A comparison of non-integrating reprogramming methods

Schlaeger, Thorsten M.; Dahéron, Laurence; Brickler, Thomas; Entwisle, Samuel W.; Chan, Karrie; Cianci, Amelia L.; DeVine, Alexander L.; Ettenger, Andrew; Fitzgerald, Kelly; Godfrey, Michelle; Gupta, Dipti; McPherson, Jade; Malwadkar, Prerana; Gupta, Manav; Bell, Blair; Doi, Akiko; Jung, Namyoung; Li, Xin; Lynes, Maureen M.; Brookes, Emily; Cherry, Anne; Demirbas, Didem; Tsankov, Alexander M.; Zon, Leonard I.; Rubin, Lee L.; Feinberg, Andrew P.; Meissner, Alexander; Cowan, Chad A.; Daley, George Q.

doi:10.1038/nbt.3070

Cited by 438 publications

(443 citation statements)

References 37 publications

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“…Indeed, when enhanced transgene expression was achieved by including the woodchuck hepatitis virus post-transcriptional regulatory element in the design, an improved efficiency of reprogramming in human cord blood CD34+ cells was observed [225]. Moreover, these data corroborate evidence that highlights the importance of careful screening of iPSC clones for the presence of transgene integration or persistent expression of exogenous sequences [46,186], as one of the iPSC clones (out of six screened clones) showed the integration of one of the reprogramming vectors.…”

Section: Discussion and Future Directionssupporting

confidence: 72%

“…This approximate the efficiency observed with the integrating viral vectors which have the highest efficiency of reprogramming; however, its advantage is negated by the requirement for repeated transfection [41] and the considerable failure rate in reprogramming [186]. Nevertheless, episomal vectors remain a viable option for reprogramming to pluripotency [186] that can be compatible with clinical translation [221].…”

Section: Discussion and Future Directionsmentioning

confidence: 96%

“…The rapid loss of episomal vectors may limit the expression of reprogramming factors to the early stages of reprogramming, possibly contributing to the low efficiency of this approach. Even with further enhancement of the episomal-based approach by suppressing TP53 and substituting cMyc with L-Myc, the efficiency of the system remains significantly lower than other non-integrative approaches, such as the Sendai-virus or mRNA-based reprogramming [186], although one study has reported otherwise under feeder-free conditions [221]. The role of TP53 suppression in promoting reprogramming has been documented in various studies; however, TP53 plays an important role in surveillance of the maintenance of genomic integrity [222].…”

Section: Discussion and Future Directionsmentioning

confidence: 99%

“…Alternatively, cells can be transduced with the reprogramming factors in the protein form [42,43,185]. Episomal-based reprogramming of somatic cells has been reported to be a more reliable method for iPSC derivation from fibroblasts in comparison with mRNA, result in a rapid and passive loss of the transfected 45 vector in comparison to Sendai virus-based reprogramming, and more importantly, iPSCs may be more appropriate for potential therapeutic use [186].…”

Section: Excision Of the Selection Cassettementioning

confidence: 99%

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CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

Al-Ateeq¹

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Beta thalassaemia is caused by mutations in the adult haemoglobin gene (HBB) and is one of the most prevalent monogenic blood disorders worldwide. The transplantation of haematopoietic stem cells derived from gene-corrected patient induced pluripotent stem cells (iPSCs) could provide a promising therapeutic strategy. Here, the generation and characterisation of footprint-free iPSCs from dermal fibroblasts of an individual with a homozygous beta thalassaemia-causing mutation affecting splicing is described (Chapter III). Successful gene correction of the disease-causing mutation was achieved by employing a CRISPR/Cas9 dual nickase approach and two donor design strategies aimed at reducing undesired on-target mutagenesis (Chapter IV), followed by the This study combined cutting-edge cellular reprogramming methods to generate beta thalassaemia iPSCs, a double nickase-mediated gene editing approach and piggyBac transposase-aided excision to achieve seamless gene repair, and a CRISPRa approach to model the impact of the diseasecausing mutation and demonstrate restoration of normal transcription following genetic repair.Having established a platform for precise gene correction and for validation of the restoration of the functional allele in this monogenic disease, this strategy may provide a viable avenue for iPSCiii based cell therapy of beta thalassaemic patients provided mature engraftable blood cells can be generated from hiPSCs in the future.iv

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Section: Discussion and Future Directionssupporting

confidence: 72%

Section: Discussion and Future Directionsmentioning

confidence: 96%

Section: Discussion and Future Directionsmentioning

confidence: 99%

Section: Excision Of the Selection Cassettementioning

confidence: 99%

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CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

Al-Ateeq¹

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“…The conversion efficiency of fibroblasts into an NSC state using episomal vectors is, however, relatively lower than that of retroviral vector-mediated conversion potentially due to the relatively low transfection efficiency of episomal vectors (15,25). Thus, further optimization is necessary for enhancing the reprogramming efficiency, for instance, by using small molecules as in previous iPSC studies.…”

Section: Discussionmentioning

confidence: 99%

Generation of Integration-free Induced Neural Stem Cells from Mouse Fibroblasts

Kim

Kwak³

et al. 2016

Journal of Biological Chemistry

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The viral vector-mediated overexpression of the defined transcription factors, Brn4/Pou3f4, Sox2, Klf4, and c-Myc (BSKM), could induce the direct conversion of somatic fibroblasts into induced neural stem cells (iNSCs). However, viral vectors may be randomly integrated into the host genome thereby increasing the risk for undesired genotoxicity, mutagenesis, and tumor formation. Here we describe the generation of integration-free iNSCs from mouse fibroblasts by non-viral episomal vectors containing BSKM. The episomal vector-derived iNSCs (eiNSCs) closely resemble control NSCs, and iNSCs generated by retrovirus (r-iNSCs) in morphology, gene expression profile, epigenetic status, and self-renewal capacity. The e-iNSCs are functionally mature, as they could differentiate into all the neuronal cell types both in vitro and in vivo. Our study provides a novel concept for generating functional iNSCs using a non-viral, non-integrating, plasmid-based system that could facilitate their biomedical applicability. Neural stem cells (NSCs),3 somatic stem cells of the brain and spinal cord, could differentiate into three major cell types (neurons, astrocytes, and oligodendrocytes) in the central nervous system (CNS) in response to relevant signaling pathways (1). A number of in vitro transplantation studies with disease animal models have demonstrated that the engrafted NSCs can functionally rescue disease-related phenotypes, proving their therapeutic potential (2-5). Thus, NSCs have long been considered as a potential source for replacing damaged cells and tissues often found in various neurodegenerative diseases (2-5). However, major concerns associated with these cells may preclude expediting advances into their therapeutic application. First, the accessibility of NSCs is largely restricted in vitro, due to their limited origins. Second, the autologous cell transplantation is not feasible using NSCs from their in vivo origins, and furthermore, allogeneic transplantation of NSCs may raise the potential for immune rejection. Finally, with current culture conditions, it is technically challenging to homogeneously maintain human NSCs in vitro (6, 7). Thus, NSC-like cells generated from easily accessible patient somatic cell types such as fibroblasts and blood cells could serve as an autologous source for therapeutic applications, thereby overcoming current obstacles to NSC-mediated translation research.We and others have demonstrated the direct conversion of somatic fibroblasts into self-renewing and multipotent induced neural stem cells (iNSCs) or induced neural progenitor cells (iNPCs) by the forced expression of different sets of transcription factors (8 -12). Recently, Thier et al. (12) have shown that the restricted expression of Oct4 by a tetracycline-dependent lentiviral vector, together with retrovirus-mediated overexpression of Sox2, Klf4, and c-Myc, could elicit the conversion of fibroblasts into iNSCs. Simultaneously, we have generated multipotent and self-renewing iNSCs from mouse fibroblasts by retrovirus-mediated ...

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Tissue Engineering Biomaterials

Peter

Eyster

Doleyres

2018

Encyclopedia of Polymer Science and Technology

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Tissue engineering has shown great promise in developing novel therapies for treating injured tissue and organ failure. Scaffolds play a pivotal role in tissue engineering. Biodegradable polymers are the primary choice of material for tissue engineering scaffolds, due to their numerous advantageous properties. In this article, the principles of tissue engineering are discussed in relation to polymer science and engineering. The most frequently used polymers in tissue engineering are briefly reviewed. These include synthetic and natural polymers commonly used in constructing both porous and hydrogel scaffolds. Their structure and critical properties in relation to scaffold function are discussed in depth, along with a detailed review of the important roles functionalized materials and controlled release play in tissue engineering. Important polymer processing techniques in the context of scaffold fabrication are also reviewed. These include the textile technologies, electrospinning, particulate‐leaching techniques, phase‐separation techniques, rapid prototyping, and other novel three‐dimensional (3D) fabrication techniques. In this update, the impact of pluripotent stem cells, conductive polymers, controlled drug release, 3D printing, and nanofibrous topology on tissue engineering are also discussed.

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A comparison of non-integrating reprogramming methods

Cited by 438 publications

References 37 publications

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

CRISPR/Cas9-mediated traceless gene correction and activation of the HBB locus in iPSCs with the beta thalassaemia mutation

Generation of Integration-free Induced Neural Stem Cells from Mouse Fibroblasts

Tissue Engineering Biomaterials

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