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.

http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Stem-Cell-Research/Induced-Pluripotent-Stem-Cells/Sendai-Virus-Reprogramming.html

http://www.lifetechnologies.com/us/en/home.html


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)





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.





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





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





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





Pluripotent Gene Expression is comparable to that of hESCs





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.





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





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





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





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.


http://www.ncbi.nlm.nih.gov/pubmed/22056989






























































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




Appendix






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)


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









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




generation of transgene-free ipscs with cytotune™ ips sendai cell reprogramming kit

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