Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix
(2017) In Journal of visualized experiments : JoVE- Abstract
Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have... (More)
Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.
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- author
- Uhlin, Elias ; Marin Navarro, Ana ; Rönnholm, Harriet ; Day, Kelly ; Kele, Malin and Falk, Anna LU
- publishing date
- 2017-07-07
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Cell Culture Techniques/methods, Cellular Reprogramming/genetics, Humans, Induced Pluripotent Stem Cells/cytology, Laminin/metabolism
- in
- Journal of visualized experiments : JoVE
- issue
- 125
- pages
- 8 pages
- publisher
- JoVE
- external identifiers
-
- scopus:85024366374
- pmid:28715399
- ISSN
- 1940-087X
- DOI
- 10.3791/56146
- language
- English
- LU publication?
- no
- id
- d14307dc-ab13-4d2a-928e-2472af643ea7
- alternative location
- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609321/pdf/jove-125-56146.pdf
- date added to LUP
- 2021-08-09 16:33:47
- date last changed
- 2024-04-20 09:38:30
@article{d14307dc-ab13-4d2a-928e-2472af643ea7,
abstract = {{<p>Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.</p>}},
author = {{Uhlin, Elias and Marin Navarro, Ana and Rönnholm, Harriet and Day, Kelly and Kele, Malin and Falk, Anna}},
issn = {{1940-087X}},
keywords = {{Cell Culture Techniques/methods; Cellular Reprogramming/genetics; Humans; Induced Pluripotent Stem Cells/cytology; Laminin/metabolism}},
language = {{eng}},
month = {{07}},
number = {{125}},
publisher = {{JoVE}},
series = {{Journal of visualized experiments : JoVE}},
title = {{Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix}},
url = {{http://dx.doi.org/10.3791/56146}},
doi = {{10.3791/56146}},
year = {{2017}},
}