Pectin-based bioinks for 3D models of neural tissue produced by a pH-controlled kinetics

Introduction: In the view of 3D-bioprinting with cell models representative of neural cells, we produced inks to mimic the basic viscoelastic properties of brain tissue. Moving from the concept that rheology provides useful information to predict ink printability, this study improves and expands the potential of the previously published 3D-reactive printing approach by introducing pH as a key parameter to be controlled, together with printing time. Methods: The viscoelastic properties, printability, and microstructure of pectin gels crosslinked with CaCO3 were investigated and their composition was optimized (i.e., by including cell culture medium, HEPES buffer, and collagen). Different cell models representative of the major brain cell populations (i.e., neurons, astrocytes, microglial cells, and oligodendrocytes) were considered. Results and Discussion: The outcomes of this study propose a highly controllable method to optimize the printability of internally crosslinked polysaccharides, without the need for additives or post-printing treatments. By introducing pH as a further parameter to be controlled, it is possible to have multiple (pH-dependent) crosslinking kinetics, without varying hydrogel composition. In addition, the results indicate that not only cells survive and proliferate following 3D-bioprinting, but they can also interact and reorganize hydrogel microstructure. Taken together, the results suggest that pectin-based hydrogels could be successfully applied for neural cell culture

Bone Marrow Mesenchymal Stem Cells Expanded Inside the Nichoid Micro-Scaffold: a Focus on Anti-Inflammatory Response

Purpose Mesenchymal stem cells (MSCs) represent a promising source for stem cell therapies in numerous diseases, including pediatric respiratory system diseases. Characterized by low immunogenicity, high anti-inflammatory, and immunoregulatory features, MSCs demonstrated an excellent therapeutic profile in numerous in vitro and preclinical models. MSCs reside in a specialized physiologic microenvironment, characterized by a unique combination of biophysical, biochemical, and cellular properties. The exploitation of the 3D micro-scaffold Nichoid, which simulates the native niche, enhanced the anti-inflammatory potential of stem cells through mechanical stimulation only, overcoming the limitation of biochemical and xenogenic growth factors application.Materials and Methods In this work, we expanded pediatric bone marrow MSCs (BM-MSCs) inside the Nichoid and performed a complete cellular characterization with different approaches including viability assays, immunofluorescence analyses, RNA sequencing, and gene expression analysis.Results We demonstrated that BM-MSCs inside the scaffold remain in a stem cell quiescent state mimicking the condition of the in vivo environment. Moreover, the gene expression profile of these cells shows a significant up-regulation of genes involved in immune response when compared with the flat control.Conclusion The significant changes in the expression profile of anti-inflammatory genes could potentiate the therapeutic effect of BM-MSCs, encouraging the possible clinical translation for the treatment of pediatric congenital and acquired pulmonary disorders, including post-COVID lung manifestations

Live-cell p53 single-molecule binding is modulated by C-terminal acetylation and correlates with transcriptional activity

Live-cell microscopy has highlighted that transcription factors bind transiently to chromatin but it is not clear if the duration of these binding interactions can be modulated in response to an activation stimulus, and if such modulation can be controlled by post-translational modifications of the transcription factor. We address this question for the tumor suppressor p53 by combining live-cell single-molecule microscopy and single cell in situ measurements of transcription and we show that p53-binding kinetics are modulated following genotoxic stress. The modulation of p53 residence times on chromatin requires C-terminal acetylation - a classical mark for transcriptionally active p53 - and correlates with the induction of transcription of target genes such as CDKN1a. We propose a model in which the modification state of the transcription factor determines the coupling between transcription factor abundance and transcriptional activity by tuning the transcription factor residence time on target sites

Live Cell Single Molecule Binding of Transcription Factors in Living Cells : Characterizing p53 Latency

The binding of transcription factors (TFs) to their regulatory sites on DNA determines how much and how timely a particular gene will be expressed, and ultimately how the cell respond to external cues. TF binding is typically studied by bulk biochemical experiments as chromatin immunoprecipitation (ChIP) As these methods provide limited temporal resolution and they are unable to provide information about these interactions at the single cell level, the interpretation of ChIP results can be challenging when dealing with TFs exhibiting rapid turnover, or with cells and tissues exhibiting a patterned nonhomogeneous transcriptional response to an external stimulus. Here we describe a microscopy-based single molecule imaging approach which can be used to obtain direct information on the TF binding kinetics to chromatin with the sub-second temporal resolution at the individual live-cell level [1,2]. We apply this method to characterize the binding of the tumor suppressor p53 both in basal, non-stimulated conditions and upon its activation by genotoxic stress induced by ionizing radiation: we show that p53 binds transiently to DNA (timescale of seconds), and that this interaction is modulated following the induction of damage, Importantly, more stable interactions are associated to higher transcription rates of p53 target genes, indicating that p53 acts as a latent TF, reviving an hypothesis initially derived from in-vitro studies [3], but later challenged by low temporal resolution ChIP experiments [4]