FAQs Epi5 Episomal iPSC Reprogramming Kit Final
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1. embryoid body formation By Day 7 you can use the TaqMan hPSC Scorecard Panel and or at Day 21 you can perform staining Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support h technologies Epi5 Episomal iPSC Reprogramming Kit Q S 37 Can I cryopreserve the iPSCs You can cryopreserve iPSCs just as you would any other pluripotent stem cells We recommend using a growth medium that contains 10 DMSO for freezing References Hong H Takahashi K Ichisaka T Aoi T Kanagawa O Nakagawa M Okita K and Yamanaka S 2009 Suppression of induced pluripotent stem cell generation by the p53 p21 pathway Nature 460 1132 1135 Kaji K Norrby K Paca A Mileikovsky M Mohseni P and Woltjen K 2009 Virus free induction of pluripotency and subsequent excision of reprogramming factors Nature 458 771 775 Kawamura T Suzuki J Wang Y V Menendez S Morera L B Raya A Wahl G M and Izpisua Belmonte J C 2009 Linking the p53 tumour suppressor pathway to somatic cell reprogramming Nature 460 1140 1144 Okita K Matsumara Y Sato Y Okada A Morizane A Okamoto S Hong H Nakagawa M Tanabe K Tezuka K Shibata T Kunisada T Takahashi M Takahashi J Saji H and Yamanaka S 2011 A more efficient method to generate integration free human i2. recommend keeping freeze thaw cycles to a minimum 2 3 cycles by dispensing the vectors into aliquots and storing them at 15 to 25 C How many experiments can be performed with one kit Each kit provides enough material to process 20 samples when transfecting 100 000 cells using 10 uL per reaction in a 6 well plate Each vial of vectors in the kit contains 20 uL and you will use 1 uL from each vial per experiment Can I omit the p53 dominant negative mutant and EBNA plasmids from my reprogramming experiment We recommend that you use both the EBNA and p53 dominant mutant plasmids in your experiment Our data using fibroblasts on Geltrex matrix coated plates show more colonies are produced when the vectors include both the p53 mutant and EBNA If you omit the p53 mutant and EBNA plasmids efficiency will be significantly reduced Number of Colonies Plasmids 1 3 Plasmids EBNA 1 3 Plasmids p53 mutant 36 Plasmids EBNA p53 mutant 69 3 Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support f technologies Epi5 Episomal iPSC Reprogramming Kit Q S 16 How many cells do I need to start my reprogramming experiment with the Epi5 Kit For each reprogramming experiment using fibroblasts on Geltrex matrix coated plates and CD34 cells each transfection requires at least 1x10
3. the genome causing problems with unrelated developmental processes 9 What cell types have been successfully reprogrammed with the Epi5 Kit iPSCs have been generated from a range of somatic cells including fibroblasts and CD34 cells Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support f technologies Epi5 Episomal iPSC Reprogramming Kit Qs 10 11 Can you reprogram mouse cells with this kit The Epi5 kit was designed to reprogram human cells not mouse cells We currently only have data with this kit on reprogramming human cells which aligns with the use of EBNA oriP episomal vectors mainly in primate cells Although rodent cells lack the oriP replicon and are not permissive some studies replaced or modified oriP with other sequences to mediate replication and some literature reported the use of unmodified EBNA oriP in certain hamster and mouse lines Are there safety concerns with the Epi5 Kit This product is safe for research purpose use only How to use the Epi5 Episomal iPSC Reprogramming Kit 12 13 14 15 How should I store the Epi5 Kit Upon receipt store the kit at 15 to 25 C For long term storage store plasmid vials at 68 to 85 C Can the Epi5 plasmids go through multiple freeze thaw cycles The vectors can be frozen and re thawed however we
4. 6 cells We recommend the following for fibroblasts but this may vary with other cell types Two to four days before transfection plate cells into a T75 flask Cells should be approximately 75 90 confluent on the day of transfection Day 0 Depending on the seeding density and culture conditions the cells may take up to 5 days to reach 75 90 confluency Since overconfluency results in decreased transfection efficiency we recommend replating your cells to achieve 75 90 confluency if they become overconfluent during culturing 17 If I need to use a dish smaller than a T75 flask what seeding density do I need to use When seeding and transfecting fewer cells both the number of cells and the volume of Epi5 vectors must be scaled accordingly Because this can involve pipetting exceedingly small volumes we recommend preparing a vector master mix to reduce pipetting error For example to transfect 50 000 fibroblasts transfer 1 uL from Tube A and 1 uL from Tube B to a sterile vial for the vector master mix and then pipette 1 uL of the master mix to your cells 18 What is the optimal passage number for reprogramming patient fibroblasts We recommend reprogramming patient cells at the earliest passage possible However it is important to have the cells growing and healthy which can take between 1 4 weeks The cells are usually ready to reprogram after completing a total of 3 4 passages 19 Are small molecules required for reprogramm
5. PS cells Nature Methods 8 409 412 Spike B T and Wahl G M 2011 p53 Stem Cells and Reprogramming Tumor Suppression beyond Guarding the Genome Genes Cancer 2 404 419 Thyagarajan B Scheying K Xue H Fontes A Chesnut J Rao M and Lakshimipathy U 2009 A single EBV based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells Regenerative Medicine 4 2 239 250 For Research Use Only Last updated 06 Dec 2013 2013 Life Technologies Corporation All rights reserved The Trademarks mentioned herein are the property of Life Technologies Corporation and or its affiliate s or their respective owners TaqMan is a registered trademark of Roche Molecular Systems Inc used under permission and license Amaxa is a registered trademark of and Nucleofector is a trademark of Lonza Cologne AG Essential 8 is a trademark of Cellular Dynamics International Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support
6. en transfecting with Neon Transfection System Cell death can be highly variable depending on cell type and cell health prior to transfection We observe approximately 25 cell death post transfection with human neonatal foreskin fibroblasts strain BJ in the recommended conditions Can I use different electroporation systems TM We have successfully tested the Amaxa Nucleofector scale 10x for their 100 uL cuvette and plating cells on a 10 cm dish We recommend following the II system for electroporation by increasing the OEM instructions if you choose to use a different electroporation system What is the recommended medium to use for my reprogrammed cells For iPSCs we recommend a feeder free environment in Essential 8 Medium on a Geltrex matrix coated dishes or Vitronectin coated dishes Are the episomal vectors selected out Episomal vectors subsequently can be removed from cells by culturing the cells resulting in a gradual loss of cellular episomal vectors from proliferating cells How can I verify the absence of episomal plasmids in my iPS clones You can detect the presence of vectors in reprogrammed iPSC colonies by endpoint PCR using the primers listed below The EBNA 1 primer set can detect all 5 episomal plasmids in the kit the oriP primer set can detect all but the pCXB EBNA1 plasmid which lacks the OriP gene Primers Transgene Primers Sequence Expected Si
7. es age of the donor patient and the type and number of reprogramming factors 33 How will my cells look after I use the Epi5 Kit Visible morphology changes Groups of cube round shaped cells cells appear unhealthy become visible throughout the plate iPSC colonies emerge Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support f technologies Epi5 Episomal iPSC Reprogramming Kit Q S Expected morphological changes as iPSC colonies emerge from human neonatal foreskin fibroblast cells strain BJ that were reprogrammed using the Epi5 Kit are shown above The phase contrast images were obtained at 5X magnification on Day 2 Day 6 and Day 21 post transfection 34 How can I identify iPSCs The images below show the expected morphology of emerging iPSCs generated from human neonatal foreskin fibroblast cells strain BJ In these 5X magnification images many large nested colonies are visible 35 How do I know when to passage the iPSCs To maintain a healthy culture iPSCs must be monitored and growth medium must be replaced daily In general iPSC colonies should be passaged when the cells reach 75 90 confluence or when most of the colonies are larger than 700 um 36 How can I verify that my iPSCs are pluripotent To verify pluripotency we recommend performing random differentiation using
8. f technologies Epi5 Episomal iPSC Reprogramming Kit Q S Product Qty Cat No Epi5 Episomal iPSC Reprogramming Kit 1 kit A15960 About Epi5 Episomal iPSC Reprogramming Kit 1 What are induced pluripotent stem cells iPSCs iPSCs are genetically reprogrammed somatic cells which exhibit a pluripotent stem cell like state similar to embryonic stem cells iPSCs can be derived by inducing selected gene expression via various methods 2 What is the Epi5 Episomal iPSC Reprogramming Kit The Epi5 Episomal iPSC Reprogramming Kit provides an easy to use highly efficient set of 5 episomal vectors designed by Dr Keisuke Okita in the laboratory of Professor Shinya Yamanaka at the Center for iPS Cell Research and Application CiRA Kyoto University This system produces transgene free virus free human induced pluripotent stem cells iPSCs with efficiencies in the range of 0 04 to 0 3 depending upon the cell type being reprogrammed 3 What vectors are included in the Epi5 Kit The Kit contains two tubes of vectors One tube contains a set of 3 episomal plasmids that carry the reprogramming factors Oct3 4 Sox2 KIf4 L myc and Lin28 and the second tube contains 2 plasmids that express the p53 dominant negative mutant mp53DD and EBNA1 genes 4 How does the Epi5 Kit work The Epi5 Kit vectors are introduced into the cell by electroporation These non integrating vectors replicate o
9. f4 L myc and Lin28 and vectors that enhance iPSC generation by suppressing p53 expression Okita et al 2011 The Episomal iPSC Reprogramming Vectors contain vectors that express the Thompson factors Oct4 Sox2 KIf4 L Myc Lin28 and Nanog 7 What are the benefits of using a viral free reprogramming method The Epi5 Kit is a well described system for producing transgene free virus free iPSCs providing a source of iPSCs Thyagarajan et al 2009 Traditionally direct reprogramming using multiple viral vectors generates iPSCs that contain many integrations of the viral vector which could affect genetic function Kaji et al 2009 In general DNA vectors are more stable than viral vectors can be conveniently thawed and re frozen through several usages prevent unwanted vector integrations and offer a less hazardous method that is more amenable to an end goal involving regenerative medicine 8 What are the benefits of using an integration free reprogramming method Integration free reprogramming methods generate iPSCs that do not contain detectable vectors or transgenes Traditional technologies used for reprogramming e g lentivirus retrovirus integrate into the genome of the target cells The resulting iPSCs and cells differentiated from those iPSCs will contain foreign DNA and could be unsafe and problematic for use in cell therapy and drug discovery applications Furthermore the integration could occur in a critical region of
10. ing with the Epi5 Kit No small molecules are not required Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support f technologies Epi5 Episomal iPSC Reprogramming Kit Q S 20 What is the workflow for reprogramming fibroblasts with the Epi5 Kit The major steps required for reprogramming human neonatal foreskin fibroblast cells are shown below Note that the timeline is provided as a guideline for experimental planning the actual timeline can vary based on the cell type and experimental conditions PSC colonies ready tor transte Electroporate i lonies ready tor trar Plate cells Change Change 4 hie ree Replace spent medium os iium Replace spent medium Day 4 2 0 1 3 5 7 9 11 13 15 17 19 21 Fibroblast Medium N2827 Medium bFGF Essential 8 Medium Geltrex Matrix Day 4 to 2 Plate human fibroblasts into a T75 flask in Fibroblast Medium so that they are 75 90 confluent on the day of transfection Day 0 Day 0 Transfect the cells using the Neon Transfection System Plate transfected cells onto Geltrex matrix coated culture dishes and incubate them overnight in complete Fibroblast Medium Day1to14 Change the medium to N2B27 Medium supplemented with 100 ng mL bFGF replace the spent medium every other day Day 15 Change the medium to Essential 8 Medium and mo
11. m containing cytokines Day 1 Change the medium to N2B27 Medium supplemented with 100 ng mL bFGF replace the spent medium every other day Day 2 to 6 Replenish the culture every day by adding fresh medium Day 7 to 8 Replace the spent medium with fresh medium every day Day 9 Change the medium to Essential 8 Medium and monitor the culture vessels for the emergence of iPSC colonies Replace the spent medium every other day Day 15 to 21 Pick and transfer undifferentiated iPSCs onto fresh Geltrex Matrix coated culture dishes for expansion 22 Can I do reprogramming on feeders While the Epi5 Kit is optimized for feeder free reprogramming the user manual provides the steps required for reprogramming StemPro CD34 cells to generate iPSCs using the kit and the subsequent culture of the reprogrammed cells on MEF feeder layers 23 What is the expected transfection efficiency when using Neon Transfection System Depending on the cell line type the expected transfection efficiency is between 0 04 and 0 3 24 Can I use lipids for transfection We currently do not offer a lipid transfection protocol Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support h technologies Epi5 Episomal iPSC Reprogramming Kit Qs 25 26 27 28 29 What is the expected percentage of cell death wh
12. nitor the culture vessels for the emergence of iPSC colonies Day 21 Pick and transfer undifferentiated iPSCs onto fresh Geltrex matrix coated culture dishes for expansion Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support e technologies FA Qs Epi5 Episomal iPSC Reprogramming Kit 21 What is the workflow for reprogramming CD34 with the Epi5 Kit The major steps required for reprogramming StemPro CD34 cellsare shown below Note that the timeline is provided as a guideline for experimental planning the actual timeline can vary based on the cell type and experimental conditions Electroporate Replace IPSC colonies ready for transfer Add fresh spent Plate medium Change Add fresh medium medium Change Replace spent medium cells k medium H medium zeae So 2 ce aoe ee ee ee ee Da 3 2 1 0 12 3 45 67 8 9 11 13 15 17 19 21 StemPro 34SFM N2B27 Medium bFGF Essential 8 Medium Geltrex Matrix Day 3 Plate StemPro CD34 cells into a 24 well plate in complete StemPro 34 medium containing cytokines i e SCF IL 3 and GM CSF Day 0 Transfect the cells using the Neon Transfection System Plate transfected cells onto Geltrex matrix coated culture dishes and incubate them overnight in complete StemPro 34 mediu
13. nly once per cell cycle with activation of replication by binding of multiple EBNA 1 homodimers to oriP within the nucleus Thyagarajan et al 2009 Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support f technologies Epi5 Episomal iPSC Reprogramming Kit Q S 5 What do the p53 dominant negative mutant and EBNA plasmids do The p53 protein is highly involved in cell cycle regulation and tumor suppression With the activation of cytotoxic responses p53 expression results in cell cycle arrest or cell death to prevent further complications within the system Knockdown of p53 has been shown to improve reprogramming efficiencies as well as to prevent differentiation via the introduction of a variety of knockdown agents Hong et al 2009 Spike amp Wahl 2011 The mp53DD is a dominant negative mutation of the p53 protein that provides higher efficiency knockdown than traditional shRNA systems Kawamura et al 2009 The presence of this gene in an episomal system allows for transient expression of the dominant negative mutant over an extended period of time diluting out with normal passaging of iPSCs 6 What is the difference between the Epi5 Episomal iPSC Reprogramming Kit A15960 and the Episomal iPSC Reprogramming Vectors A14703 The Epi5 Kit contains vectors that express the Yamanaka factors Oct3 4 Sox2 KI
14. ze dae pEP4 SF1 oriP 5 TTC CAC GAG GGT AGT GAA CC 3 544 bp pEP4 SR1 oriP 5 TCG GGG GTG TTA GAG ACA AC 3 pEP4 SF2 oriP 5 ATC GTC AAA GCT GCA CAC AG 3 EBNA 1 666 bp pEP4 SR2 oriP 5 CCC AGG AGT CCC AGT AGT CA 3 PCR Conditions Step Temperature Time Cycles Initial Denaturation 94 C 2 minutes Denaturation 94 C 30 seconds Annealing 55 C 30 seconds 35 40 Elongation 72 C 1 minute Final Elongation 72 C 7 minutes Visit lifetechnologies com epi5 for more product information data protocols and troubleshooting tips or contact stemcell help lifetech com for support f technologies Epi5 Episomal iPSC Reprogramming Kit Q S 30 Should differentiated material be removed prior to passaging Newly derived PSC lines may contain a fair amount of differentiation through passage 4 It is not necessary to remove differentiated material prior to passaging By propagating or splitting the cells the overall culture health should improve throughout the early passages Expected results with the Epi5 Kit 31 How long does it take to see the iPSC colonies You should be able to identify iPSC colonies by morphology roughly from 2 to 3 weeks post transfection 32 Why is there a wide range in the number of colonies obtained using this derivation technique The wide range is due to the variability in the source of fibroblasts neonatal vs adult commercial sources neonatal vs adult patient biopsi
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