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Platform technology for scalable assembly of instantaneously functional mosaic tissues.

ABSTRACT: Engineering mature tissues requires a guided assembly of cells into organized three-dimensional (3D) structures with multiple cell types. Guidance is usually achieved by microtopographical scaffold cues or by cell-gel compaction. The assembly of individual units into functional 3D tissues is often time-consuming, relying on cell ingrowth and matrix remodeling, whereas disassembly requires an invasive method that includes either matrix dissolution or mechanical cutting. We invented Tissue-Velcro, a bio-scaffold with a microfabricated hook and loop system. The assembly of Tissue-Velcro preserved the guided cell alignment realized by the topographical features in the 2D scaffold mesh and allowed for the instant establishment of coculture conditions by spatially defined stacking of cardiac cell layers or through endothelial cell coating. The assembled cardiac 3D tissue constructs were immediately functional as measured by their ability to contract in response to electrical field stimulation. Facile, on-demand tissue disassembly was demonstrated while preserving the structure, physical integrity, and beating function of individual layers.

SUBMITTER: Zhang B

PROVIDER: S-EPMC4643798 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Platform technology for scalable assembly of instantaneously functional mosaic tissues.

Zhang Boyang B Montgomery Miles M Davenport-Huyer Locke L Korolj Anastasia A Radisic Milica M

Science advances 20150821 7

Engineering mature tissues requires a guided assembly of cells into organized three-dimensional (3D) structures with multiple cell types. Guidance is usually achieved by microtopographical scaffold cues or by cell-gel compaction. The assembly of individual units into functional 3D tissues is often time-consuming, relying on cell ingrowth and matrix remodeling, whereas disassembly requires an invasive method that includes either matrix dissolution or mechanical cutting. We invented Tissue-Velcro, ...[more]

PMID: 26601234

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Project description:Functional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naive and cultivation-independent exploration of metagenomes. However, preparation of these libraries is often limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, mosaic ends tagmentation (METa) assembly, for highly efficient functional metagenomic library preparation. We applied tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergized with homology-based assembly cloning to result in a 300-fold increase in cloning efficiency compared to traditional blunt-cloning-based protocols. We show that compared to published libraries prepared by state-of-the-art protocols, METa assembly is on average ca. 20- to 200-fold more efficient and can prepare gigabase-sized libraries with as little as 200 ng of input DNA. We show the usefulness of METa assembly first by using a normative 5-μg mass of soil metagenomic DNA to prepare a 700-Gb library that allowed us to discover novel nourseothricin resistance genes and a potentially new mode of resistance to this antibiotic and second by using only 300 ng of goose fecal metagenomic DNA to prepare a 27-Gb library that captured numerous tetracycline and colistin resistance genes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low-biomass or scarce microbiomes. IMPORTANCE Medically and industrially important genes can be recovered from microbial communities by high-throughput sequencing, but precise annotation is often limited to characterized genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library, directly connecting genes to functions, is a sequence-naive and cultivation-independent method to discover novel genes. The process of preparing these libraries is DNA greedy and inefficient, however. Here, we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and a goose fecal microbiome and allowed us to discover new antibiotic resistance genes and mechanisms. This library preparation method will potentially allow the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such as biomedical swabs or exotic samples.

Project description:BackgroundPathogenic TNNT2 variants are a cause of hypertrophic and dilated cardiomyopathies, which promote heart failure by incompletely understood mechanisms. The precise functional significance for 87% of TNNT2 variants remains undetermined, in part, because of a lack of functional genomics studies. The knowledge of which and how TNNT2 variants cause hypertrophic and dilated cardiomyopathies could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, and provide new insights into cardiomyopathy pathogenesis, as well.MethodsWe created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study TNNT2 variant pathogenicity and pathophysiology. Using human induced pluripotent stem cell-derived cardiomyocytes in cardiac microtissue and single-cell assays, we functionally interrogated 51 TNNT2 variants, including 30 pathogenic/likely pathogenic variants and 21 variants of uncertain significance. We used RNA sequencing to determine the transcriptomic consequences of pathogenic TNNT2 variants and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction of TNNT2 variant pathogenicity. We also studied variant-specific pathophysiology using a thin filament-directed calcium reporter to monitor changes in myofilament calcium affinity.ResultsHypertrophic cardiomyopathy-associated TNNT2 variants caused increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction. TNNT2 variant-dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK (mitogen-activated protein kinase) signaling targets, HOPX, and NPPB. We distinguished pathogenic TNNT2 variants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenous NPPB locus. On the basis of a combination of NPPB reporter activity and cardiac microtissue contraction, our study provides experimental support for the reclassification of 2 pathogenic/likely pathogenic variants and 2 variants of uncertain significance.ConclusionsOur study found that hypertrophic cardiomyopathy-associated TNNT2 variants increased cardiac microtissue contraction, whereas dilated cardiomyopathy-associated variants decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including NPPB levels, directly correlated with sarcomere function and can be used to predict TNNT2 variant pathogenicity.

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Platform technology for scalable assembly of instantaneously functional mosaic tissues.

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Platform technology for scalable assembly of instantaneously functional mosaic tissues.

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