Generation of Transgene-free iPSCs with CytoTune™ iPS Sendai Cell Reprogramming Kit
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Transcript of Generation of Transgene-free iPSCs with CytoTune™ iPS Sendai Cell Reprogramming Kit
Highly Efficient Generation of Transgene-free iPSCs
November, 2011
2 12/2/2011 | Life Technologies™ Proprietary and confidential
iPSC Experimental Workflow: Challenges and Solutions
Reprogram: Efficient Methods
Current bottleneck
Reprogramming Technologies
• Integrating viral methods
• Non-integrating viral methods
• Non-integrating nonviral methods
Efficiency
<0.1%
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
3 12/2/2011 | Life Technologies™ Proprietary and confidential
Somatic Cell Intermediate iPSC Colony
Current Methods for Reprogramming
Adenovirus
Retrovirus
Lentivirus Excisable Lentivirus
RNA
Transposon
Episomal Vector
Small Molecule
Protein
SAFETY
EF
FIC
IEN
CY
(Modified from Gonzalez et al., 2011)
miRNA
CytoTuneTM
(Sendai virus)
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
4 12/2/2011 | Life Technologies™ Proprietary and confidential
CytoTuneTM -iPS Reprogramming Kit Uses Sendai Virus
Sendai Virus
• A respiratory mouse parainfluenza type I virus of
mouse and rat, belonging to the Paramyxoviridae
family
• Genome of virus is RNA (minus sense)
• Replicates exclusively in the cytoplasm
• Non-pathogenic to humans
No possibility of altering host chromosomes.
Sendai Virus Vectors
• Fusion (F) gene deleted for retention of infectivity but
cannot produce infectious particles
• Capable of transducing a wide range of animal cells with
a short contact time (~24 hours)
• High transduction efficiency with low multiplicity of
infection (MOI) yields a high level of transgene expression
Non-integrating: vectors and transgenes are
eliminated from host cells.
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
5 12/2/2011 | Life Technologies™ Proprietary and confidential
Comparison of Reprogramming Efficiency, Integration and Ease-of-Use
1. Up to 100-fold higher reprogramming efficiency than lentiviral methods2. Sendai is a non integrating virus, and the number of non-iPS colonies are
significantly reduced (Low background)3. Only one round of transduction is required for most cell types
Sendai
virus
Lentivirus/
Retrovirus
Adenovirus Episomal/
Minicircle
Protein Modified
mRNAs
Efficiency 0.01-1% 0.001-0.01% 0.0001% 0.0001% 0.00001% >1%
Integration No Yes No (DNA)No
(DNA)No No
Multiple
TransductionsNo No No No/Yes Yes Yes
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
6 12/2/2011 | Life Technologies™ Proprietary and confidential
Day 1Day -1 Day 8 Day 14
Emerging
Colonies
CytoTune™ (Sendai virus)
Plate
Cells
Day 28
Add transforming reagent
(virus or mRNA)Passage cells onto fresh
MEFsEmerging colonies Efficiency: 1.56
Day 1Day -1 Day 9 Day 14
Emerging
Colonies
Lentivirus
Plate
Cells
Day 21
Perform live
staining
Efficiency:0.003
Simplified and High Efficiency Reprogramming with CytoTune™
Day
1
**Note: Media Preparation Required (Pluriton Conditioned Medium) (7 Days)
Day 17Day -1
Plate
Cells
Change
Medium
Emerging
Colonies
mRNA
Day 28Day 12
Efficiency:0.02
Alkaline Phosphatase
Staining
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
7 12/2/2011 | Life Technologies™ Proprietary and confidential
CytoTune™-mediated iPSC Clones are Integration-free and Pluripotent
Anti-Sendai Antibody
Negative: with passagePositive: Early clone
Sendai Vector
TaqMan® Protein Assay
• Presence of residual Sendai virus in early clones is lost with passage
• PCR analysis confirms absence of the Sendai vector and expression of pluripotent markers
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
8 12/2/2011 | Life Technologies™ Proprietary and confidential
Pluripotent Gene Expression is comparable to that of hESCs
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
9 12/2/2011 | Life Technologies™ Proprietary and confidential
Expanded iPSC Clones Maintain Pluripotency
and Differentiation Potential
Tra-1-60
Tra-1-81
SSEA4
SMA
bIIITub
AFP
Oct4
Sox2
Nanog
Dapi
Dapi
Dapi
Dapi
Dapi
Dapi
Dapi
Dapi
Dapi
Isolated clones expand, express pluripotent markers, and can be spontaneously differentiated.
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
10 12/2/2011 | Life Technologies™ Proprietary and confidential
Derivation CytoTuneTM-mediated iPSCs in Feeder-free Conditions
StemPro® hESC SFM
on hESC-qualified Geltrex™
Expression of pluripotent markers
Differentiation potential – Expression of lineage markers
AFP SMA Beta-III
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
11 12/2/2011 | Life Technologies™ Proprietary and confidential
Tra-1-81
Derivation of CytoTuneTM-mediated iPSCs in Xeno-free Conditions
Xeno-free Conditions
KO-DMEM, KSR-XF, GF
Cocktail w/ Human Feeders
Phase Nanog
SSEA4
DAPI
Sox2 Tra1-60
Expression of pluripotent markers
Differentiation potential – Expression of lineage markers
AFP SMA Beta-III
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
12 12/2/2011 | Life Technologies™ Proprietary and confidential
Derivation of CytoTuneTM-mediated iPSCs from CD34+ Cells
CD34+
CytoTune Sendai Virus
StemPro 34
Day 20Phase Tra1-60
Day 19
CD34+ Colony formation
AP-Day 24
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
13 12/2/2011 | Life Technologies™ Proprietary and confidential
Sendai Virus References
Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson's disease.
Kriks S, Shim JW, Piao J, Ganat YM, Wakeman DR, Xie Z, Carrillo-Reid L, Auyeung G, Antonacci C, Buch A, Yang L, Beal MF, Surmeier DJ, Kordower JH, Tabar
V, Studer L.
Nature. 2011 Nov 6. doi: 10.1038/nature10648
Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.
Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu PQ, Paschon DE, Miranda E, Ordóñez A, Hannan NR, Rouhani FJ, Darche S, Alexander G, Marciniak SJ,
Fusaki N, Hasegawa M, Holmes MC, Di Santo JP, Lomas DA, Bradley A, Vallier L.
Nature. 2011 Oct 12;478(7369):391-4. doi: 10.1038/nature10424.
Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors.
Ban H, Nishishita N, Fusaki N, Tabata T, Saeki K, Shikamura M, Takada N, Inoue M, Hasegawa M, Kawamata S, Nishikawa S.
Proc Natl Acad Sci U S A. 2011 Aug 23;108(34):14234-9. Epub 2011 Aug 5.
Development of defective and persistent Sendai virus vector: a unique gene delivery/expression system ideal for cell reprogramming.
Nishimura K, Sano M, Ohtaka M, Furuta B, Umemura Y, Nakajima Y, Ikehara Y, Kobayashi T, Segawa H, Takayasu S, Sato H, Motomura K, Uchida E, Kanayasu-
Toyoda T, Asashima M, Nakauchi H, Yamaguchi T, Nakanishi M.
J Biol Chem. 2011 Feb 11;286(6):4760-71. Epub 2010 Dec 7.
Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells.
Seki T, Yuasa S, Oda M, Egashira T, Yae K, Kusumoto D, Nakata H, Tohyama S, Hashimoto H, Kodaira M, Okada Y, Seimiya H, Fusaki N, Hasegawa M, Fukuda
K.
Cell Stem Cell. 2010 Jul 2;7(1):11-4.
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.
Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M.
Proc Jpn Acad Ser B Phys Biol Sci. 2009;85(8):348-62.
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
14 12/2/2011 | Life Technologies™ Proprietary and confidential
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
TaqMan is a registered trademark of Roche Molecular Systems, Inc.
© 2011 Life Technologies Corporation. All rights reserved.
The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
Pauline Lieu
Chad MacArthur
Andrew Fontes
Jasmeet Kaur
Mohan Vemuri
Uma Lakshmipathy
Upinder Singh
Rene Quintanilla
Scot Grecian
Kyle Gee
David Welch
Kate Wagner
Alaine Maxwell
Jennifer Taylor
Fiona Coats
Mark Powers
Acknowledgements
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
Appendix
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
16 12/2/2011 | Life Technologies™ Proprietary and confidential
Invitrogen™ CytoTune™ -iPS Reprogramming Kit
This kit contains Sendai virus particles of four genes (Oct4, Sox2, Klf4, c-Myc) used to reprogram somatic cells to induced pluripotent stem cells (iPSCs)
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
Cat. No. Unit Size
A13780-01 1 pack (1 of each gene)
A13780-02 3 pack (3 of each gene)
** High efficiency **Non-integrating **Single application required
For the full protocol, visit the CytoTune™ page on the Life Technologies website.
17 12/2/2011 | Life Technologies™ Proprietary and confidential
Less Background with Sendai vs. Retro
Phase + Tra1-81Tra1-81
Phase + Tra1-81Tra1-81
Retrovirus –high background, hard to distinguish iPSC colonies.
Sendai virus
Sendai – low background, easier to distinguish iPSC colonies.
Retro-virus
For the full protocol, visit the CytoTune™ page on the Life Technologies website.