Engineering Complex Tissues

Atala, Anthony; Kasper, F. Kurtis; Mikos, Antonios G.

doi:10.1126/scitranslmed.3004890

Cited by 482 publications

(355 citation statements)

References 275 publications

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“…To circumvent this, future studies may be directed toward the replacement of the constitutive EF-1α promoter with a drug-inducible promoter (61), which could confer engineered control of the duration and magnitude of transgene expression. Moreover, the technique of biomaterial-mediated gene delivery may be applied toward engineering complex tissues or tissue interfaces for regenerative medicine (62)(63)(64).…”

Section: Discussionmentioning

confidence: 99%

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage

Brunger

Huynh

Guenther

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

116

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The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(e-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffoldmediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(e-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.biomaterials | regenerative medicine | genetic engineering | gene therapy | chondrocyte

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Section: Discussionmentioning

confidence: 99%

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage

Brunger

Huynh

Guenther

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

116

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“…36 Aiming at musculoskeletal repair, the cultivation of autologous human MSCs on 3D matrices is a basic prerequisite. 3 Fluorescence-based qPCR is the most commonly used molecular technology for the quantification of gene expression.…”

Section: Discussionmentioning

confidence: 99%

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Rauh

Jacobi

Stiehler

2015

Tissue Engineering Part C: Methods

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The principles of tissue engineering (TE) are widely used for bone regeneration concepts. Three-dimensional (3D) cultivation of autologous human mesenchymal stromal cells (MSCs) on porous scaffolds is the basic prerequisite to generate newly formed bone tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive analytical tool for the measurement of mRNA-levels in cells or tissues.For an accurate quantification of gene expression levels, stably expressed reference genes (RGs) are essential to obtain reliable results. Since the 3D environment can affect a cell's morphology, proliferation, and gene expression profile compared with two-dimensional (2D) cultivation, there is a need to identify robust RGs for the quantification of gene expression. So far, this issue has not been adequately investigated. The aim of this study was to identify the most stably expressed RGs for gene expression analysis of 3D-cultivated human bone marrow-derived MSCs (BM-MSCs). For this, we analyzed the gene expression levels of n = 31 RGs in 3D-cultivated human BM-MSCs from six different donors compared with conventional 2D cultivation using qRT-PCR. MSCs isolated from bone marrow aspirates were cultivated on human cancellous bone cube scaffolds for 14 days. Osteogenic differentiation was assessed by cell-specific alkaline phosphatase (ALP) activity and expression of osteogenic marker genes. Expression levels of potential reference and target genes were quantified using commercially available TaqMan Ò assays. mRNA expression stability of RGs was determined by calculating the coefficient of variation (CV) and using the algorithms of geNorm and NormFinder. Using both algorithms, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the most stably expressed RGs in 3D-cultivated BMMSCs. Notably, genes that are routinely used as RGs, for example, beta actin (ACTB) and ribosomal protein L37a (RPL37A), were among the least stable genes. We recommend the combined use of TBP, TFRC, and HPRT1 for the accurate and robust normalization of qRT-PCR data of 3D-cultivated human BM-MSCs.

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“…[4][5][6][7][8] Because they are patient-specific, iPSC lines can be used in a wide range of biomedical applications. [9][10][11] However, the extent of the similarity between iPSCs and the "gold standard" of pluripotency, human ESCs, is still unclear. Indeed, the tetraploid complementation approach can be used to determine this similarity for mouse iPSCs; however, it is not applicable to humans and other species.…”

Section: Introductionmentioning

confidence: 99%

An integrative analysis of reprogramming in human isogenic system identified a clone selection criterion

et al. 2016

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Kiselev (2016) An integrative analysis of reprogramming in human isogenic system identified a clone selection criterion, Cell Cycle, 15:7, 986-997, DOI: 10.1080/15384101.2016 ABSTRACTThe pluripotency of newly developed human induced pluripotent stem cells (iPSCs) is usually characterized by physiological parameters; i.e., by their ability to maintain the undifferentiated state and to differentiate into derivatives of the 3 germ layers. Nevertheless, a molecular comparison of physiologically normal iPSCs to the "gold standard" of pluripotency, embryonic stem cells (ESCs), often reveals a set of genes with different expression and/or methylation patterns in iPSCs and ESCs. To evaluate the contribution of the reprogramming process, parental cell type, and fortuity in the signature of human iPSCs, we developed a complete isogenic reprogramming system. We performed a genome-wide comparison of the transcriptome and the methylome of human isogenic ESCs, 3 types of ESC-derived somatic cells (fibroblasts, retinal pigment epithelium and neural cells), and 3 pairs of iPSC lines derived from these somatic cells. Our analysis revealed a high input of stochasticity in the iPSC signature that does not retain specific traces of the parental cell type and reprogramming process. We showed that 5 iPSC clones are sufficient to find with 95% confidence at least one iPSC clone indistinguishable from their hypothetical isogenic ESC line. Additionally, on the basis of a small set of genes that are characteristic of all iPSC lines and isogenic ESCs, we formulated an approach of "the best iPSC line" selection and confirmed it on an independent dataset.

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Engineering Complex Tissues

Abstract: Advances in tissue engineering technologies will enable regeneration of complex tissues and organs.

Cited by 482 publications

References 275 publications

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

An integrative analysis of reprogramming in human isogenic system identified a clone selection criterion

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