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UNIT 4A.4Using MicroRNAs to Enhance theGeneration of Induced Pluripotent StemCellsZhonghan Li1and Tariq M. Rana11Program for RNA Biology, Sanford-Burnham Medical Research Institute, La Jolla,CaliforniaABSTRACTSomatic cells reprogrammed to acquire an ES-like state are termed iPS cells. In this unit,a protocol to use microRNAs as enhancers to increase the reprogramming efficiency isdescribed. Mouse embryonic fibroblasts (MEFs) are isolated from E13.5 mouse embryosand seeded for reprogramming by defined factors. microRNA mimics are transfectedinto MEFs at two time points during this process to enhance the overall reprogrammingefficiency. Two standard protocols for characterization of these miR-iPSCs, embryoidbody formation and teratoma formation, are also provided. By using this method, theinvestigators can obtain a significantly higher number of bona-fide iPSC colonies andmiR-iPSCs can be derived at a faster rate than with non-treated cells. Curr. Protoc. StemCell Biol. 20:4A.4.1-4A.4.14.C2012 by John Wiley & Sons, Inc.Keywords: reprogramminga114microRNAa114iPSCa114embryonic bodya114teratomaINTRODUCTIONSince their first discovery in 2006 (Takahashi and Yamanaka, 2006), somatic cells fromboth mouse and human can be reprogrammed to become ES-like cells called inducedpluripotent stem cells (iPSCs) (Meissner et al., 2007; Takahashi et al., 2007; Yu et al.,2007; Aoi et al., 2008; Park et al., 2008; Giorgetti et al., 2009). These iPSCs havebeen shown to possess great potential for stem cellbased therapeutics as they can bedifferentiated into any lineage, and the derivatives are valuable sources for studying thepathogenesis of human diseases (Marchetto et al., 2010; Itzhaki et al., 2011; Yazawaet al., 2011; Zhang et al., 2011). However, the reprogramming process suffers fromextremely low efficiency when using viral integration vectors (Takahashi and Yamanaka,2006; Aoi et al., 2008; Nakagawa et al., 2008). Recent studies have identified barriers forthis process and better methods have been developed to enhance the efficiency of iPSCgeneration (Ichida et al., 2009; Lyssiotis et al., 2009; Maherali and Hochedlinger, 2009;Shietal.,2010).microRNAsaresmallnon-codingRNAswith18to24nucleotides,whichare usually associated with a protein complex called RNA-induced silencing complex(RISC). microRNAs have been shown to be involved in many important processes andcanregulatehundredsoftargetgenes(Wangetal.,2008;LatronicoandCondorelli,2009;Xu et al., 2009). For iPSC generation, a number of miRNAs have been identified thatcould enhance reprogramming efficiency (Judson et al., 2009; Li et al., 2011). Here aprotocol for high-efficiency derivation of iPSCs by applying microRNAs as enhancersof the reprogramming process is described. By using this method, a significant increasein the number of iPSC colonies can be achieved and miR-iPSCs can be derived at afaster rate than cells from the traditional reprogramming experiments. Two standardprocedures, embryoid body formation and teratoma formation assay, are also includedto help investigators characterize the derived iPSC lines.Current Protocols in Stem Cell Biology 4A.4.1-4A.4.14Published online March 2012 in Wiley Online Library ().DOI: 10.1002/9780470151808.sc04a04s20CopyrightC2012 John Wiley & Sons, Inc.Manipulation ofPotency4A.4.1Supplement 20MicroRNAsEnhanceGeneration ofPluripotent StemCells4A.4.2Supplement 20 Current Protocols in Stem Cell BiologyNOTE: All protocols using live animals must first be reviewed and approved by an Insti-tutional Animal Care and Use Committee (IACUC) and must follow officially approvedprocedures for the care and use of laboratory animals.BASICPROTOCOL 1PREPARATION OF MOUSE EMBRYONIC FIBROBLASTS (MEFs)This protocol is used to efficiently obtain mouse embryonic fibroblasts (MEFs) for thereprogramming experiment. The derived MEFs could be used for both iPSC generationand providing feeder cells for established embryonic stem cells or iPSCs.MaterialsFemale mice, B6;129S4-Pou5f1tm2Jae/J (13 to 14 days gestation)Phosphate buffered saline (PBS; Invitrogen, cat. no. 10010023)0.25% trypsin (Invitrogen, cat. no. 25200)MEF derivation medium (see recipe)MEF culture medium (see recipe)Cryopreservation medium (see recipe)Liquid nitrogen tankAbsorbent paper towelsAlcohol padsDissecting scissors, sterileSterile, disposable petri dishes (Sarstedt, cat. no. 82.1473.001)Watchmakers forcepsSterile, disposable 10-ml pipets (Costar, cat. no. 4488)Castro-Viejo scissors37C, 5% CO2incubator50-ml conical tubes (Costar, cat. no. 430828)75-cm2flasks (BD Falcon, cat. no. 353136)1.7-ml cryopreservation tubes (cryovial, Corning, cat. no. 430488)Isopropanol freezing containerHarvest E13.5 post-coitum embryos1. In a sterile tissue culture hood, sacrifice B6;129S4-Pou5f1tm2Jae/J female mice (time-mated) by cervical dislocation and place onto absorbent paper towels with belly up.This strain of mice has GFP expression under control of the endogenous Pou5f1 promoter,which serves as the marker to select fully reprogrammed cells.2. Saturate the mouse abdomen using alcohol pads and cut the skin with sterile scissorsto expose the peritoneum.Saturate abdomen well because the fur of the mouse makes cleaning very difficult andcould cause contamination in derived cells. Applying enough alcohol to saturate theregion will strongly reduce the risk.3. Cut peritoneal wall with new sterile scissors and expose uterine horns.4. Dissect out uterine horns with new sterile scissors and place them into a steriledisposable petri dish containing 20 to 30 ml of PBS.5. Wash horns three times with PBS (15 ml each time), transferring the horns to newpetri dishes for each wash.The purpose of the washes is to remove blood cells.6. Open the sacs using sterile watchmakers forceps, and release the embryos.Manipulation ofPotency4A.4.3Current Protocols in Stem Cell Biology Supplement 207. TransfertheembryosintoanewpetridishcontainingPBSusingsterilewatchmakersforceps and wash three times, changing the dishes for each wash.It is sufficient to swirl the embryos in the PBS for each wash.8. Separatethevisceraltissue(darkredpartofabdomenofembryos)fromtheembryos.Transfer the dissected embryos into a new petri dish.9. Wash embryos three times with PBS, transferring them to new dishes for each wash.Count the number of embryos.Separate MEFs from embryo tissue10. Transfer embryos into a new petri dish and remove PBS with a 10-ml pipet.11. Cut embryos into minimal sized pieces (1-mm diameter) with Castro-Viejoscissors.This process will take 5 to 10 min. Large tissue pieces will be resistant to efficientdigestion.12. Dilute 0.25% trypsin to 0.05% in PBS and add 2 ml of 0.05% trypsin to the dish.Continue cutting for an additional 5 min.13. Add an additional 5 ml of 0.05% trypsin and incubate dish 30 min in a 37C, 5%CO2incubator.14. Transfer digested tissues with a 10-ml pipet into a 50-ml conical tube and pipet upand down vigorously to destroy the remaining tissue. Add 20 ml of MEF derivationmedium to neutralize the trypsin.15. Allow the mixture to sit for 10 min at room temperature, after which the remainingtissue debris will collect onto the bottom of the tube.16. Into each 75-cm2flask, transfer the supernatant from three of the embryos (threeembryos per 75-cm2flask).17. Keep flasks overnight in the 37C, 5% CO2tissue culture incubator.18. Refresh with MEF derivation medium on the next day.Harvest MEFs and establish frozen stocks19. Two days after refreshing medium, harvest cells.20. Aspirate the medium and wash cells by swirling flask once with PBS (10 ml per75-cm2flask).21. Add 5 ml of 0.25% trypsin and incubate 5 min at 37C.22. Dislodge the cells by tapping side of flask and pipet up and down to obtain a single-cell suspension.23. Add 5 to 10 ml of MEF culture medium per flask to neutralize the trypsin.24. Collect the cell suspension in a 50-ml conical tube and centrifuge 5 min at 150 g,room temperature.25. Resuspend cells in 1.5 ml MEF culture medium per 75-cm2flask and add an equalvolume of 2 cryopreservation medium.26. Add 1 ml suspension per 1.7-ml cryovial and place them in an isopropanol freezingcontainer.27. Place the container overnight at 80C and transfer to a liquid nitrogen tank on thenext day.MicroRNAsEnhanceGeneration ofPluripotent StemCells4A.4.4Supplement 20 Current Protocols in Stem Cell BiologyBASICPROTOCOL 2USE microRNAs AS AN ENHANCER TO REPROGRAM MOUSEEMBRYONIC FIBROBLASTSThis protocol is to enhance iPSCs generation with microRNAs. Typically, microRNAmimics are transfected into MEFs by lipisome based method. Transfected cells are thentransduced with freshly-made retrovirus encoding four transgenes (Oct4/Pou5f1, Sox2,Klf4,andcMyc) and cultured for 10 to 12 days to become fully reprogrammed. GFPexpression is used as the marker to monitor the progression of reprogramming and toidentify ideal colonies to derive iPSC lines from (Fig. 4A.4.1).MaterialsPLAT-E cells (Cell Biolabs, cat. no. RV-101)10% FBS medium without selection drug (see recipe)PLAT-E culture medium (see recipe)pMX vectors (Oct4, Sox2, Klf4, cMyc; Addgene)Lipofectamine (Invitrogen, cat. no. 18324012)PLUS reagent (Invitrogen, cat. no. 11514015)Basal DMEM medium0.1% (w/v) gelatin solutionMEFs (CF-1, Oct4-GFP, etc.; see Basic Protocol 1)microRNA mimics (miR-93, 106b, Dharmacon; Li et al., 2011)Opti-MEM (Invitrogen, cat. no. 37985070)Lipofectamine 2000 (Invitrogen, cat. no. 11668019)MEF culture medium (see recipe)mES culture medium (see recipe)15-ml conical tubes (Costar, cat. no. 430790)37C water bathBench top centrifuge150-cm2tissue culture flasks (BD Biosciences, cat. no. 355001)37C, 5% CO2incubator10-cm tissue culture plates (Corning, cat. no. 430167)Syringe filters, 0.22- and 0.45-m (Millipore, cat. nos. SLGP033RS andSLHV033RS)12-well tissue culture plates (BD Biosciences, cat. no. 353043)Thaw PLAT-E cells1. Thaw one vial of PLAT-E cells from frozen stock in a 15-ml conical tube in a 37Cwater bath.2. Add 5 ml of fresh 10% FBS medium and centrifuge 5 min at 150 g, roomtemperature.3. Remove the supernatant by vacuum, transfer cells to a 150-cm2flask, and resus-pend cells in 15 ml fresh 10% FBS medium. Incubate overnight in 37C, 5% CO2incubator.4. On the next day, change the medium with 15 ml PLAT-E cell culture.The cells will be ready for seeding for virus production when they reach 80% to 90%confluency.Prepare retrovirus for four transcription factors5. Seed 5.5 106PLAT-E cells per 10-cm plate for transfection (day 0) in 10 ml of10% FBS medium without selection drug. Incubate overnight at 37C.Manipulation ofPotency4A.4.5Current Protocols in Stem Cell Biology Supplement 20ABday 5mES medium H11001 LIFMEF mediummicroRNAs4F virusday 3 day 11 day 15day 0Figure 4A.4.1 Scheme for iPSC generatioin with microRNAs as enhancers. (A) microRNAs were transfected both onday 0 and day 5 at a final concentration of 50 nM. Infected MEFs were first cultured in MEF medium until day 3 post-4Ftransduction and then switched to mES culture medium containing LIF supplement. Typically, for 4F-transduced cells,Oct4-GFP-positive colonies could be readily picked around day 11 to establish iPSC lines, and for OSK-transduced cells,GFP-positive colonies could be picked around day 15. (B) Derived miR-iPSC clone. miR-93 iPSC no. 5 clone was derivedas mentioned in A and the cells have acquired typical mouse embryonic stem cell morphology. They have also turned onthe endogenous locus for Oct4 and thus become GFP-positive.6. On the next day, transfect cells with 9 g each of pMXs-Oct4, Sox2, Klf4, andcMyc vectors together with 25 l lipofectamine and 20 lPLUSreagent(day1)asfollows:a. Individually dilute 9 g of DNA in basal DMEM medium and add 20 lPLUSreagent for a final volume of 450 l. Vortex and incubate 15 min at roomtemperature.b. Prepare 25 l lipofectamine in a final volume of 450 l in basal DMEM mediumand incubate 5 min at room temperature.c. Mix solutions from a and b together and incubate an additional 15 min beforeadding to the cells. Add the 900 l mixture to cells and incubate overnight in37C, 5% CO2incubator.The cells are in 10% FBS medium without selection drug (from step 5).MicroRNAsEnhanceGeneration ofPluripotent StemCells4A.4.6Supplement 20 Current Protocols in Stem Cell Biology7. One day after transfection, change the medium to fresh 10% FBS medium withoutselection drugs (day 2).8. Harvest viral supernatant and mix the four viruses together (or three if only OSKviruses are used). Centrifuge 5 min at 2000 g, room temperature, and filter througha 0.45-m syringe filter to remove cell debris. Viruses are ready for use.The viruses will be used immediately thus do not need to be stored. No quantification isneeded. The virus titer is fairly consistent (107IFU/ml) (day 3). OSK refers to Oct4,Sox2, and Klf4 transgenes.Transfect MEFs with microRNAs and infect with OSKMOSKM refers to Oct4, Sox2, Klf4, and cMyc transgenes.9. Coat 12-well tissue culture plates with 0.1% gelatin for at least 20 min in a 37C,5% CO2incubator.10. Seed 4 104cells/well in the 12-well plates (day 2).11. Transfect MEFs with 50 nM microRNA mimics at a final volume of 400 linOpti-MEM as follows.a. Dilute microRNA mimics in 40 l Opti-MEM and separately dilute 2 l Lipofec-tamine 2000 in 40 l Opti-MEM, incubate 5 min at room temperature.b. Mix the solutions together and incubate an additional 20 min before adding intothe cells.c. While solutions are incubating, wash MEFs with 0.5 ml Opti-MEM per well oncewithswirlingandadd320 lOpti-MEMintoeachwellof12-wellplates.Incubate3hrina37C, 5% CO2incubator.12. Three hours after transfection, add 0.8 ml of filtered virus supernatants (from step 8)per well to transduce the cells. Incubate overnight in a 37C, 5% CO2incubator.13. One day after transfection and transduction, change the medium with fresh MEFculture medium.14. Three days post-initial transduction, change the medium to mES culture mediumwith LIF.15. At day 5 after transduction, transfect the cells again as in step 11 with microRNAmimics. (No virus infection is needed.) Three hours later, add 0.8 ml mES culturemedium to each well instead of 4F virus. Incubate overnight.16. On the next day, refresh the medium and change it every other day until colonies areready to be picked.BASICPROTOCOL 3DERIVATION AND CHARACTERIZATION OF miR-iPSC COLONIESThis protocol is to establish the iPSC clones and characterize the quality of expandedcolonies. In general, fully reprogrammed iPSC colonies are ready to be picked at 12to 14 days post-OSKM transduction. Picking and expanding the colonies is extremelytime consuming. For each experimental condition, picking 10 to 20 colonies is rec-ommended, and after the first round of picking, about five of the picks may yield pureGFP-expressing cells in the culture dish. For dishes with mixed populations, a secondround of picking is usually necessary to establish a pure iPSC line. After establishingdifferent lines, they can then be cultured in larger amounts for both the embroid bodyformation assay and teratoma injection in athymic nude mice.Manipulation ofPotency4A.4.7Current Protocols in Stem Cell Biology Supplement 20MaterialsMEFs (CF-1, Oct4-GFP, etc.; see Basic Protocol 1)0.25% trypsin/EDTAMEF culture medium (see recipe)PBS0.1% (w/v) gelatin solutionmES culture medium (see recipe)15% ES-screened FBS, 10% DMSO in DMEM (Invitrogen, cat. no. 11995065)mEB formation medium (see recipe)4% paraformaldehyde4- to 6-week-old female athymus nude miceAvertinAlcohol padsZinc formalin solution (Fisher, cat. no. 23313096)150-cm2flasks50-ml conical tubesIrradiator (RS2000, Rad Source)12-well tissue culture plates (BD Biosciences, cat. no. 353043)MicroscopePasteur pipets96-well tissue culture plates20-l Gilson pipet37C incubator10-cm petri dishes (SARSTED, cat. no. 821473001)6-well tissue culture plates (BD Biosciences, cat. no. 353046)Additional reagents and equipment for MEFs (see Basic Protocol 1)Prepare irradiated MEFs as feeder cells for iPSC culture1. Derive MEFs from the CF-1 mouse strain as described in Basic Protocol 1 andculture in 150-cm2flasks.2. When CF-1 MEFs reach 90% confluency, remove medium with vacuum. Add10 ml PBS and swirl to cover cells. Remove PBS by vacuum, add 5 ml of 0.25%trypsin/EDTA. Incubate 5 min in a 37C, 5% CO2incubator.3. Neutralize trypsin by adding 10 ml fresh MEF culture medium and collect cells in a50-ml conical tube.4. Centrifuge cells 5 min at 150 g, room temperature.5. Remove supernatant by vacuum. Resuspend the cells in 50-ml conical tubes with10mlfreshMEFmediumandquantifythecellconcentrationusingahemacytometer.6. Transfer the conical tubes into an irradiator and use a dose of 7000 rad to block theproliferation of treated feeder cells.7. Store cells in liquid nitrogen or seed in tissue culture dish for immediate use.Typically, 1 106cells are seeded per 12-well plate.Pick colonies and establish iPSC clones8. When the iPSC colonies are big enough to pick, mark the desired colonies under amicroscope.MicroRNAsEnhanceGeneration ofPluripotent StemCells4A.4.8Supplement 20 Current Protocols in Stem Cell BiologyWhen the cells reach a fully reprogrammed state, endogenous Oct4 expression will beactivated. Since GFP expression in the (B6;129S4-Pou5f1tm2Jae/J) transgenic MEFs isdriven by the Oct4 promoter, the iPSCs will be GFP positive. GFP+ colonies usuallystart to show up around day 9 when using 4F virus and the number of colonies willcontinue to go up every day when the proper culture condition is provided.For iPSCs generated from three factors (OSK), the first appearance of GFP+ colonieswill be a few days delayed compared with 4F transduction. Transfection of microRNAs(miR-106b for example) will both increase the colony formation as well as shorten theoverall timing of reprogramming, in which case the onset of GFP+ colonies will be atleast 2 to 3 days earlier than siControl transfected cells.9. Prepare colony p
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