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

被引：91

作者：

Beers, Jeanette ^{[1
]}

Linask, Kaari L. ^{[1
]}

Chen, Jane A. ^{[1
]}

Siniscalchi, Lauren I. ^{[1
]}

Lin, Yongshun ^{[1
]}

Zheng, Wei ^{[2
]}

Rao, Mahendra ^{[3
]}

Chen, Guokai ^{[1
,4
]}

机构：

[1] NHLBI, Ctr Mol Med, Bethesda, MD 20892 USA

[2] NIH, Natl Ctr Adv Translat Sci, Bethesda, MD 20892 USA

[3] NIH, Ctr Regenerat Med, Bethesda, MD 20892 USA

[4] Univ Macau, Fac Hlth Sci, Macau, Peoples R China

来源：

SCIENTIFIC REPORTS | 2015年 / 5卷

基金：

美国国家卫生研究院;

关键词：

GENERATION; INDUCTION; BIOLOGY; VECTOR;

D O I：

10.1038/srep11319

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页数：9

共 13 条

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

Beers, Jeanette ;

Gulbranson, Daniel R. ;

George, Nicole ;

Siniscalchi, Lauren I. ;

Jones, Jeffrey ;

Thomson, James A. ;

Chen, Guokai .

NATURE PROTOCOLS, 2012, 7 (11) :2029-2040

[2]

Burridge PW, 2014, NAT METHODS, V11, P855, DOI [10.1038/NMETH.2999, 10.1038/nmeth.2999]

[3] HTqPCR: high-throughput analysis and visualization of quantitative real-time PCR data in R [J].

Dvinge, Heidi ;

Bertone, Paul .

BIOINFORMATICS, 2009, 25 (24) :3325-3326

[4] Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome [J].

Fusaki, Noemi ;

Ban, Hiroshi ;

Nishiyama, Akiyo ;

Saeki, Koichi ;

Hasegawa, Mamoru .

PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES, 2009, 85 (08) :348-362

[5] Bioconductor: open software development for computational biology and bioinformatics [J].

Gentleman, RC ;

Carey, VJ ;

Bates, DM ;

Bolstad, B ;

Dettling, M ;

Dudoit, S ;

Ellis, B ;

Gautier, L ;

Ge, YC ;

Gentry, J ;

Hornik, K ;

Hothorn, T ;

Huber, W ;

Iacus, S ;

Irizarry, R ;

Leisch, F ;

Li, C ;

Maechler, M ;

Rossini, AJ ;

Sawitzki, G ;

Smith, C ;

Smyth, G ;

Tierney, L ;

Yang, JYH ;

Zhang, JH .

GENOME BIOLOGY, 2004, 5 (10)

[6] Chemical Approaches to Stem Cell Biology and Therapeutics [J].

Li, Wenlin ;

Li, Ke ;

Wei, Wanguo ;

Ding, Sheng .

CELL STEM CELL, 2013, 13 (03) :270-283

[7]

Lieu Pauline T, 2013, Methods Mol Biol, V997, P45, DOI 10.1007/978-1-62703-348-0_5

[8] Generation of Transgene-Free Lung Disease-Specific Human Induced Pluripotent Stem Cells Using a Single Excisable Lentiviral Stem Cell Cassette [J].

Somers, Aba ;

Jean, Jyh-Chang ;

Sommer, Cesar A. ;

Omari, Amel ;

Ford, Christopher C. ;

Mills, Jason A. ;

Ying, Lei ;

Sommer, Andreia Gianotti ;

Jean, Jenny M. ;

Smith, Brenden W. ;

Lafyatis, Robert ;

Demierre, Marie-France ;

Weiss, Daniel J. ;

French, Deborah L. ;

Gadue, Paul ;

Murphy, George J. ;

Mostoslavsky, Gustavo ;

Kotton, Darrell N. .

STEM CELLS, 2010, 28 (10) :1728-1740

[9] Induction of pluripotent stem cells from adult human fibroblasts by defined factors [J].

Takahashi, Kazutoshi ;

Tanabe, Koji ;

Ohnuki, Mari ;

Narita, Megumi ;

Ichisaka, Tomoko ;

Tomoda, Kiichiro ;

Yamanaka, Shinya .

CELL, 2007, 131 (05) :861-872

[10] Induced pluripotent stem cells in medicine and biology [J].

Takahashi, Kazutoshi ;

Yamanaka, Shinya .

DEVELOPMENT, 2013, 140 (12) :2457-2461

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