A cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells in chemically defined culture

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Abstract

Factors limiting the adoption of iPSC technology include the cost of developing lines and the time period that it takes to characterize and bank them, particularly when integration free, feeder free, and Xeno-free components are used. In this manuscript we describe our optimization procedure that enables a single technician to make 20–40 lines at a time in a 24–96 well format in a reliable and reproducible fashion. Improvements spanned the entire workflow and included using RNA virus, reducing cytotoxicity of reagents, developing improved transfection and freezing efficiencies, modifying the manual colony picking steps, enhancing passaging efficiency and developing early criteria of success. These modifications allowed us to make more than two hundred well-characterized lines per year.

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Human induced pluripotent stem cells free of vector and transgene sequences.

Junying Yu, Kejin Hu, Kim Smuga-Otto … (2009)

Reprogramming differentiated human cells to induced pluripotent stem (iPS) cells has applications in basic biology, drug development, and transplantation. Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications. We describe the derivation of human iPS cells with the use of nonintegrating episomal vectors. After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem (ES) cells in proliferative and developmental potential. These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors and removes one obstacle to the clinical application of human iPS cells.

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Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.

Daniel Gulbranson, Guokai Chen, Andrew J. Thomson … (2012)

This protocol describes an EDTA-based passaging procedure to be used with chemically defined E8 medium that serves as a tool for basic and translational research into human pluripotent stem cells (PSCs). In this protocol, passaging one six-well or 10-cm plate of cells takes about 6-7 min. This enzyme-free protocol achieves maximum cell survival without enzyme neutralization, centrifugation or drug treatment. It also allows for higher throughput, requires minimal material and limits contamination. Here we describe how to produce a consistent E8 medium for routine maintenance and reprogramming and how to incorporate the EDTA-based passaging procedure into human induced PSC (iPSC) derivation, colony expansion, cryopreservation and teratoma formation. This protocol has been successful in routine cell expansion, and efficient for expanding large-volume cultures or a large number of cells with preferential dissociation of PSCs. Effective for all culture stages, this procedure provides a consistent and universal approach to passaging human PSCs in E8 medium.

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Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.

Audrey French, Paul Gadue, Jyh-Chang Jean … (2010)

The development of methods to achieve efficient reprogramming of human cells while avoiding the permanent presence of reprogramming transgenes represents a critical step toward the use of induced pluripotent stem cells (iPSC) for clinical purposes, such as disease modeling or reconstituting therapies. Although several methods exist for generating iPSC free of reprogramming transgenes from mouse cells or neonatal normal human tissues, a sufficiently efficient reprogramming system is still needed to achieve the widespread derivation of disease-specific iPSC from humans with inherited or degenerative diseases. Here, we report the use of a humanized version of a single lentiviral "stem cell cassette" vector to accomplish efficient reprogramming of normal or diseased skin fibroblasts obtained from humans of virtually any age. Simultaneous transfer of either three or four reprogramming factors into human target cells using this single vector allows derivation of human iPSC containing a single excisable viral integration that on removal generates human iPSC free of integrated transgenes. As a proof of principle, here we apply this strategy to generate >100 lung disease-specific iPSC lines from individuals with a variety of diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, including cystic fibrosis, α-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease. Moreover, we demonstrate that human iPSC generated with this approach have the ability to robustly differentiate into definitive endoderm in vitro, the developmental precursor tissue of lung epithelia.

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Journal

Journal ID (nlm-ta): Sci Rep

Journal ID (iso-abbrev): Sci Rep

Title: Scientific Reports

Publisher: Nature Publishing Group

ISSN (Electronic): 2045-2322

Publication date (Electronic): 11 June 2015

Publication date Collection: 2015

Volume: 5

Electronic Location Identifier: 11319

Affiliations

[1 ]Center for Molecular Medicine, National Heart, Lung and Blood Institute , Bethesda, Maryland

[2 ]National Center for Advancing Translational Sciences, National Institutes of Health , Bethesda, Maryland

[3 ]Center for Regenerative Medicine, National Institutes of Health , Bethesda, Maryland

[4 ]Faculty of Health Sciences, University of Macau , China

Author notes

[a ] GuokaiChen@ 123456umac.edu

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Publisher Item ID: srep11319

DOI: 10.1038/srep11319

PMC ID: 4464084

PubMed ID: 26066579

SO-VID: a8011972-c6a6-41d9-a2d3-26eb50a49922

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This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

A cost-effective and efficient reprogramming platform for large-scale production of integration-free human induced pluripotent stem cells in chemically defined culture

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Most cited references 7

Human induced pluripotent stem cells free of vector and transgene sequences.

Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions.

Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette.

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