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1.
ACS Biomater Sci Eng ; 7(4): 1344-1360, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33750112

RESUMO

Human pluripotent stem cells harbor an unlimited capacity to generate therapeutically relevant cells for applications in regenerative medicine. However, to utilize these cells in the clinic, scalable culture systems that activate defined receptors and signaling pathways to sustain stem cell self-renewal are required; and synthetic materials offer considerable promise to meet these needs. De novo development of materials that target novel pathways has been stymied by a limited understanding of critical receptor interactions maintaining pluripotency. Here, we identify peptide agonists for the human pluripotent stem cell (hPSC) laminin receptor and pluripotency regulator, α6-integrin, through unbiased, library-based panning strategies. Biophysical characterization of adhesion suggests that identified peptides bind hPSCs through α6-integrin with sub-µM dissociation constants similar to laminin. By harnessing a high-throughput microculture platform, we developed predictive guidelines for presenting these integrin-targeting peptides alongside canonical binding motifs at optimal stoichiometries to generate nascent culture surfaces. Finally, when presented as self-assembled monolayers, predicted peptide combinations supported hPSC expansion, highlighting how unbiased screens can accelerate the discovery of targeted biomaterials.


Assuntos
Células-Tronco Pluripotentes , Proliferação de Células , Autorrenovação Celular , Humanos , Laminina , Peptídeos
2.
Antibodies (Basel) ; 9(4)2020 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-33172091

RESUMO

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.

3.
J Biomed Mater Res A ; 108(8): 1736-1746, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32270584

RESUMO

The decrease in contractility in myocardium adjacent (border zone; BZ) to a myocardial infarction (MI) is correlated with an increase in reactive oxygen species (ROS). We hypothesized that injection of a thermoresponsive hydrogel, with ROS scavenging properties, into the MI would decrease ROS and improve BZ function. Fourteen sheep underwent antero-apical MI. Seven sheep had a comb-like copolymer synthesized from N-isopropyl acrylamide (NIPAAm) and 1500 MW methoxy poly(ethylene glycol) methacrylate, (NIPAAm-PEG1500), injected (20 × 0.5 mL) into the MI zone 40 min after MI (MI + NIPAAm-PEG1500) and seven sheep were MI controls. Cardiac MRI was performed 2 weeks before and 6 weeks after MI + NIPAAm-PEG1500. BZ wall thickness at end systole was significantly higher for MI + NIPAAm-PEG1500 (12.32 ± 0.51 mm/m2 MI + NIPAAm-PEG1500 vs. 9.88 ± 0.30 MI; p = .023). Demembranated muscle force development for BZ myocardium 6 weeks after MI was significantly higher for MI + NIPAAm-PEG1500 (67.67 ± 2.61 mN/m2 MI + NIPAAm-PEG1500 vs. 40.53 ± 1.04 MI; p < .0001) but not significantly different from remote myocardium or BZ or non-operated controls. Levels of ROS in BZ tissue were significantly lower in the MI + NIPAAm-PEG1500 treatment group (hydroxyl p = .0031; superoxide p = .0182). We conclude that infarct injection of the NIPAAm-PEG1500 hydrogel with ROS scavenging properties decreased ROS and improved contractile protein function in the border zone 6 weeks after MI.


Assuntos
Sequestradores de Radicais Livres/farmacologia , Hidrogéis/farmacologia , Contração Miocárdica/efeitos dos fármacos , Acrilamidas/administração & dosagem , Acrilamidas/farmacologia , Animais , Sequestradores de Radicais Livres/administração & dosagem , Hidrogéis/administração & dosagem , Injeções , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ovinos
4.
Adv Healthc Mater ; 7(12): e1800225, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29717823

RESUMO

Human pluripotent stem cells (hPSCs) offer considerable potential for biomedical applications including drug screening and cell replacement therapies. Clinical translation of hPSCs requires large quantities of high quality cells, so scalable methods for cell culture are needed. However, current methods are limited by scalability, the use of animal-derived components, and/or low expansion rates. A thermoresponsive 3D hydrogel for scalable hPSC expansion and differentiation into several defined lineages is recently reported. This system would benefit from increased control over material properties to further tune hPSC behavior, and here a scalable 3D biomaterial with the capacity to tune both the chemical and the mechanical properties is demonstrated to promote hPSC expansion under defined conditions. This 3D biomaterial, comprised of hyaluronic acid and poly(N-isopropolyacrylamide), has thermoresponsive properties that readily enable mixing with cells at low temperatures, physical encapsulation within the hydrogel upon elevation at 37 °C, and cell recovery upon cooling and reliquefaction. After optimization, the resulting biomaterial supports hPSC expansion over long cell culture periods while maintaining cell pluripotency. The capacity to modulate the mechanical and chemical properties of the hydrogel provides a new avenue to expand hPSCs for future therapeutic application.


Assuntos
Resinas Acrílicas , Técnicas de Cultura de Células/métodos , Ácido Hialurônico , Hidrogéis , Células-Tronco Pluripotentes/metabolismo , Resinas Acrílicas/química , Resinas Acrílicas/farmacologia , Linhagem Celular , Temperatura Alta , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Hidrogéis/química , Hidrogéis/farmacologia , Células-Tronco Pluripotentes/citologia
5.
Drug Deliv Transl Res ; 5(2): 160-7, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25787741

RESUMO

Gene delivery provides a powerful tool for regulating tissue regeneration by activating or inhibiting specific genes associated with targeted signaling pathways. Up-regulating bone morphogenetic protein-2 (BMP-2) or silencing GNAS and Noggin gene expression in stem cells has been shown to enhance osteogenic differentiation and bone tissue formation. However, few studies have examined how such gene delivery would influence other differentiated cell types residing in the bone. In this study, we examined the effects of DNA delivery of BMP-2 and siRNA delivery of GNAS or Noggin on a widely used human fetal osteoblast cell line (hFOB1.19) using biomaterials-mediated gene delivery. Our results showed that both GNAS and Noggin siRNA delivery increased cell death in hFOB1.19 in a dose-dependent manner. In particular, groups treated with the highest doses of BMP-2, siGNAS or siNoggin showed a more than 50% decline in cell proliferation and a 90% decline in cell viability compared to untransfected and sham DNA/siRNA-transfected controls. TUNEL staining showed that BMP-2, siGNAS or siNoggin induced cell apoptosis in hFOBs. In contrast, cells transfected using sham DNA or siRNA showed no noticeable cell death or apoptosis. These results elucidate the nuanced responses of progenitor and immortalized cell populations to the delivery of exogenous osteoinductive genes. In particular, they highlight the differences between immortalized and primary cell lines and underscore the importance of targeted gene delivery mechanisms in the regeneration of injured bone tissue.


Assuntos
Proteína Morfogenética Óssea 2/genética , Proteínas de Transporte/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Técnicas de Transferência de Genes , Morte Celular , Linhagem Celular , Cromograninas , DNA/genética , Feto , Humanos , Osteoblastos/metabolismo , RNA Interferente Pequeno/genética
6.
ACS Synth Biol ; 3(12): 929-31, 2014 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-25409531

RESUMO

In microbial communities, bacterial populations are commonly controlled using indiscriminate, broad range antibiotics. There are few ways to target specific strains effectively without disrupting the entire microbiome and local environment. Here, we use conjugation, a natural DNA horizontal transfer process among bacterial species, to deliver an engineered CRISPR interference (CRISPRi) system for targeting specific genes in recipient Escherichia coli cells. We show that delivery of the CRISPRi system is successful and can specifically repress a reporter gene in recipient cells, thereby establishing a new tool for gene regulation across bacterial cells and potentially for bacterial population control.


Assuntos
Sistemas CRISPR-Cas/genética , Conjugação Genética/genética , Engenharia Genética/métodos , Interferência de RNA , Escherichia coli/genética , Transferência Genética Horizontal , Modelos Genéticos , Biologia Sintética
7.
Pharm Res ; 28(6): 1328-37, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21424160

RESUMO

PURPOSE: To assess the effects of co-delivering osteoinductive DNA and/or small interfering RNA in directing the osteogenic differentiation of human adipose-derived stem cells (hADSCs) using a combinatorial, non-viral gene delivery approach. METHODS: hADSCs were transfected using combinations of the following genes: BMP2, siGNAS and siNoggin using poly(ß-amino esters) or lipid-like molecules. A total of 15 groups were evaluated by varying DNA doses, timing of treatment, and combinations of signals. All groups were cultured in osteogenic medium for up to 37 days, and outcomes were measured using gene expression, biochemical assays, and histology. RESULTS: Biomaterials-mediated gene delivery led to a dose-dependent up-regulation of BMP2 and significant gene silencing of GNAS and Noggin in hADSCs. BMP2 alone slightly up-regulates osteogenic marker expression in hADSCs. In contrast, co-delivery of BMP2 and siGNAS or siNoggin significantly accelerates the hADSC differentiation towards osteogenic differentiation, with marked increase in bone marker expression and mineralization. CONCLUSIONS: We report a combinatorial platform for identifying synergistic interactions among multiple genetic signals associated with osteogenic differentiation of hADSCs. Our results suggest that inductive or suppressive genetic switches interact in a complex manner, and highlight the promise of combinatorial approaches towards rapidly identifying optimal signals for promoting desired stem cell differentiation.


Assuntos
Adipócitos/citologia , Técnicas de Transferência de Genes , Osteogênese/genética , Células-Tronco/citologia , Materiais Biocompatíveis/administração & dosagem , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Expressão Gênica , Marcadores Genéticos/genética , Humanos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Regulação para Cima
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