Generation of Transgene-free iPSCs with CytoTune™ iPS Sendai Cell Reprogramming Kit

Highly Efficient Generation of
Transgene-free iPSCs
November, 2011

iPSC Experimental Workflow: Challenges and Solutions

Efficiency
<0.1%

Current bottleneck

Reprogram: Efficient Methods

Reprogramming Technologies
• Integrating viral methods
• Non-integrating viral methods
• Non-integrating nonviral methods

2
For the full protocol, visit the CytoTune™ page on the Life Technologies website. and confidential
12/2/2011 | Life Technologies™ Proprietary

Current Methods for Reprogramming

Somatic Cell Intermediate iPSC Colony

miRNA
CytoTuneTM
Retrovirus (Sendai virus) RNA
EFFICIENCY

Lentivirus Excisable Lentivirus
Transposon
Episomal Vector

Small Molecule
Adenovirus
Protein
SAFETY
(Modified from Gonzalez et al., 2011)

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CytoTuneTM -iPS Reprogramming Kit Uses Sendai Virus

Sendai Virus Sendai Virus Vectors

• A respiratory mouse parainfluenza type I virus of • Fusion (F) gene deleted for retention of infectivity but
mouse and rat, belonging to the Paramyxoviridae cannot produce infectious particles
family • Capable of transducing a wide range of animal cells with
• Genome of virus is RNA (minus sense) a short contact time (~24 hours)

• Replicates exclusively in the cytoplasm • High transduction efficiency with low multiplicity of
infection (MOI) yields a high level of transgene expression
• Non-pathogenic to humans
Non-integrating: vectors and transgenes are
No possibility of altering host chromosomes. eliminated from host cells.

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Comparison of Reprogramming Efficiency, Integration and Ease-of-Use

Sendai Lentivirus/ Adenovirus Episomal/ Protein Modified
virus Retrovirus Minicircle mRNAs

Efficiency 0.01-1% 0.001-0.01% 0.0001% 0.0001% 0.00001% >1%

No
Integration No Yes No (DNA) No No
(DNA)

Multiple
No No No No/Yes Yes Yes
Transductions

1. Up to 100-fold higher reprogramming efficiency than lentiviral methods
2. 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

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Simplified and High Efficiency Reprogramming with CytoTune™
CytoTune™ (Sendai virus) Alkaline Phosphatase
Staining
Day -1 Day 1 Day 8 Day 14 Day 28

Plate
Cells

Add transforming reagent Passage cells onto fresh
Emerging
(virus or mRNA) MEFs Colonies Emerging colonies Efficiency: 1.56

Lentivirus
Day -1 Day 1 Day 9 Day 14 Day 21

Plate Perform live
Cells staining
Emerging
Colonies
Efficiency:0.003
mRNA
**Note: Media Preparation Required (Pluriton Conditioned Medium) (7 Days)

Day -1 Day Day 12 Day 17 Day 28
1

Change
Plate Medium
Cells Emerging Efficiency:0.02
Colonies

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CytoTune™-mediated iPSC Clones are Integration-free and Pluripotent
Anti-Sendai Antibody
TaqMan® Protein Assay

Positive: Early clone Negative: with passage

Sendai Vector

• 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

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Pluripotent Gene Expression is comparable to that of hESCs

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Expanded iPSC Clones Maintain Pluripotency
and Differentiation Potential
Dapi Dapi
Dapi
Oct4 SMA
Tra-1-60

Dapi Dapi Dapi
SSEA4 Sox2 bIIITub

Dapi Dapi Dapi
Tra-1-81 Nanog AFP

Isolated clones expand, express pluripotent markers, and can be spontaneously differentiated.

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Derivation CytoTuneTM-mediated iPSCs in Feeder-free
Conditions
Expression of pluripotent markers

Differentiation potential – Expression of lineage markers
AFP SMA Beta-III
StemPro® hESC SFM
on hESC-qualified Geltrex™

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Derivation of CytoTuneTM-mediated iPSCs in Xeno-free Conditions
Expression of pluripotent markers
Phase Nanog DAPI

SSEA4 Sox2 Tra1-60

Xeno-free Conditions
KO-DMEM, KSR-XF, GF
Cocktail w/ Human Feeders
Differentiation potential – Expression of lineage markers
AFP SMA Beta-III

Tra-1-81

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Derivation of CytoTuneTM-mediated iPSCs from CD34+ Cells

Phase Day 20 Tra1-60

CD34+

CytoTune Sendai Virus AP-Day 24
StemPro 34

CD34+ Colony formation

Day 19
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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.

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Acknowledgements

Pauline Lieu Uma Lakshmipathy David Welch
Chad MacArthur Upinder Singh Kate Wagner
Andrew Fontes Rene Quintanilla Alaine Maxwell
Jasmeet Kaur Scot Grecian Jennifer Taylor
Mohan Vemuri Kyle Gee Fiona Coats
Mark Powers

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.

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Appendix

For the full protocol, visit the CytoTune™ page on the Life Technologies website.

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 ** High efficiency
**Non-integrating
A13780-01 1 pack (1 of each gene) **Single application required
A13780-02 3 pack (3 of each gene)

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Less Background with Sendai vs. Retro

Retrovirus –
high background, hard
Retro- to distinguish iPSC
virus colonies.

Tra1-81 Phase + Tra1-81

Sendai – low
background, easier to
Sendai distinguish iPSC
virus colonies.

Tra1-81 Phase + Tra1-81

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Generation of Transgene-free iPSCs with CytoTune™ iPS Sendai Cell Reprogramming Kit

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Generation of Transgene-free iPSCs with CytoTune™ iPS Sendai Cell Reprogramming Kit