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1.
MRS Commun ; : 1-9, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34513262

RESUMO

In vitro thrombogenicity test systems require co-cultivation of endothelial cells and platelets under blood flow-like conditions. Here, a commercially available perfusion system is explored using plasma-treated cyclic olefin copolymer (COC) as a substrate for the endothelial cell layer. COC was characterized prior to endothelialization and co-cultivation with platelets under static or flow conditions. COC exhibits a low roughness and a moderate hydrophilicity. Flow promoted endothelial cell growth and prevented platelet adherence. These findings show the suitability of COC as substrate and the importance of blood flow-like conditions for the assessment of the thrombogenic risk of drugs or cardiovascular implant materials. Supplementary Information: The online version contains supplementary material available at 10.1557/s43579-021-00072-6.

3.
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
4.
Sci Rep ; 11(1): 13455, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188099

RESUMO

Biophysical cues such as osmotic pressure modulate proliferation and growth arrest of bacteria, yeast cells and seeds. In tissues, osmotic regulation takes place through blood and lymphatic capillaries and, at a single cell level, water and osmoregulation play a critical role. However, the effect of osmotic pressure on single cell cycle dynamics remains poorly understood. Here, we investigate the effect of osmotic pressure on single cell cycle dynamics, nuclear growth, proliferation, migration and protein expression, by quantitative time-lapse imaging of single cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2). Single cell data reveals that under hyperosmotic stress, distinct cell subpopulations emerge with impaired nuclear growth, delayed or growth arrested cell cycle and reduced migration. This state is reversible for mild hyperosmotic stress, where cells return to regular cell cycle dynamics, proliferation and migration. Thus, osmotic pressure can modulate the reversible growth arrest and reactivation of human metastatic cells.

5.
Biofabrication ; 13(4)2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34111862

RESUMO

The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harshin vivoenvironments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.

6.
J Bone Miner Res ; 36(8): 1621-1635, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33905594

RESUMO

Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl- /H+ -exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).


Assuntos
Células-Tronco Pluripotentes Induzidas , Osteopetrose , Canais de Cloreto/genética , Humanos , Leucócitos Mononucleares , Mutação , Osteoclastos , Osteopetrose/genética
7.
Int J Mol Sci ; 22(2)2021 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-33478148

RESUMO

Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells.


Assuntos
Pesquisa Biomédica , Doenças Cardiovasculares/terapia , Células Endoteliais da Veia Umbilical Humana/citologia , Artérias Umbilicais/citologia , Implantes Absorvíveis , Citoesqueleto de Actina/metabolismo , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Doenças Cardiovasculares/etiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Terapia Baseada em Transplante de Células e Tecidos/tendências , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Medicina Regenerativa/métodos , Medicina Regenerativa/tendências , Engenharia Tecidual/métodos , Engenharia Tecidual/tendências , Artérias Umbilicais/metabolismo , Fator de von Willebrand/metabolismo
8.
J Mol Med (Berl) ; 98(12): 1767-1779, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33146744

RESUMO

Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level.

9.
J Transl Med ; 18(1): 437, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208161

RESUMO

BACKGROUND: Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes. METHODS: LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices. RESULTS: Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices. CONCLUSIONS: We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases.


Assuntos
Dependovirus , Terapia Genética , Animais , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Camundongos , Sorogrupo , Transdução Genética
10.
Sci Rep ; 10(1): 4181, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144280

RESUMO

Monocytes and macrophages are key players in maintaining immune homeostasis. Identifying strategies to manipulate their functions via gene delivery is thus of great interest for immunological research and biomedical applications. We set out to establish conditions for mRNA transfection in hard-to-transfect primary human monocytes and monocyte-derived macrophages due to the great potential of gene expression from in vitro transcribed mRNA for modulating cell phenotypes. mRNA doses, nucleotide modifications, and different carriers were systematically explored in order to optimize high mRNA transfer rates while minimizing cell stress and immune activation. We selected three commercially available mRNA transfection reagents including liposome and polymer-based formulations, covering different application spectra. Our results demonstrate that liposomal reagents can particularly combine high gene transfer rates with only moderate immune cell activation. For the latter, use of specific nucleotide modifications proved essential. In addition to improving efficacy of gene transfer, our findings address discrete aspects of innate immune activation using cytokine and surface marker expression, as well as cell viability as key readouts to judge overall transfection efficiency. The impact of this study goes beyond optimizing transfection conditions for immune cells, by providing a framework for assessing new gene carrier systems for monocyte and macrophage, tailored to specific applications.


Assuntos
Macrófagos/metabolismo , Monócitos/metabolismo , RNA Mensageiro/metabolismo , Células Cultivadas , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Leucócitos Mononucleares/metabolismo , Microscopia de Fluorescência , Nucleotídeos/metabolismo , RNA Mensageiro/genética , Transfecção
11.
Sci Rep ; 10(1): 1664, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32015441

RESUMO

The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step.


Assuntos
Anticorpos Monoclonais/biossíntese , Células Produtoras de Anticorpos/imunologia , Hibridomas/imunologia , Animais , Especificidade de Anticorpos , Antígenos/genética , Antígenos/imunologia , Biotinilação , Fusão Celular , Linhagem Celular Tumoral , Epitopos/genética , Epitopos/imunologia , Receptores ErbB/genética , Receptores ErbB/imunologia , Hemaglutininas/genética , Hemaglutininas/imunologia , Humanos , Hibridomas/citologia , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Ovalbumina/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transfecção
12.
Stem Cell Res ; 35: 101367, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30763735

RESUMO

Autosomal recessive osteopetrosis (ARO) is a genetic bone disease that can be caused by mutations in the CLCN7 gene preventing osteoclast-mediated bone resorption. We generated a human induced pluripotent stem cell (hiPSC) line, BIHi002-A, from peripheral blood mononuclear cells of an ARO patient carrying the CLCN7 mutations c.875G>A and c.1208G>A using Sendai viral vectors. The pluripotent identity of the BIHi002-A line was confirmed by their expression of typical markers for undifferentiated hiPSCs, their capacity to differentiate into cells of the three germ layers and by PluriTest analysis. The BIHi002-A line provides a tool for disease modelling and therapy development.


Assuntos
Linhagem Celular , Canais de Cloreto , Células-Tronco Pluripotentes Induzidas , Leucócitos Mononucleares , Mutação , Osteopetrose , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Lactente , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Osteopetrose/genética , Osteopetrose/metabolismo , Osteopetrose/patologia
13.
FASEB J ; 33(1): 49-70, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30188756

RESUMO

To date, no viable therapeutic options exist for the effective and sustained reversal of cardiac failure, other than heart transplantation and mechanical circulatory assist devices. Therefore, divergent strategies aiming at the de novo formation of contractile tissue, as a prerequisite for the restoration of cardiac pump function, are currently being pursued. Clinical trials involving the transplantation of somatic progenitor cells failed. The search for alternative cell-based strategies to combat the consequences of ischemic injury has sparked widespread interest in the genetic and pharmacologic reprogramming of fibroblasts into cardiomyocytes, harnessing the abundant in vivo pool of cardiac fibroblasts. Here, we provide a comprehensive overview of in vitro and in vivo cardiac reprogramming studies identified in an extensive literature search. We systematically review and evaluate feasibility, efficiency, and reproducibility of the different technologies currently being explored. Finally, we discuss potential safety issues deduced from preclinical studies and identify obstacles that must be overcome before clinical translation.-Klose, K., Gossen, M., Stamm, C. Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies.


Assuntos
Transdiferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos/métodos , Reprogramação Celular , Fibroblastos/citologia , Insuficiência Cardíaca/terapia , Miócitos Cardíacos/citologia , Regeneração , Animais , Humanos
14.
Sci Transl Med ; 10(452)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-30068569

RESUMO

Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV+ patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3+ T, CD8+ T, CD4+ T, and FOXP3+ regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening.


Assuntos
Teste em Amostras de Sangue Seco , Epigênese Genética , Testes Imunológicos/métodos , Contagem de Células , Estudos de Coortes , Metilação de DNA/genética , Loci Gênicos , Infecções por HIV/diagnóstico , Infecções por HIV/imunologia , Humanos , Recém-Nascido , Triagem Neonatal , Sulfitos , Subpopulações de Linfócitos T/metabolismo
15.
Thorac Cardiovasc Surg ; 66(1): 53-62, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29216651

RESUMO

For more than 20 years, tremendous efforts have been made to develop cell-based therapies for treatment of heart failure. However, the results of clinical trials using somatic, nonpluripotent stem or progenitor cells have been largely disappointing in both cardiology and cardiac surgery scenarios. Surgical groups were among the pioneers of experimental and clinical myocyte transplantation ("cellular cardiomyoplasty"), but little translational progress was made prior to the development of cellular reprogramming for creation of induced pluripotent stem cells (iPSC). Ever since, protocols have been developed which allow for the derivation of large numbers of autologous cardiomyocytes (CMs) from patient-specific iPSC, moving translational research closer toward clinical pilot trials. However, compared with somatic cell therapy, the technology required for safe and efficacious pluripotent stem cell (PSC)-based therapies is extremely complex and requires tremendous resources and close interactions between basic scientists and clinicians. This review summarizes PSC sources, strategies to derive CMs, current cardiac tissue engineering approaches, concerns regarding immunogenicity and cellular maturity, and highlights the contributions made by surgical groups.


Assuntos
Doenças Cardiovasculares/cirurgia , Células-Tronco Embrionárias/transplante , Miocárdio/patologia , Miócitos Cardíacos/transplante , Células-Tronco Pluripotentes/transplante , Regeneração , Medicina Regenerativa/métodos , Animais , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Doenças Cardiovasculares/fisiopatologia , Linhagem da Célula , Reprogramação Celular , Técnicas de Reprogramação Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/transplante , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Fenótipo , Células-Tronco Pluripotentes/metabolismo , Recuperação de Função Fisiológica , Transdução de Sinais , Resultado do Tratamento
16.
Sci Rep ; 7(1): 17771, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29259215

RESUMO

Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use.


Assuntos
Proteína 9 Associada à CRISPR/genética , Cromossomos/genética , DNA Nucleotidiltransferases/genética , Mutagênese Insercional/genética , Recombinação Genética/genética , Animais , Células CHO , Sistemas CRISPR-Cas/genética , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Cricetulus , Células HEK293 , Humanos , Transgenes/genética
17.
Clin Hemorheol Microcirc ; 67(3-4): 267-278, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28869459

RESUMO

Controlling mesenchymal stem cells (MSCs) behavior is necessary to fully exploit their therapeutic potential. Various approaches are employed to effectively influence the migration capacity of MSCs. Here, topographic microstructures with different microscale roughness were created on polystyrene (PS) culture vessel surfaces as a feasible physical preconditioning strategy to modulate MSC migration. By analyzing trajectories of cells migrating after reseeding, we demonstrated that the mobilization velocity of human adipose derived mesenchymal stem cells (hADSCs) could be promoted by and persisted after brief preconditioning with the appropriate microtopography. Moreover, the elevated activation levels of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) in hADSCs were also observed during and after the preconditioning process. These findings underline the potential enhancement of in vivo therapeutic efficacy in regenerative medicine via transplantation of topographic microstructure preconditioned stem cells.


Assuntos
Células-Tronco Mesenquimais/metabolismo , Animais , Humanos , Imuno-Histoquímica , Células-Tronco Mesenquimais/citologia , Ratos
18.
Biomacromolecules ; 18(11): 3819-3833, 2017 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-28954190

RESUMO

The rational design of a polyplex gene carrier aims to balance maximal effectiveness of nucleic acid transfection into cells with minimal adverse effects. Depsipeptide blocks with an Mn ∼ 5 kDa exhibiting strong physical interactions were conjugated with PEI moieties (2.5 or 10 kDa) to di- and triblock copolymers. Upon nanoparticle formation and complexation with DNA, the resulting polyplexes (sizes typically 60-150 nm) showed remarkable stability compared to PEI-only or lipoplex and facilitated efficient gene delivery. Intracellular trafficking was visualized by observing fluorescence-labeled pDNA and highlighted the effective cytoplasmic uptake of polyplexes and release of DNA to the perinuclear space. Specifically, a triblock copolymer with a middle depsipeptide block and two 10 kDa PEI swallowtail structures mediated the highest levels of transgenic VEGF secretion in mesenchymal stem cells with low cytotoxicity. These nanocarriers form the basis for a delivery platform technology, especially for gene transfer to primary human cells.


Assuntos
DNA/genética , Depsipeptídeos/química , Técnicas de Transferência de Genes , Nanopartículas/química , Sobrevivência Celular/genética , DNA/química , Depsipeptídeos/genética , Humanos , Plasmídeos/química , Plasmídeos/genética , Polietilenoglicóis/química , Polietilenoimina/química , Cultura Primária de Células , Transfecção/métodos
19.
Cell Signal ; 29: 23-30, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27713089

RESUMO

Patients with Fibrodysplasia Ossificans Progressiva (FOP) suffer from ectopic bone formation, which progresses during life and results in dramatic movement restrictions. Cause of the disease are point mutations in the Activin A receptor type 1 (ACVR1), with p.R206H being most common. In this study we compared the signalling responses of ACVR1WT and ACVR1R206H to different ligands. ACVR1WT, but not ACVR1R206H inhibited BMP signalling of BMP2 or BMP4 in a ligand binding domain independent manner. Likewise, the basal BMP signalling activity of the receptor BMPR1A or BMPR1B was inhibited by ACVR1WT, but enhanced by ACVR1R206H. In comparison, BMP6 or BMP7 activated ACVR1WT and caused a hyper-activation of ACVR1R206H. These effects were dependent on an intact ligand binding domain. Finally, the neofunction of Activin A in FOP was tested and found to depend on the ligand binding domain for activating ACVR1R206H. We conclude that the FOP mutation ACVR1R206H is more sensitive to a number of natural ligands. The mutant receptor apparently lost some essential inhibitory interactions with its ligands and co-receptors, thereby conferring an enhanced ligand-dependent signalling and stimulating ectopic bone formation as observed in the patients.


Assuntos
Receptores de Ativinas Tipo I/genética , Mutação/genética , Miosite Ossificante/genética , Ativinas/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Células NIH 3T3 , Osteogênese/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo
20.
J Mater Chem B ; 5(35): 7415-7425, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-32264191

RESUMO

Fine tuning of the substrate properties to modulate the function of mesenchymal stem cells (MSCs) has emerged as an attractive strategy to optimize their therapeutic potential. In the context of the mechanotransduction process, the conformational change of integrin (integrin activation) plays a critical role in perceiving and transmitting various signals. In this study, polymeric cell culture inserts with defined bottom roughness were fabricated as a model system for cell cultivation. We showed that the conformational change of integrin and its downstream signaling cascade of human adipose-derived mesenchymal stem cells (hADSCs) could be modulated by the curvature of the cell-material interface. The curvature of the substrate surface with a roughness in the size range of a single cell could strongly increase the high-affinity ß1 integrin level of hADSCs without alteration of the total ß1 integrin level. Further, the integrin downstream FAK/ERK and Rho/ROCK pathways were activated and resulted in upregulated VEGF secretion of hADSCs. A conditioned medium on such a surface exhibited a strong pro-angiogenic effect, with an increased formation of the tubular structure, a higher migration velocity of endothelial cells and an enhanced blood vessel density in an ex vivo hen's egg test-chorioallantoic membrane (HET-CAM). These results highlighted the clinical potential to manipulate the topographic features of the cell culture substrate, whereby to regulate integrin affinity states and further control MSC functions.

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