Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency

Hotta, Akitsu; Cheung, Aaron; Farra, Natalie; Vijayaragavan, Kausalia; Séguin, Cheryle A.; Draper, Jonathan S.; Pasceri, Peter; Maksakova, Irina A.; Mager, Dixie L.; Rossant, Janet; Bhatia, Mickie; Ellis, James

doi:10.1038/nmeth.1325

Cited by 284 publications

(270 citation statements)

References 30 publications

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“…The cDNA for TGF-β3 was obtained from HEK-293T cells via RT-PCR and cloned into a lentiviral transfer vector [Addgene Plasmid 12250 (70) modified with a custom multiple-cloning site downstream of the EF-1α promoter]. Virus driving overexpression of eGFP was produced using either Addgene Plasmid 21320 (71) or 11645 (72) modified to contain an EF1-α-eGFP cassette. To produce VSV-G pseudotyped LV, 4.2e6 HEK-293T cells were plated onto each 10-cm dish in DMEM-high glucose supplemented with L-glutamine, sodium pyruvate, and 10% FBS (D-10 medium).…”

Section: Methodsmentioning

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: Methodsmentioning

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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“…Rett syndrome results from mutations in the X-chromosome-linked MeCP2 gene, which regulates gene expression through modulating DNA methylation (Akbarian, 2003;Chahrour and Zoghbi, 2007). Fibroblasts of patients with Rett syndrome have been successfully reprogrammed into iPSCs (Hotta et al, 2009;Marchetto et al, 2010a). In Marchetto et al's study, the phenotypes of Rett syndrome were recapitulated in neurons differentiated from patient-derived iPSCs, and have subsequently been extensively studied.…”

Section: Modeling Of Neuronal Disorders With Nscsmentioning

confidence: 99%

Neural stem cells: mechanisms and modeling

Yao

Gage

2012

Protein Cell

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In the adult brain, neural stem cells have been found in two major niches: the hippocampus and the olfactory bulb. Neurons derived from these stem cells contribute to learning, memory, and the autonomous repair of the brain under pathological conditions. Hence, the physiology of adult neural stem cells has become a significant component of research on synaptic plasticity and neuronal disorders. In addition, the recently developed induced pluripotent stem cell technique provides a powerful tool for researchers engaged in the pathological and pharmacological study of neuronal disorders. In this review, we briefly summarize the research progress in neural stem cells in the adult brain and in the neuropathological application of the induced pluripotent stem cell technique.

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“…Surprisingly, the specificity of this miRNA regulatory strategy was closely comparable to a knock-in reporter system (Oct4-GFP) in the mouse setting. Recently, an artificial promoter generated by combining a strong LTR promoter with repetitive Oct4 and Sox2 core enhancer elements was shown to be active mostly in pluripotent cells [35]. Our system which is purely based on miRNA regulation reaches a similar level of specificity while transcription is driven by a ubiquitous housekeeping promoter.…”

Section: Discussionmentioning

confidence: 95%

A microRNA-Based System for Selecting and Maintaining the Pluripotent State in Human Induced Pluripotent Stem Cells

et al. 2011

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Induced pluripotent stem cell (iPSC) technology has provided researchers with a unique tool to derive disease-specific stem cells for the study and possible treatment of degenerative disorders with autologous cells. The low efficiency and heterogeneous nature of reprogramming is a major impediment to the generation of personalized iPSC lines. Here, we report the generation of a lentiviral system based on a microRNA-regulated transgene that enables for the efficient selection of mouse and human pluripotent cells. This system relies on the differential expression pattern of the mature form of microRNA let7a in pluripotent versus committed or differentiated cells. We generated microRNA responsive green fluorescent protein and Neo reporters for specific labeling and active selection of the pluripotent cells in any culture condition. We used this system to establish Rett syndrome and Parkinson's disease human iPSCs. The presented selection procedure represents a straightforward and powerful tool for facilitating the derivation of patient-specific iPSCs.

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Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency

Cited by 284 publications

References 30 publications

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage

Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage

Neural stem cells: mechanisms and modeling

A microRNA-Based System for Selecting and Maintaining the Pluripotent State in Human Induced Pluripotent Stem Cells

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