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1.
Nat Commun ; 11(1): 4043, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792475

RESUMO

Genetically fusing protein domains to Cas9 has yielded several transformative technologies; however, the genetic modifications are limited to natural polypeptide chains at the Cas9 termini, which excludes a diverse array of molecules useful for gene editing. Here, we report chemical modifications that allow site-specific and multiple-site conjugation of a wide assortment of molecules on both the termini and internal sites of Cas9, creating a platform for endowing Cas9 with diverse functions. Using this platform, Cas9 can be modified to more precisely incorporate exogenously supplied single-stranded oligonucleotide donor (ssODN) at the DNA break site. We demonstrate that the multiple-site conjugation of ssODN to Cas9 significantly increases the efficiency of precision genome editing, and such a platform is compatible with ssODNs of diverse lengths. By leveraging the conjugation platform, we successfully engineer INS-1E, a ß-cell line, to repurpose the insulin secretion machinery, which enables the glucose-dependent secretion of protective immunomodulatory factor interleukin-10.


Assuntos
Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/metabolismo , Engenharia Celular/métodos , Biologia Sintética/métodos , Proteína 9 Associada à CRISPR/genética , Linhagem Celular , Edição de Genes , Humanos , Sistemas de Infusão de Insulina
2.
Plast Reconstr Surg ; 146(2): 309-320, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32740581

RESUMO

BACKGROUND: Adipose-derived stem cells are considered as candidate cells for regenerative plastic surgery. Measures to influence cellular properties and thereby direct their regenerative potential remain elusive. Hyperbaric oxygen therapy-the exposure to 100% oxygen at an increased atmospheric pressure-has been propagated as a noninvasive treatment for a multitude of indications and presents a potential option to condition cells for tissue-engineering purposes. The present study evaluates the effect of hyperbaric oxygen therapy on human adipose-derived stem cells. METHODS: Human adipose-derived stem cells from healthy donors were treated with hyperbaric oxygen therapy at 2 and 3 atm. Viability before and after each hyperbaric oxygen therapy, proliferation, expression of surface markers and protein contents of transforming growth factor (TGF)-ß, tumor necrosis factor-α, hepatocyte growth factor, and epithelial growth factor in the supernatants of treated adipose-derived stem cells were measured. Lastly, adipogenic, osteogenic, and chondrogenic differentiation with and without use of differentiation-inducing media (i.e., autodifferentiation) was examined. RESULTS: Hyperbaric oxygen therapy with 3 atm increased viability, proliferation, and CD34 expression and reduced the CD31/CD34/CD45 adipose-derived stem cell subset and endothelial progenitor cell population. TGF-ß levels were significantly decreased after two hyperbaric oxygen therapy sessions in the 2-atm group and decreased after three hyperbaric oxygen therapy sessions in the 3-atm group. Hepatocyte growth factor secretion remained unaltered in all groups. Although the osteogenic and chondrogenic differentiation were not influenced, adipogenic differentiation and autodifferentiation were significantly enhanced, with osteogenic autodifferentiation significantly alleviated by hyperbaric oxygen therapy with 3 atm. CONCLUSION: Hyperbaric oxygen therapy with 3 atm increases viability and proliferation of adipose-derived stem cells, alters marker expression and subpopulations, decreases TGF-ß secretion, and skews adipose-derived stem cells toward adipogenic differentiation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.


Assuntos
Adipogenia/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Engenharia Celular/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Oxigênio/administração & dosagem , Tecido Adiposo/citologia , Adulto , Biomarcadores/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Células-Tronco Mesenquimais/fisiologia , Pessoa de Meia-Idade , Pressão , Cultura Primária de Células/métodos
3.
Nat Rev Drug Discov ; 19(7): 463-479, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32612263

RESUMO

Naturally occurring stem cells isolated from humans have been used therapeutically for decades. This has primarily involved the transplantation of primary cells such as haematopoietic and mesenchymal stem cells and, more recently, derivatives of pluripotent stem cells. However, the advent of cell-engineering approaches is ushering in a new generation of stem cell-based therapies, greatly expanding their therapeutic utility. These next-generation stem cells are being used as 'Trojan horses' to improve the delivery of drugs and oncolytic viruses to intractable tumours and are also being engineered with angiogenic, neurotrophic and anti-inflammatory molecules to accelerate the repair of injured or diseased tissues. Moreover, gene therapy and gene editing technologies are being used to create stem cell derivatives with improved functionality, specificity and responsiveness compared with their natural counterparts. Here, we review these engineering approaches and areas in which they will help broaden the utility and clinical applicability of stem cells.


Assuntos
Engenharia Celular/métodos , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Animais , Sistemas de Liberação de Medicamentos , Edição de Genes , Terapia Genética/métodos , Humanos , Terapia Viral Oncolítica/métodos
4.
Nat Commun ; 11(1): 3659, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32694598

RESUMO

As synthetic biocircuits become more complex, distributing computations within multi-strain microbial consortia becomes increasingly beneficial. However, designing distributed circuits that respond predictably to variation in consortium composition remains a challenge. Here we develop a two-strain gene circuit that senses and responds to which strain is in the majority. This involves a co-repressive system in which each strain produces a signaling molecule that signals the other strain to down-regulate production of its own, orthogonal signaling molecule. This co-repressive consortium links gene expression to ratio of the strains rather than population size. Further, we control the cross-over point for majority via external induction. We elucidate the mechanisms driving these dynamics by developing a mathematical model that captures consortia response as strain fractions and external induction are varied. These results show that simple gene circuits can be used within multicellular synthetic systems to sense and respond to the state of the population.


Assuntos
Engenharia Celular/métodos , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Consórcios Microbianos/genética , Percepção de Quorum/genética , Redes Reguladoras de Genes , Transdução de Sinais/genética , Biologia Sintética/métodos
5.
Nat Commun ; 11(1): 3778, 2020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32728076

RESUMO

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.


Assuntos
Engenharia Celular/métodos , Edição de Genes , Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Hemofilia A/terapia , Humanos , Doenças Metabólicas/terapia , Camundongos , Regiões Promotoras Genéticas/genética , Transplante Autólogo/métodos , Transplante Heterólogo , alfa-Globinas/genética , alfa-Globinas/metabolismo
6.
Nat Commun ; 11(1): 2708, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32488070

RESUMO

Although many animals have evolved intrinsic transparency for the purpose of concealment, the development of dynamic, that is, controllable and reversible, transparency for living human cells and tissues has remained elusive to date. Here, by drawing inspiration from the structures and functionalities of adaptive cephalopod skin cells, we design and engineer human cells that contain reconfigurable protein-based photonic architectures and, as a result, possess tunable transparency-changing and light-scattering capabilities. Our findings may lead to the development of unique biophotonic tools for applications in materials science and bioengineering and may also facilitate an improved understanding of a wide range of biological systems.


Assuntos
Engenharia Celular/métodos , Cefalópodes , Óptica e Fotônica , Animais , Técnicas de Cultura de Células , Feminino , Engenharia Genética , Células HEK293 , Humanos , Proteínas/química , Pele , Biologia Sintética/métodos
7.
Proc Natl Acad Sci U S A ; 117(22): 12041-12049, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32424098

RESUMO

Split inteins are privileged molecular scaffolds for the chemical modification of proteins. Though efficient for in vitro applications, these polypeptide ligases have not been utilized for the semisynthesis of proteins in live cells. Here, we biochemically and structurally characterize the naturally split intein VidaL. We show that this split intein, which features the shortest known N-terminal fragment, supports rapid and efficient protein trans-splicing under a range of conditions, enabling semisynthesis of modified proteins both in vitro and in mammalian cells. The utility of this protein engineering system is illustrated through the traceless assembly of multidomain proteins whose biophysical properties render them incompatible with a single expression system, as well as by the semisynthesis of dual posttranslationally modified histone proteins in live cells. We also exploit the domain swapping function of VidaL to effect simultaneous modification and translocation of the nuclear protein HP1α in live cells. Collectively, our studies highlight the VidaL system as a tool for the precise chemical modification of cellular proteins with spatial and temporal control.


Assuntos
Inteínas/fisiologia , Biossíntese de Proteínas/fisiologia , Engenharia de Proteínas/métodos , Processamento de Proteína/fisiologia , Engenharia Celular/métodos
8.
PLoS One ; 15(4): e0231664, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32302338

RESUMO

Natural killer (NK) cells are innate lymphocytes with functions that include target cell killing, inflammation and regulation. NK cells integrate incoming activating and inhibitory signals through an array of germline-encoded receptors to gauge the health of neighbouring cells. The reactive potential of NK cells is influenced by microRNA (miRNA), small non-coding sequences that interfere with mRNA expression. miRNAs are highly conserved between species, and a single miRNA can have hundreds to thousands of targets and influence entire cellular programs. Two miRNA species, miR-155-5p and miR-146a-5p are known to be important in controlling NK cell function, but research to best understand the impacts of miRNA species within NK cells has been bottlenecked by a lack of techniques for altering miRNA concentrations efficiently and without off-target effects. Here, we describe a non-viral and straightforward approach for increasing or decreasing expression of miRNA in primary human NK cells. We achieve >90% transfection efficiency without off-target impacts on NK cell viability, education, phenotype or function. This opens the opportunity to study and manipulate NK cell miRNA profiles and their impacts on NK cellular programs which may influence outcomes of cancer, inflammation and autoimmunity.


Assuntos
Engenharia Celular/métodos , Células Matadoras Naturais/metabolismo , MicroRNAs/genética , Transfecção/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Meios de Cultura Livres de Soro/farmacologia , Voluntários Saudáveis , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/transplante , MicroRNAs/agonistas , MicroRNAs/antagonistas & inibidores , Cultura Primária de Células
10.
Nat Commun ; 11(1): 1193, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132536

RESUMO

The last decade has seen bacteria at the forefront of biotechnological innovation, with applications including biomolecular computing, living therapeutics, microbiome engineering and microbial factories. These emerging applications are all united by the need to precisely control complex microbial dynamics in spatially extended environments, requiring tools that can bridge the gap between intracellular and population-level coordination. To address this need, we engineer an inducible quorum sensing system which enables precise tunability of bacterial dynamics both at the population and community level. As a proof-of-principle, we demonstrate the advantages of this system when genetically equipped for cargo delivery. In addition, we exploit the absence of cross-talk with respect to the majority of well-characterized quorum sensing systems to demonstrate inducibility of multi-strain communities. More broadly, this work highlights the unexplored potential of remotely inducible quorum sensing systems which, coupled to any gene of interest, may facilitate the translation of circuit designs into applications.


Assuntos
Engenharia Celular/métodos , Escherichia coli/fisiologia , Microbiota/fisiologia , Percepção de Quorum/genética , Estudo de Prova de Conceito , Biologia Sintética/métodos
11.
J Biotechnol ; 312: 11-22, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32114154

RESUMO

An increasing number of engineered therapeutic recombinant proteins with unpredictable manufacturability are currently filling industrial cell line development pipelines. These proteins can be "difficult-to-express" (DTE) in that production of a sufficient quantity of correctly processed recombinant product by engineered mammalian cells is difficult to achieve. In these circumstances, identification of appropriate cell engineering strategies to increase yield is difficult as constraints are cell line and product-specific. Here we describe and validate the development of a high-throughput microscale platform for multiparallel testing of multiple functional genetic components at varying stoichiometry followed by assessment of their effect on cell functional performance. The platform was used to compare and identify optimal cell engineering solutions for both transient and stable production of a model DTE IgG1 monoclonal antibody. We simultaneously tested the functional effect of 32 genes encoding discrete ER or secretory pathway components, each at varying levels of expression and utilized in different combinations. We show that optimization of functional gene load and relative stoichiometry is critical and optimal cell engineering solutions for stable and transient production contexts are significantly different. Our analysis indicates that cell engineering workflows should be cell line, protein product and production-process specific; and that next-generation cell engineering technology that enables precise control of the relative expression of multiple functional genetic components is necessary to achieve this.


Assuntos
Células CHO , Engenharia Celular/métodos , Engenharia Genética/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/genética , Células CHO/metabolismo , Técnicas de Cultura de Células , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Ensaios de Triagem em Larga Escala , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Via Secretória/genética , Via Secretória/fisiologia
12.
PLoS One ; 15(2): e0228112, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040512

RESUMO

Neoantigens can be predicted and in some cases identified using the data obtained from the whole exome sequencing and transcriptome sequencing of tumor cells. These sequencing data can be coupled with single-cell RNA sequencing for the direct interrogation of the transcriptome, surfaceome, and pairing of αß T-cell receptors (TCRαß) from hundreds of single T cells. Using these 2 large datasets, we established a platform for identifying antigens recognized by TCRαßs obtained from single T cells. Our approach is based on the rapid expression of cloned TCRαß genes as Sleeping Beauty transposons and the determination of the introduced TCRαßs' antigen specificity and avidity using a reporter cell line. The platform enables the very rapid identification of tumor-reactive TCRs for the bioengineering of T cells with redirected specificity.


Assuntos
Engenharia Celular/métodos , Clonagem Molecular/métodos , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Expressão Gênica , Biblioteca Gênica , Genes MHC Classe I/genética , Genes MHC da Classe II/genética , Células HEK293 , Humanos , Cinética , Receptores de Antígenos de Linfócitos T alfa-beta/genética
13.
Nat Biotechnol ; 38(4): 460-470, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32094658

RESUMO

Generation of pancreatic ß cells from human pluripotent stem cells (hPSCs) holds promise as a cell replacement therapy for diabetes. In this study, we establish a link between the state of the actin cytoskeleton and the expression of pancreatic transcription factors that drive pancreatic lineage specification. Bulk and single-cell RNA sequencing demonstrated that different degrees of actin polymerization biased cells toward various endodermal lineages and that conditions favoring a polymerized cytoskeleton strongly inhibited neurogenin 3-induced endocrine differentiation. Using latrunculin A to depolymerize the cytoskeleton during endocrine induction, we developed a two-dimensional differentiation protocol for generating human pluripotent stem-cell-derived ß (SC-ß) cells with improved in vitro and in vivo function. SC-ß cells differentiated from four hPSC lines exhibited first- and second-phase dynamic glucose-stimulated insulin secretion. Transplantation of islet-sized aggregates of these cells rapidly reversed severe preexisting diabetes in mice at a rate close to that of human islets and maintained normoglycemia for at least 9 months.


Assuntos
Engenharia Celular/métodos , Citoesqueleto/metabolismo , Células Secretoras de Insulina/citologia , Células-Tronco Pluripotentes/citologia , Actinas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Diabetes Mellitus/terapia , Endoderma/citologia , Endoderma/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/transplante , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco Pluripotentes/metabolismo , Tiazolidinas/farmacologia , Transativadores/metabolismo , Moduladores de Tubulina/farmacologia
14.
Nat Commun ; 11(1): 304, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949141

RESUMO

Biological processes in development and disease are controlled by the abundance, localization and modification of cellular proteins. We have developed versatile tools based on recombinant E3 ubiquitin ligases that are controlled by light or drug induced heterodimerization for nanobody or DARPin targeted depletion of endogenous proteins in cells and organisms. We use this rapid, tunable and reversible protein depletion for functional studies of essential proteins like PCNA in DNA repair and to investigate the role of CED-3 in apoptosis during Caenorhabditis elegans development. These independent tools can be combined for spatial and temporal depletion of different sets of proteins, can help to distinguish immediate cellular responses from long-term adaptation effects and can facilitate the exploration of complex networks.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Técnicas Citológicas , Luz , Ubiquitina-Proteína Ligases/efeitos dos fármacos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/efeitos da radiação , Animais , Apoptose , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/efeitos da radiação , Caspases/efeitos dos fármacos , Caspases/metabolismo , Caspases/efeitos da radiação , Engenharia Celular/métodos , Dano ao DNA , DNA Ligase Dependente de ATP , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Lamina Tipo A/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética
15.
Nat Biotechnol ; 38(2): 233-244, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31907405

RESUMO

Despite the global rapid increase in the number of clinical trials employing chimeric antigen receptors (CARs), no comprehensive survey of their scope, targets and design exists. In this study, we present an interactive CAR clinical trial database, spanning 64 targets deployed in T cells (CAR-T), natural killer cells (CAR-NK) or mixtures (CAR-NK/T) from over 500 clinical trials in 20 countries, encompassing >20,000 patients. By combining these data with transcriptional and proteomic data, we create a 'targetable landscape' for CAR cell therapies based on 13,206 proteins and RNAs across 78 tissues, 124 cell types and 20 cancer types. These data suggest a landscape of over 100 single targets and over 100,000 target pairs using logical switches for CAR cell engineering. Our analysis of the CAR cellular therapeutic landscape may aid the design of future therapies, improve target-to-patient matching, and ultimately help inform our understanding of CAR therapy's safety and efficacy.


Assuntos
Engenharia Celular/métodos , Receptores de Antígenos Quiméricos/metabolismo , Terapia Baseada em Transplante de Células e Tecidos , Ensaios Clínicos como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Neoplasias/metabolismo
16.
ACS Appl Mater Interfaces ; 12(3): 4163-4173, 2020 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-31891476

RESUMO

Engineering of cell surfaces holds promise in manipulating cellular activities in a physicochemical route as a complement to the biological approach. Mediated by Ca2+, a quick and convenient yet cytocompatible method is used to achieve surface engineering, by which polydopamine nanostructures can be in situ grown onto dendritic cell (DC) surfaces within 10 min. Ca2+, as the physical bridge between the negative cell surface and polydopamine, avoids the direct chemical polymerization of polydopamine onto the cell surface, critically important to maintain the cell viability. As a proof of concept in potential applications, this cell surface engineering shows a good control toward DC maturation. Upon surface polydopamine engineering, bone-marrow-derived DC exhibits a unique bidirectional control of maturation. The polydopamine structure enables effective suppression of DC activation by acting as an efficient scavenger of reactive oxygen species, a key signal during maturation. Conversely, an 808 nm laser irradiation can remotely relieve the suppressed state and effectively activate DC maturation by the photoheat effect of polydopamine (39 °C). The work provides an easily implemented, straightforward approach to achieve cell surface engineering, through which the DC maturation can be controlled.


Assuntos
Cálcio/metabolismo , Engenharia Celular/métodos , Células Dendríticas/citologia , Indóis/química , Polímeros/química , Animais , Diferenciação Celular , Engenharia Celular/instrumentação , Sobrevivência Celular , Dendritos/metabolismo , Células Dendríticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Polimerização , Espécies Reativas de Oxigênio/metabolismo
17.
Arch Physiol Biochem ; 126(2): 139-156, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-30445857

RESUMO

Anchorage of a subset of cell surface proteins in eukaryotic cells is mediated by a glycosylphosphatidylinositol (GPI) moiety covalently attached to the carboxy-terminus of the protein moiety. Experimental evidence for the potential of GPI-anchored proteins (GPI-AP) of being released from cells into the extracellular environment has been accumulating, which involves either the loss or retention of the GPI anchor. Release of GPI-AP from donor cells may occur spontaneously or in response to endogenous or environmental signals. The experimental evidence for direct insertion of exogenous GPI-AP equipped with the complete anchor structure into the outer plasma membrane bilayer leaflets of acceptor cells is reviewed as well as the potential underlying molecular mechanisms. Furthermore, promiscuous transfer of certain GPI-AP between plasma membranes of different cells in vivo under certain (patho)physiological conditions has been reported. Engineering of target cell surfaces using chimeric GPI-AP with complete GPI anchor may be useful for therapeutic applications.


Assuntos
Engenharia Celular/métodos , Glicosilfosfatidilinositóis/metabolismo , Proteínas Ligadas a Lipídeos/uso terapêutico , Doenças Metabólicas/terapia , Neoplasias/terapia , Doenças Priônicas/terapia , Transtornos da Coagulação Sanguínea/terapia , Membrana Celular/química , Membrana Celular/metabolismo , Células Eucarióticas/citologia , Células Eucarióticas/metabolismo , Glicosilfosfatidilinositóis/química , Hemoglobinúria Paroxística/terapia , Humanos , Imunoterapia/métodos , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/metabolismo , Transporte Proteico , Técnicas de Reprodução Assistida , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia
18.
ACS Appl Mater Interfaces ; 11(51): 47720-47729, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31793283

RESUMO

Regulating cell behavior and cell fate are of great significance for basic biological research and cell therapy. Carbohydrates, as the key biomacromolecules, play a crucial role in regulating cell behavior. Herein, "modular" glycopolymers were synthesized by reversible addition-fragmentation chain transfer polymerization. These glycopolymers contain sugar units (glucose), anchoring units (cholesterol), "guest" units (adamantane) for host-guest interaction, and fluorescent labeling units (fluorescein). It was demonstrated that these glycopolymers can insert into cell membranes with high efficiency and their residence time on the membranes can be regulated by controlling their cholesterol content. Furthermore, the behavior of the engineered cells can be controlled by modifying with different functional ß-cyclodextrins (CD-X) via host-guest interactions with the adamantane units. Host-guest interactions with the modular polymers were demonstrated using CD-RBITC (X = a rhodamine B isothiocyanate). The glycopolymers were modified with CD-S (X = seven sulfonate groups) and CD-M (X = seven mannose groups) and were then attached, respectively, to the surfaces of mouse embryonic stem cells for the promotion of neural differentiation and to the surfaces of cancer cells for the enhancement of the immune response. The combination of multiple anchors and host-guest interactions provides a widely applicable cell membrane modification platform for a variety of applications.


Assuntos
Neurônios/citologia , Polímeros/química , Antígeno B7-1/metabolismo , Antígeno B7-2/metabolismo , Diferenciação Celular/efeitos dos fármacos , Engenharia Celular/métodos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citometria de Fluxo , Células HeLa , Humanos , Neurônios/efeitos dos fármacos , Polimerização , Polímeros/farmacologia , beta-Ciclodextrinas/química
19.
PLoS One ; 14(12): e0225222, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31790444

RESUMO

Cellular rejection of liver transplant allografts remains a concern despite immunosuppressant use. Existing transplant biomarkers are often not sensitive enough to detect acute or chronic rejection at an early enough stage to allow successful clinical intervention. We herein developed a cell-based sensor that can potentially be used for monitoring local events following liver transplantation. Utilizing a machine perfusion system as a platform to engraft the cells into a donor liver, we effectively established the biocompatibility of the biosensor cells and confirmed efficient delivery of cells distributed throughout the organ. This work proves an innovative concept of integrating synthetic reporter cells ex vivo into organs as a transplant-within-a-transplant during functional organ preservation with a vision to use cell biosensors as a broad way to monitor and treat tissue transplants.


Assuntos
Engenharia Celular/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Fibroblastos , Engenharia Genética/métodos , Transplante de Fígado/métodos , Perfusão/métodos , Transplantes , Animais , Linhagem Celular , Vetores Genéticos , Rejeição de Enxerto/prevenção & controle , Doadores Vivos , Masculino , Ratos , Ratos Endogâmicos Lew
20.
Exp Hematol ; 80: 1-10, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31765673

RESUMO

The processes generating cells of adaptive immunity render them less amenable to the single cytokine signals used so effectively to regenerate myeloid cells. T-cell neogenesis begins in the bone marrow, where specific sets of late osteolineage cells govern the specification of hematopoietic cells capable of migrating to the thymus where differentiation is completed. Osteocalcin-expressing bone marrow stromal cells producing Dll4 serve as a progenitor niche enabling this T-competent cell production. Biocompatible alginate-based cryogels containing bone morphogenetic proteins (BMP-2) and the Notch ligand Dll4 were engineered to recapitulate the endogenous niche. These cryogels are highly pliable and can be injected under the skin of animals undergoing bone marrow transplantation. The result in mice is an ectopic niche fostering T-competent progenitor generation that results in improved T-cell numbers and receptor diversity. The recipients can generate neoantigen vaccine responses while having improved tolerance manifest by reduced graft-versus-host disease upon allogeneic transplant. Through emerging details of niches in the bone marrow, therapeutics more complex than those necessary for myeloid reconstitution are possible. Niche biology-guided bioengineered design offers the possibility of regenerative therapies for T lymphoid cells.


Assuntos
Engenharia Celular/métodos , Linfopoese , Medicina Regenerativa/métodos , Nicho de Células-Tronco/fisiologia , Subpopulações de Linfócitos T/citologia , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Adulto , Fatores Etários , Alginatos , Animais , Atrofia , Austrália , Distinções e Prêmios , Medula Óssea/fisiologia , Proteína Morfogenética Óssea 2/farmacologia , Proteínas de Ligação ao Cálcio/farmacologia , Linhagem da Célula , Criogéis/farmacologia , Xenoenxertos , Humanos , Camundongos , Osteocalcina/biossíntese , Sociedades Científicas , Pesquisa com Células-Tronco , Subpopulações de Linfócitos T/transplante , Timo/patologia
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