Cellular stress promotes NOD1/2-dependent inflammation via the endogenous metabolite sphingosine-1-phosphate.

Cellular stress has been associated with inflammation, yet precise underlying mechanisms remain elusive. In this study, various unrelated stress inducers were employed to screen for sensors linking altered cellular homeostasis and inflammation. We identified the intracellular pattern recognition receptors NOD1/2, which sense bacterial peptidoglycans, as general stress sensors detecting perturbations of cellular homeostasis. NOD1/2 activation upon such perturbations required generation of the endogenous metabolite sphingosine-1-phosphate (S1P). Unlike peptidoglycan sensing via the leucine-rich repeats domain, cytosolic S1P directly bound to the nucleotide binding domains of NOD1/2, triggering NF-κB activation and inflammatory responses. In sum, we unveiled a hitherto unknown role of NOD1/2 in surveillance of cellular homeostasis through sensing of the cytosolic metabolite S1P. We propose S1P, an endogenous metabolite, as a novel NOD1/2 activator and NOD1/2 as molecular hubs integrating bacterial and metabolic cues.

14-3-3 and erlin proteins differentially interact with RIPK2 complexes.

The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for signal transduction induced by the pattern recognition receptors NOD1 and NOD2 (referred to collectively as NOD1/2). Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells. Here, we identified the molecular composition of these complexes. Infection with Shigella flexneri to activate NOD1-RIPK2 revealed that RIPK2 formed dynamic interactions with several cellular proteins, including A20 (also known as TNFAIP3), erlin-1, erlin-2 and 14-3-3. Whereas interaction of RIPK2 with 14-3-3 proteins was strongly reduced upon infection with Shigella, erlin-1 and erlin-2 (erlin-1/2) specifically bound to RIPK2 complexes. The interaction of these proteins with RIPK2 was validated using protein binding assays and immunofluorescence staining. Beside bacterial activation of NOD1/2, depletion of the E3 ubiquitin ligase XIAP and treatment with RIPK2 inhibitors also led to the formation of RIPK2 cytosolic complexes. Although erlin-1/2 were recruited to RIPK2 complexes following XIAP inhibition, these proteins did not associate with RIPK2 structures induced by RIPK2 inhibitors. While the specific recruitment of erlin-1/2 to RIPK2 suggests a role in innate immune signaling, the biological response regulated by the erlin-1/2-RIPK2 association remains to be determined.

Influence of Human Jaw Periosteal Cells Seeded ß-Tricalcium Phosphate Scaffolds on Blood Coagulation.

Tissue engineering offers auspicious opportunities in oral and maxillofacial surgery to heal bone defects. For this purpose, the combination of cells with stability-providing scaffolds is required. Jaw periosteal cells (JPCs) are well suited for regenerative therapies, as they are easily accessible and show strong osteogenic potential. In this study, we analyzed the influence of uncoated and polylactic-co-glycolic acid (PLGA)-coated ß-tricalcium phosphate (ß-TCP) scaffolds on JPC colonization and subsequent osteogenic differentiation. Furthermore, interaction with the human blood was investigated. This study demonstrated that PLGA-coated and uncoated ß-TCP scaffolds can be colonized with JPCs and further differentiated into osteogenic cells. On day 15, after cell seeding, JPCs with and without osteogenic differentiation were incubated with fresh human whole blood under dynamic conditions. The activation of coagulation, complement system, inflammation, and blood cells were analyzed using ELISA and scanning electron microscopy (SEM). JPC-seeded scaffolds showed a dense cell layer and osteogenic differentiation capacity on both PLGA-coated and uncoated ß-TCP scaffolds. SEM analyses showed no relevant blood cell attachment and ELISA results revealed no significant increase in most of the analyzed cell activation markers (ß-thromboglobulin, Sc5B-9, polymorphonuclear (PMN)-elastase). However, a notable increase in thrombin-antithrombin III (TAT) complex levels, as well as fibrin fiber accumulation on JPC-seeded ß-TCP scaffolds, was detected compared to the scaffolds without JPCs. Thus, this study demonstrated that besides the scaffold material the cells colonizing the scaffolds can also influence hemostasis, which can influence the regeneration of bone tissue.

Generation of iPSCs from Jaw Periosteal Cells Using Self-Replicating RNA.

Jaw periosteal cells (JPCs) represent a suitable stem cell source for bone tissue engineering (BTE) applications. However, challenges associated with limited cell numbers, stressful cell sorting, or the occurrence of cell senescence during in vitro passaging and the associated insufficient osteogenic potential in vitro of JPCs and other mesenchymal stem/stromal cells (MSCs) are main hurdles and still need to be solved. In this study, for the first time, induced pluripotent stem cells (iPSCs) were generated from human JPCs to open up a new source of stem cells for BTE. For this purpose, a non-integrating self-replicating RNA (srRNA) encoding reprogramming factors and green fluorescent protein (GFP) as a reporter was used to obtain JPC-iPSCs with a feeder- and xeno-free reprogramming protocol to meet the highest safety standards for future clinical applications. Furthermore, to analyze the potential of these iPSCs as a source of osteogenic progenitor cells, JPC-iPSCs were differentiated into iPSC-derived mesenchymal stem/stromal like cells (iMSCs) and further differentiated to the osteogenic lineage under xeno-free conditions. The produced iMSCs displayed MSC marker expression and morphology as well as strong mineralization during osteogenic differentiation.

Concise Review: Application of In Vitro Transcribed Messenger RNA for Cellular Engineering and Reprogramming: Progress and Challenges.

Several diseases are caused by missing or defective synthesis of proteins due to genetic or acquired disorders. In recent years, in vitro transcribed (IVT) messenger RNA (mRNA)-based therapy for de novo protein expression in cells has increased in importance. Thereby, desired proteins can be produced in cells by exogenous delivery of IVT mRNA, which does not integrate into the host genome and results in transient production of target proteins. Due to the lack of genomic integration, the risk of mutation and tumor development is minimized. Different approaches using IVT mRNA have been applied to alter the expression profiles of cells by the production of proteins. IVT mRNAs encoding transcription factors have led to the highly efficient induction of pluripotency in somatic cells and generated induced pluripotent stem cells that are free of viral vector components. Furthermore, specific IVT mRNA cocktails containing more than one specific IVT mRNA can be used to directly induce the differentiation into a desired cell type. In theory, every desired mRNA can be produced in vitro and used to enable extrinsic biosynthesis of target proteins in each cell type. Cells can be engineered by IVT mRNA to express antigens on dendritic cells for vaccination and tumor treatment, surface receptors on stem cells for increased homing to distinct areas, and to produce industrial grade human growth factors. In this review, we focus on the progress and challenges in mRNA-based cell engineering approaches. Stem Cells 2017;35:68-79.

Incorporation of Synthetic mRNA in Injectable Chitosan-Alginate Hybrid Hydrogels for Local and Sustained Expression of Exogenous Proteins in Cells.

The application of synthetic messenger RNA (mRNA) exhibits various advantages, such as expression of desired proteins in cells without genomic integration. In the field of tissue engineering, synthetic mRNAs could be also used to modulate the protein expression in implanted cells. Therefore, in this study, we incorporated synthetic humanized Gaussia luciferase (hGLuc) mRNA into alginate, chitosan, or chitosan-alginate hybrid hydrogels and analyzed the release of hGLuc mRNA from these hydrogels. After 3 weeks, 79% of the incorporated mRNA was released from alginate hydrogels, approximately 42% was released from chitosan hydrogels, and about 70% was released from chitosan-alginate hydrogels. Due to the injectability, chitosan-alginate hybrid hydrogels were selected for further investigation of the bioactivity of embedded hGLuc mRNA and the stability of these hydrogels was examined after the incorporation of synthetic mRNA by rheometric analysis. Therefore, HEK293 cells were incorporated into chitosan-alginate hydrogels containing mRNA transfection complexes and the luciferase activity in the supernatants was detected for up to 3 weeks. These results showed that the biodegradable chitosan-alginate hybrid hydrogels are promising delivery systems for sustained delivery of synthetic mRNAs into cells. Since chitosan-alginate hybrid hydrogels are injectable, the hydrogels can be simultaneously loaded with cells and the desired synthetic mRNA for exogenous protein synthesis and can be administered by minimally invasive local injection for tissue engineering applications.

Rapid Complexation of Aptamers by Their Specific Antidotes.

Nucleic acid ligands, aptamers, harbor the unique characteristics of small molecules and antibodies. The specificity and high affinity of aptamers enable their binding to different targets, such as small molecules, proteins, or cells. Chemical modifications of aptamers allow increased bioavailability. A further great benefit of aptamers is the antidote (AD)-mediated controllability of their effect. In this study, the AD-mediated complexation and neutralization of the thrombin binding aptamer NU172 and Toll-like receptor 9 (TLR9) binding R10-60 aptamer were determined. Thereby, the required time for the generation of aptamer/AD-complexes was analyzed at 37 °C in human serum using gel electrophoresis. Afterwards, the blocking of aptamers' effects was analyzed by determining the activated clotting time (ACT) in the case of the NU172 aptamer, or the expression of immune activation related genes IFN-1ß, IL-6, CXCL-10, and IL-1ß in the case of the R10-60 aptamer. Gel electrophoresis analyses demonstrated the rapid complexation of the NU172 and R10-60 aptamers by complementary AD binding after just 2 min of incubation in human serum. A rapid neutralization of anticoagulant activity of NU172 was also demonstrated in fresh human whole blood 5 min after addition of AD. Furthermore, the TLR9-mediated activation of PMDC05 cells was interrupted after the addition of the R10-60 AD. Using these two different aptamers, the rapid antagonizability of the aptamers was demonstrated in different environments; whole blood containing numerous proteins, cells, and different small molecules, serum, or cell culture media. Thus, nucleic acid ADs are promising molecules, which offer several possibilities for different in vivo applications, such as antagonizing aptamer-based drugs, immobilization, or delivery of oligonucleotides to defined locations.

Assaying RIPK2 Activation by Complex Formation.

The receptor-interacting serine/threonine-protein kinase-2 (RIPK2, RIP2) is a key player in downstream signaling of nuclear oligomerization domain (NOD)-like receptor (NLR)-mediated innate immune response against bacterial infections. RIPK2 is recruited following activation of the pattern recognition receptors (PRRs) NOD1 and NOD2 by sensing bacterial peptidoglycans leading to activation of NF-κB and MAPK pathways and the production of pro-inflammatory cytokines. Upon NOD1/2 activation, RIPK2 forms complexes in the cytoplasm of human cells, also called RIPosomes. These can be induced by Shigella flexneri or by the inhibition of RIPK2 by small compounds, such as GSK583 and gefitinib.In this chapter, we describe fluorescent light microscopic and Western blot approaches to analyze the cytoplasmic aggregation of RIPK2 upon infection with the invasive, Gram-negative bacterial pathogen Shigella flexneri, or by the treatment with RIPK2 inhibitors. This method is based on HeLa cells stably expressing eGFP-tagged RIPK2 and describes a protocol to induce and visualize RIPosome formation. The described method is useful to study the deposition of RIPK2 in speck-like structures, also in living cells, using live cell imaging and can be adopted for the study of other inhibitory proteins or to further analyze the process of RIPosome structure assembly.

Homing of mRNA-Modified Endothelial Progenitor Cells to Inflamed Endothelium.

Endothelial progenitor cells (EPCs) are one of the most important stem cells for the neovascularization of tissues damaged by ischemic diseases such as myocardial infarction, ischemic stroke, or critical limb ischemia. However, their low homing efficiency in the treatment of ischemic tissues limits their potential clinical applications. The use of synthetic messenger RNA (mRNA) for cell engineering represents a novel and promising technology for the modulation of cell behavior and tissue regeneration. To improve the therapeutic potential of EPCs, in this study, murine EPCs were engineered with synthetic mRNAs encoding C-X-C chemokine receptor 4 (CXCR4) and P-selectin glycoprotein ligand 1 (PSGL-1) to increase the homing and migration efficiency of EPCs to inflamed endothelium. Flow cytometric measurements revealed that the transfection of EPCs with CXCR4 and PSGL-1 mRNA resulted in increased expressions of CXCR4 and PSGL-1 on the cell surface compared with the unmodified EPCs. The transfection of EPCs with mRNAs did not affect cell viability. CXCR4-mRNA-modified EPCs showed significantly higher migration potential than unmodified cells in a chemotactic migration assay. The binding strength of the EPCs to inflamed endothelium was determined with single-cell atomic force microscopy (AFM). This showed that the mRNA-modified EPCs required a three-fold higher detachment force to be released from the TNF-α-activated endothelium than unmodified EPCs. Furthermore, in a dynamic flow model, significantly increased binding of the mRNA-modified EPCs to inflamed endothelium was detected. This study showed that the engineering of EPCs with homing factors encoding synthetic mRNAs increases the homing and migration potentials of these stem cells to inflamed endothelium. Thus, this strategy represents a promising strategy to increase the therapeutic potential of EPCs for the treatment of ischemic tissues.

An alternative in vivo model to evaluate pluripotency of patient-specific iPSCs.

The generation of autologous human induced pluripotent stem cells (hiPSCs) from a patient's somatic cells and the sub­sequent differentiation of these cells into desired cell types offer innovative treatment options for tissue regeneration. The hiPSCs obtained are usually implanted in immunodeficient mice, and teratoma formation is analyzed after 4 to 6 weeks to assess the cells' pluripotency. In this study, an alternative in vivo model based on chicken egg chorioallantoic membrane (CAM) was established to analyze the pluripotency of newly created hiPSCs. 0.5, 1, 2, 4 x 106 hiPSCs gen­erated from urine-derived renal epithelial cells were seeded on CAM and incubated for 9 days. Teratoma formation was detected in 70% of eggs inoculated with 2 x 106 hiPSCs and in 100% of eggs inoculated with 4 x 106 hiPSCs. All teratomas exhibited vascular structures. The robustness of the CAM model was confirmed using two additional hiPSC lines derived from human fibroblasts (NuFFs) or jaw periosteal cells. The presence of all three germ layers within the teratomas was successfully verified by histochemical and immunofluorescence staining and gene expression analysis of germ layer-specific markers. Urine-derived renal epithelial cells were used as negative control and showed no teratoma formation. The CAM-based in vivo model provides an optimal in vivo test environment for the pluripotency evaluation of newly generated hiPSC lines. This simple, fast, inexpensive and reproducible method reduces the suffering of animals and thus implements the principles of the 3Rs (replacement, reduction, and refinement).

Delivery of synthetic mRNAs for tissue regeneration.

In recent years, nucleic acid-based therapeutics have gained increasing importance as novel treatment options for disease prevention and treatment. Synthetic messenger RNAs (mRNAs) are promising nucleic acid-based drugs to transiently express desired proteins that are missing or defective. Recently, synthetic mRNA-based vaccines encoding viral proteins have been approved for emergency use against COVID-19. Various types of vehicles, such as lipid nanoparticles (LNPs) and liposomes, are being investigated to enable the efficient uptake of mRNA molecules into desired cells. In addition, the introduction of novel chemical modifications into mRNAs increased the stability, enabled the modulation of nucleic acid-based drugs, and increased the efficiency of mRNA-based therapeutic approaches. In this review, novel and innovative strategies for the delivery of synthetic mRNA-based therapeutics for tissue regeneration are discussed. Moreover, with this review, we aim to highlight the versatility of synthetic mRNA molecules for various applications in the field of regenerative medicine and also discuss translational challenges and required improvements for mRNA-based drugs.

Hydrojet-based delivery of footprint-free iPSC-derived cardiomyocytes into porcine myocardium.

The reprogramming of patient´s somatic cells into induced pluripotent stem cells (iPSCs) and the consecutive differentiation into cardiomyocytes enables new options for the treatment of infarcted myocardium. In this study, the applicability of a hydrojet-based method to deliver footprint-free iPSC-derived cardiomyocytes into the myocardium was analyzed. A new hydrojet system enabling a rapid and accurate change between high tissue penetration pressures and low cell injection pressures was developed. Iron oxide-coated microparticles were ex vivo injected into porcine hearts to establish the application parameters and the distribution was analyzed using magnetic resonance imaging. The influence of different hydrojet pressure settings on the viability of cardiomyocytes was analyzed. Subsequently, cardiomyocytes were delivered into the porcine myocardium and analyzed by an in vivo imaging system. The delivery of microparticles or cardiomyocytes into porcine myocardium resulted in a widespread three-dimensional distribution. In vitro, 7 days post-injection, only cardiomyocytes applied with a hydrojet pressure setting of E20 (79.57 ± 1.44%) showed a significantly reduced cell viability in comparison to the cells applied with 27G needle (98.35 ± 5.15%). Furthermore, significantly less undesired distribution of the cells via blood vessels was detected compared to 27G needle injection. This study demonstrated the applicability of the hydrojet-based method for the intramyocardial delivery of iPSC-derived cardiomyocytes. The efficient delivery of cardiomyocytes into infarcted myocardium could significantly improve the regeneration.

Generation of iPSCs by Nonintegrative RNA-Based Reprogramming Techniques: Benefits of Self-Replicating RNA versus Synthetic mRNA.

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is gaining in importance in the fields of regenerative medicine, tissue engineering, and disease modeling. Patient-specific iPSCs have as an unlimited cell source a tremendous potential for generating various types of autologous cells. For the future clinical applicability of these iPSC-derived cells, the generation of iPSCs via nongenome integrating methods and the efficient reprogramming of patients' somatic cells are required. In this study, 2 different RNA-based footprint-free methods for the generation of iPSCs were compared: the use of synthetic modified messenger RNAs (mRNAs) or self-replicating RNAs (srRNAs) encoding the reprogramming factors and GFP. Using both RNA-based methods, integration-free iPSCs without genomic alterations were obtained. The pluripotency characteristics identified by specific marker detection and the in vitro and in vivo trilineage differentiation capacity were comparable. Moreover, the incorporation of a GFP encoding sequence into the srRNA enabled a direct and convenient monitoring of the reprogramming procedure and the successful detection of srRNA translation in the transfected cells. Nevertheless, the use of a single srRNA to induce pluripotency was less time consuming, faster, and more efficient than the daily transfection of cells with synthetic mRNAs. Therefore, we believe that the srRNA-based approach might be more appropriate and efficient for the reprogramming of different types of somatic cells for clinical applications.

Reprogramming of Urine-Derived Renal Epithelial Cells into iPSCs Using srRNA and Consecutive Differentiation into Beating Cardiomyocytes.

The generation of induced pluripotent stem cells (iPSCs) from patient's somatic cells and the subsequent differentiation into desired cell types opens up numerous possibilities in regenerative medicine and tissue engineering. Adult cardiomyocytes have limited self-renewal capacity; thus, the efficient, safe, and clinically applicable generation of autologous cardiomyocytes is of great interest for the treatment of damaged myocardium. In this study, footprint-free iPSCs were successfully generated from urine-derived renal epithelial cells through a single application of self-replicating RNA (srRNA). The expression of pluripotency markers and the in vitro as well as in vivo trilineage differentiation were demonstrated. Furthermore, the resulting iPSCs contained no residual srRNA, and the karyotyping analysis demonstrated no detectable anomalies. The cardiac differentiation of these iPSCs resulted in autologous contracting cardiomyocytes after 10 days. We anticipate that the use of urine as a non-invasive cell source to obtain patient cells and the use of srRNA for reprogramming into iPSCs will greatly improve the future production of clinically applicable cardiomyocytes and other cell types. This could allow the regeneration of tissues by generating sufficient quantities of autologous cells without the risk of immune rejection.

Efficient reduction of synthetic mRNA induced immune activation by simultaneous delivery of B18R encoding mRNA.

The application of synthetic modified messenger RNA (mRNA) is a promising approach for the treatment of a variety of diseases and vaccination. In the past few years, different modifications of synthetic mRNA were applied to render the mRNA more stable and less immunogenic. However, the repeated application of synthetic mRNA still requires the suppression of immune activation to avoid cell death and to allow a sufficient production of exogenous proteins. Thus, the addition of type I interferon (IFN) inhibiting recombinant protein B18R is often required to avoid IFN response. In this study, the ability of B18R encoding mRNA to prevent the immune response of cells to the delivered synthetic mRNA was analyzed. The co-transfection of enhanced green fluorescent protein (eGFP) mRNA transfected fibroblasts with B18R encoding mRNA over 7-days resulted in comparable cell viability and eGFP protein expression as in the cells transfected with eGFP mRNA and incubated with B18R protein. Using qRT-PCR, significantly reduced expression of interferon-stimulated gene Mx1 was detected in the cells transfected with B18R mRNA and stimulated with IFNß compared to the cells without B18R mRNA transfection. Thereby, it was demonstrated that the co-transfection of synthetic mRNA transfected cells with B18R encoding mRNA can reduce the IFN response-related cell death and thus, improve the protein expression.

Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs.

The application of endothelial progenitor cells (EPCs) for the revascularization of ischemic tissues, such as after myocardial infarction, stroke, and acute limb ischemia, has a huge clinical potential. However, the low retention and engraftment of EPCs as well as the poor survival of migrated stem cells in ischemic tissues still hamper the successful clinical application. Thus, in this study, we engineered, for the first time, murine EPCs with synthetic mRNAs to transiently produce proangiogenic factors vascular endothelial growth factor-A (VEGF-A), stromal cell-derived factor-1α (SDF-1α), and angiopoietin-1 (ANG-1). After the transfection of cells with synthetic mRNAs, significantly increased VEGF-A, SDF-1α, and ANG-1 protein levels were detected compared to untreated EPCs. Thereby, mRNA-engineered EPCs showed significantly increased chemotactic activity versus untreated EPCs and resulted in significantly improved attraction of EPCs. Furthermore, ANG-1 mRNA-transfected EPCs displayed a strong wound-healing capacity. Already after 12 hr, 94% of the created wound area in the scratch assay was closed compared to approximately 45% by untreated EPCs. Moreover, the transfection of EPCs with ANG-1 or SDF-1α mRNA also significantly improved the in vitro tube formation capacity; however, the strongest effect could be detected with EPCs simultaneously transfected with VEGF-A, SDF-1α, and ANG-1 mRNA. In the in vivo chicken chorioallantoic membrane (CAM) assay, EPCs transfected with ANG-1 mRNA revealed the strongest angiogenetic potential with significantly elevated vessel density and total vessel network length. In conclusion, this study demonstrated that EPCs can be successfully engineered with synthetic mRNAs encoding proangiogenic factors to improve their therapeutic angiogenetic potential in patients experiencing chronic or acute ischemic disease.

Blood-Contacting Biomaterials: In Vitro Evaluation of the Hemocompatibility.

Hemocompatibility of blood-contacting biomaterials is one of the most important criteria for their successful in vivo applicability. Thus, extensive in vitro analyses according to ISO 10993-4 are required prior to clinical applications. In this review, we summarize essential aspects regarding the evaluation of the hemocompatibility of biomaterials and the required in vitro analyses for determining the blood compatibility. Static, agitated, or shear flow models are used to perform hemocompatibility studies. Before and after the incubation of the test material with fresh human blood, hemolysis, cell counts, and the activation of platelets, leukocytes, coagulation and complement system are analyzed. Furthermore, the surface of biomaterials are evaluated concerning attachment of blood cells, adsorption of proteins, and generation of thrombus and fibrin networks.

Intradermal Delivery of Synthetic mRNA Using Hollow Microneedles for Efficient and Rapid Production of Exogenous Proteins in Skin.

In recent years, synthetic mRNA-based applications to produce desired exogenous proteins in cells have been gaining importance. However, systemic delivery of synthetic mRNA can result in unspecific uptake into undesired cells or organs and, thereby, fail to target desired cells. Thus, local and targeted delivery of synthetic mRNA becomes increasingly important to reach the desired cell types and tissues. In this study, intradermal delivery of synthetic mRNA using a hollow microneedle injection-based method was evaluated. Furthermore, an ex vivo porcine skin model was established to analyze synthetic mRNA-mediated protein expression in the skin following intradermal delivery. Using this model, highly efficient delivery of synthetic mRNA was demonstrated, which resulted in detection of high levels of secretable humanized Gaussia luciferase (hGLuc) protein encoded by the microinjected synthetic mRNA. Interestingly, synthetic mRNA injected without transfection reagent was also able to enter the cells and resulted in protein expression. The established ex vivo porcine skin model can be used to evaluate the successful production of desired proteins after intradermal delivery of synthetic mRNAs before starting with in vivo experiments. Furthermore, the use of microneedles enables patient-friendly, painless, and efficient delivery of synthetic mRNAs into the dermis; thus, this method could be applied for local treatment of different skin diseases as well as for vaccination and immunotherapy.

Generation of Large-Scale DNA Hydrogels with Excellent Blood and Cell Compatibility.

Hemocompatibility and cytocompatibility of biomaterials codetermine the success of tissue engineering applications. DNA, the natural component of our cells, is an auspicious biomaterial for the generation of designable scaffolds with tailorable characteristics. In this study, a combination of rolling circle amplification and multiprimed chain amplification is used to generate hydrogels at centimeter scale consisting solely of DNA. Using an in vitro rotation model and fresh human blood, the reaction of the hemostatic system on DNA hydrogels is analyzed. The measurements of hemolysis, platelets activation, and the activation of the complement, coagulation, and neutrophils using enzyme-linked immunosorbent assays demonstrate excellent hemocompatibility. In addition, the cytocompatibility of the DNA hydrogels is tested by indirect contact (agar diffusion tests) and material extract experiments with L929 murine fibroblasts according to the ISO 10993-5 specifications and no negative impact on the cell viability is detected. These results indicate the promising potential of DNA hydrogels as biomaterials for versatile applications in the field of regenerative medicine.

In vitro test system for evaluation of immune activation potential of new single-stranded DNA-based therapeutics.

Aptamers are synthetic single-stranded DNA (ssDNA) molecules with the ability to fold into complex three-dimensional structures. They can bind their targets with a high selectivity and affinity, thus they have an enormous potential as therapeutic agents. However, since aptamers are synthetic and especially since certain sequences can increasingly bind to the pattern recognition receptors of the immune cells when applied in vivo, they can induce an immune activation. Here, we established a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) based assay to evaluate aptamers-induced immune activation prior to in vivo studies. Human whole blood or plasmacytoid dendritic cell line (PMDC05) were incubated with CpG, R10-60 aptamer, start library, or a CpG containing aptamer. After 2 and 4 h, cytokine expression was measured using qRT-PCR to determine immune reaction against different aptamers. CpG containing a phosphorothioate backbone led to a significant up-regulation of CCL-7, IFN-1α, IFN-1ß in whole blood after 4 h. Compared to the samples without ssDNA, significantly higher TNF-α expression was detected after the R10-60 aptamer incubation for 4 h. The stimulation of PMDC05 cells with different ssDNA enabled more sensitive detection of aptamer sequence specific immune activation. After 4 h, CpG led to a significantly higher expression of CCL-8, CXCL-10, IL-1ß, IL-6, IL-8, IFN-1ß, and TNF-α. R10-60 aptamer caused a significant up-regulation of IL-1ß, IFN-1ß, and TNF-α. Negative control aptamers did not induce an immune activation. The use of this assay before starting with in vivo studies will facilitate the in vitro prediction of immune activation potential of aptamers.