Document Detail


Reprogramming of human fibroblasts to induced pluripotent stem cells under xeno-free conditions.
MedLine Citation:
PMID:  19890879     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The availability of induced pluripotent stem cells (iPSCs) has created extraordinary opportunities for modeling and perhaps treating human disease. However, all reprogramming protocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely devoid of xenobiotics. We first developed a xeno-free cell culture media that supported the long-term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno-free medium. We also derived primary cultures of human dermal fibroblasts under strict xeno-free conditions (XF-HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin (trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G-pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF-HFF cells under xeno-free conditions. A total of 10 xeno-free human iPSC lines were generated, which could be continuously passaged in xeno-free conditions and maintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency-associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSCs can be generated and maintained under strict xeno-free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC-based therapies.
Authors:
Ignasi Rodríguez-Pizà; Yvonne Richaud-Patin; Rita Vassena; Federico González; María José Barrero; Anna Veiga; Angel Raya; Juan Carlos Izpisúa Belmonte
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Stem cells (Dayton, Ohio)     Volume:  28     ISSN:  1549-4918     ISO Abbreviation:  Stem Cells     Publication Date:  2010 Jan 
Date Detail:
Created Date:  2010-01-19     Completed Date:  2010-03-01     Revised Date:  2011-12-15    
Medline Journal Info:
Nlm Unique ID:  9304532     Medline TA:  Stem Cells     Country:  United States    
Other Details:
Languages:  eng     Pagination:  36-44     Citation Subset:  IM    
Affiliation:
Center for Regenerative Medicine in Barcelona, Dr. Aiguader 88, 08003 Barcelona, Spain.
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MeSH Terms
Descriptor/Qualifier:
Animals
Biological Markers / metabolism
Cell Culture Techniques
Cell Proliferation
Cell Transdifferentiation* / genetics
Cells, Cultured
Culture Media
Epidermal Growth Factor / metabolism
Fibroblasts / metabolism*,  pathology
GPI-Linked Proteins
Gene Expression Regulation, Developmental
Homeodomain Proteins / metabolism
Humans
Induced Pluripotent Stem Cells / metabolism*,  pathology
Intercellular Signaling Peptides and Proteins
Kruppel-Like Transcription Factors / genetics,  metabolism
Male
Membrane Glycoproteins / metabolism
Mice
Mice, SCID
Neoplasm Proteins / metabolism
Nuclear Reprogramming*
Octamer Transcription Factor-3 / genetics,  metabolism
Recombinant Proteins / metabolism
SOXB1 Transcription Factors / genetics,  metabolism
Teratoma / metabolism,  pathology
Transduction, Genetic
Chemical
Reg. No./Substance:
0/Biological Markers; 0/Culture Media; 0/GKLF protein; 0/GPI-Linked Proteins; 0/Homeodomain Proteins; 0/Intercellular Signaling Peptides and Proteins; 0/Kruppel-Like Transcription Factors; 0/Membrane Glycoproteins; 0/NANOG protein, human; 0/Neoplasm Proteins; 0/Octamer Transcription Factor-3; 0/POU5F1 protein, human; 0/Pou5f1 protein, mouse; 0/Recombinant Proteins; 0/SOX2 protein, human; 0/SOXB1 Transcription Factors; 0/Sox2 protein, mouse; 0/TDGF1 protein, human; 62229-50-9/Epidermal Growth Factor

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