. Anisotropic swelling can produce zonal distributions of crosslink density, water content and viscoelasticity on the macro- and micro-scales within the hydrogel scaffold. We have found that anisotropically swelling hydrogels can be obtained by a combination of chemical crosslinks and patterned photocrosslinks within a single dual-crosslinked hydrogel. According to our method an unswollen chemically crosslinked hydrogel substrate was spatially patterned with photocrosslinks that restricted swelling at selected sites. The resulting dual-crosslinked hydrogel swelled anisotropically because of differential crosslink densities between the photopatterned and non-photopatterned regions. Anisotropic swelling permitted the hydrogel to contort and evolve a shape different from that of the unswollen hydrogel. A biodegradable hydrogel with this unique swelling behavior yields a new, unexplored type of shape-changing TE scaffold.