bims-cateng 2023-10-15 papers

Issue of 2023–10–15
six papers selected by
Chance Bowman, Dartmouth College

Polymers (Basel). 2023 Sep 29. pii: 3940. [Epub ahead of print]15(19):

Recent Progress of the Vat Photopolymerization Technique in Tissue Engineering: A Brief Review of Mechanisms, Methods, Materials, and Applications.

Ying Li, Xueqin Zhang, Xin Zhang, Yuxuan Zhang, Dan Hou.

Vat photopolymerization (VP), including stereolithography (SLA), digital light processing (DLP), and volumetric printing, employs UV or visible light to solidify cell-laden photoactive bioresin contained within a vat in a point-by-point, layer-by-layer, or volumetric manner. VP-based bioprinting has garnered substantial attention in both academia and industry due to its unprecedented control over printing resolution and accuracy, as well as its rapid printing speed. It holds tremendous potential for the fabrication of tissue- and organ-like structures in the field of regenerative medicine. This review summarizes the recent progress of VP in the fields of tissue engineering and regenerative medicine. First, it introduces the mechanism of photopolymerization, followed by an explanation of the printing technique and commonly used biomaterials. Furthermore, the application of VP-based bioprinting in tissue engineering was discussed. Finally, the challenges facing VP-based bioprinting are discussed, and the future trends in VP-based bioprinting are projected.

Keywords: biocompatibility; bioprinting; computed axial lithography; continuous liquid interface production; digital light processing; stereolithography; tissue engineering; vat photopolymerization

DOI: https://doi.org/10.3390/polym15193940

Nat Methods. 2023 Oct 09.

Compact engineered human mechanosensitive transactivation modules enable potent and versatile synthetic transcriptional control.

Barun Mahata, Alan Cabrera, Daniel A Brenner, Rosa Selenia Guerra-Resendez, Jing Li, Jacob Goell, Kaiyuan Wang, Yannie Guo, Mario Escobar, Abinand Krishna Parthasarathy, Hailey Szadowski, Guy Bedford, Daniel R Reed, Sunghwan Kim, Isaac B Hilton.

Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR-Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here, we defined and optimized minimal TADs built from human mechanosensitive transcription factors. We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS and eN3x9) and to build the CRISPR-dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific and robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II and V CRISPR systems, transcription activator-like effectors and zinc finger proteins. Further, as proofs of concept, we used dCas9-NMS to efficiently reprogram human fibroblasts into induced pluripotent stem cells and demonstrated that mechanosensitive transcription factor TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules and delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.

DOI: https://doi.org/10.1038/s41592-023-02036-1

STAR Protoc. 2023 Oct 11. pii: S2666-1667(23)00605-6. [Epub ahead of print]4(4): 102638

Protocol for generation of a time-resolved cellular interactome during tissue remodeling in adult mice.

Elena Groppa, Lin Wei Tung, Stefania Mattevi, Morten Ritso, Fabio M V Rossi, Paolo Martini.

Efficient skeletal muscle regeneration necessitates fine-tuned coordination among multiple cell types through an intricate network of intercellular communication. We present a protocol for generation of a time-resolved cellular interactome during tissue remodeling. We describe steps for isolating distinct cell populations from skeletal muscle of adult mice after acute damage and extracting RNA from purified cells prior to the generation of RNA sequencing data. We then detail procedures for generating and deciphering a time- and lineage-resolved model of intercellular crosstalk. For complete details on the use and execution of this protocol, please refer to Groppa et al. (2023).1.

Keywords: Bioinformatics; Cell Biology; Cell isolation; Computer sciences; Flow Cytometry/Mass Cytometry; Molecular Biology; RNAseq; Sequence analysis; Sequencing; Stem Cells

DOI: https://doi.org/10.1016/j.xpro.2023.102638

Int J Mol Sci. 2023 Sep 28. pii: 14701. [Epub ahead of print]24(19):

CRISPR-Cas9 Direct Fusions for Improved Genome Editing via Enhanced Homologous Recombination.

Tahmina Tabassum, Giovanni Pietrogrande, Michael Healy, Ernst J Wolvetang.

DNA repair in mammalian cells involves the coordinated action of a range of complex cellular repair machinery. Our understanding of these DNA repair processes has advanced to the extent that they can be leveraged to improve the efficacy and precision of Cas9-assisted genome editing tools. Here, we review how the fusion of CRISPR-Cas9 to functional domains of proteins that directly or indirectly impact the DNA repair process can enhance genome editing. Such studies have allowed the development of diverse technologies that promote efficient gene knock-in for safer genome engineering practices.

Keywords: CRISPR-Cas9; DNA repair; gene editing; homologous recombination

DOI: https://doi.org/10.3390/ijms241914701

Adv Mater Technol. 2023 Aug;pii: admt.202300026. [Epub ahead of print]8(15):

Spatial-Selective Volumetric 4D Printing and Single-Photon Grafting of Biomolecules within Centimeter-Scale Hydrogels via Tomographic Manufacturing.

Marc Falandt, Paulina Nuñez Bernal, Oksana Dudaryeva, Sammy Florczak, Gabriel Gröfibacher, Matthias Schweiger, Alessia Longoni, Coralie Greant, Marisa Assunção, Olaf Nijssen, Sandra van Vlierberghe, Jos Malda, Tina Vermonden, Riccardo Levato.

Conventional additive manufacturing and biofabrication techniques are unable to edit the chemicophysical properties of the printed object postprinting. Herein, a new approach is presented, leveraging light-based volumetric printing as a tool to spatially pattern any biomolecule of interest in custom-designed geometries even across large, centimeter-scale hydrogels. As biomaterial platform, a gelatin norbornene resin is developed with tunable mechanical properties suitable for tissue engineering applications. The resin can be volumetrically printed within seconds at high resolution (23.68 ± 10.75 μm). Thiol-ene click chemistry allows on-demand photografting of thiolated compounds postprinting, from small to large (bio)molecules (e.g., fluorescent dyes or growth factors). These molecules are covalently attached into printed structures using volumetric light projections, forming 3D geometries with high spatiotemporal control and ≈50 μm resolution. As a proof of concept, vascular endothelial growth factor is locally photografted into a bioprinted construct and demonstrated region-dependent enhanced adhesion and network formation of endothelial cells. This technology paves the way toward the precise spatiotemporal biofunctionalization and modification of the chemical composition of (bio)printed constructs to better guide cell behavior, build bioactive cue gradients. Moreover, it opens future possibilities for 4D printing to mimic the dynamic changes in morphogen presentation natively experienced in biological tissues.

Keywords: 4D printing; biofabrication; light-based printing; photopatterning; volumetric additive manufacturing

DOI: https://doi.org/10.1002/admt.202300026

Anal Chem. 2023 Oct 10.

Spatial Transcriptomics: Emerging Technologies in Tissue Gene Expression Profiling.

Agustín Robles-Remacho, Rosario M Sanchez-Martin, Juan J Diaz-Mochon.

In this Perspective, we discuss the current status and advances in spatial transcriptomics technologies, which allow high-resolution mapping of gene expression in intact cell and tissue samples. Spatial transcriptomics enables the creation of high-resolution maps of gene expression patterns within their native spatial context, adding an extra layer of information to the bulk sequencing data. Spatial transcriptomics has expanded significantly in recent years and is making a notable impact on a range of fields, including tissue architecture, developmental biology, cancer, and neurodegenerative and infectious diseases. The latest advancements in spatial transcriptomics have resulted in the development of highly multiplexed methods, transcriptomic-wide analysis, and single-cell resolution utilizing diverse technological approaches. In this Perspective, we provide a detailed analysis of the molecular foundations behind the main spatial transcriptomics technologies, including methods based on microdissection, in situ sequencing, single-molecule FISH, spatial capturing, selection of regions of interest, and single-cell or nuclei dissociation. We contextualize the detection and capturing efficiency, strengths, limitations, tissue compatibility, and applications of these techniques as well as provide information on data analysis. In addition, this Perspective discusses future directions and potential applications of spatial transcriptomics, highlighting the importance of the continued development to promote widespread adoption of these techniques within the research community.

DOI: https://doi.org/10.1021/acs.analchem.3c02029