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Using MicroRNAs to Enhance the Generation of Induced Pluripotent Stem Cells

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

Somatic 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 is described. Mouse embryonic fibroblasts (MEFs) are isolated from E13.5 mouse embryos and seeded for reprogramming by defined factors. microRNA mimics are transfected into MEFs at two time points during this process to enhance the overall reprogramming efficiency. Two standard protocols for characterization of these miR?iPSCs, embryoid body formation and teratoma formation, are also provided. By using this method, the investigators can obtain a significantly higher number of bona?fide iPSC colonies and miR?iPSCs can be derived at a faster rate than with non?treated cells. Curr. Protoc. Stem Cell Biol. 20:4A.4.1?4A.4.14. © 2012 by John Wiley & Sons, Inc.

Keywords: reprogramming; microRNA; iPSC; embryonic body; teratoma

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Introduction
Basic Protocol 1: Preparation of Mouse Embryonic Fibroblasts (MEFs)
Basic Protocol 2: Use microRNAs as an Enhancer to Reprogram Mouse Embryonic Fibroblasts
Basic Protocol 3: Derivation and Characterization of miR‐iPSC Colonies
Reagents and Solutions
Commentary
Literature Cited
Figures

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Basic Protocol 1: Preparation of Mouse Embryonic Fibroblasts (MEFs) Materials

Female 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 tank

Absorbent paper towels
Alcohol pads
Dissecting scissors, sterile
Sterile, disposable petri dishes (Sarstedt, cat. no. 82.1473.001)
Watchmaker's forceps
Sterile, disposable 10‐ml pipets (Costar, cat. no. 4488)
Castro‐Viejo scissors
37°C, 5% CO 2 incubator
50‐ml conical tubes (Costar, cat. no. 430828)
75‐cm2 flasks (BD Falcon, cat. no. 353136)
1.7‐ml cryopreservation tubes (cryovial, Corning, cat. no. 430488)
Isopropanol freezing container

Basic Protocol 2: Use microRNAs as an Enhancer to Reprogram Mouse Embryonic Fibroblasts Materials

PLAT‐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 medium
0.1% (w/v) gelatin solution
MEFs (CF‐1, Oct4‐GFP, etc.; see protocol 1 )
microRNA mimics (miR‐93, 106b, Dharmacon; Li et al., )
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)
37°C water bath
Bench top centrifuge
150‐cm2 tissue culture flasks (BD Biosciences, cat. no. 355001)
37°C, 5% CO 2 incubator
10‐cm tissue culture plates (Corning, cat. no. 430167)
Syringe filters, 0.22‐ and 0.45‐µm (Millipore, cat. nos. SLGP033RS and SLHV033RS)
12‐well tissue culture plates (BD Biosciences, cat. no. 353043)

Basic Protocol 3: Derivation and Characterization of miR‐iPSC Colonies Materials

MEFs (CF‐1, Oct4‐GFP, etc.; see protocol 1 )
0.25% trypsin/EDTA
MEF culture medium (see recipe )
PBS
0.1% (w/v) gelatin solution
mES culture medium (see recipe )
15% ES‐screened FBS, 10% DMSO in DMEM (Invitrogen, cat. no. 11995065)
mEB formation medium (see recipe )
4% paraformaldehyde
4‐ to 6‐week‐old female athymus nude mice
Avertin
Alcohol pads
Zinc formalin solution (Fisher, cat. no. 23313096)

150‐cm2 flasks
50‐ml conical tubes
Irradiator (RS2000, Rad Source)
12‐well tissue culture plates (BD Biosciences, cat. no. 353043)
Microscope
Pasteur pipets
96‐well tissue culture plates
20‐µl Gilson pipet
37°C incubator
10‐cm petri dishes (SARSTED, cat. no. 821473001)
6‐well tissue culture plates (BD Biosciences, cat. no. 353046)

Additional reagents and equipment for MEFs (see protocol 1 )

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Figure 4.A0.1 Scheme for iPSC generatioin with microRNAs as enhancers. (A ) microRNAs were transfected both on day 0 and day 5 at a final concentration of 50 nM. Infected MEFs were first cultured in MEF medium until day 3 post‐4F transduction 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 derived as mentioned in A and the cells have acquired typical mouse embryonic stem cell morphology. They have also turned on the endogenous locus for Oct4 and thus become GFP‐positive.

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Figure 4.A0.2 iPSC characterization by embryoid body formation assay. iPS cells were first trypsinized to become a single cell suspension and diluted to a final concentration of 1 × 105 ∼1.5 × 105 cells/ml. Cells were then seeded in 20‐µl drops onto the cover of petri dishes to reach 2000∼3000 cells/drop. Embryoid bodies (mEBs) were formed by inverted culture of the cells in petri dishes with ∼15 ml PBS, which could prevent the drops from drying. mEBs would be harvested at day 2 to 3 and they typically have universal morphology by this method. mEBs were then seeded onto tissue culture‐treated 6‐well plates to allow them attach the bottom. Attached mEBs were further maintained in mEB differentiation medium until around day 13 to 14 for immunostaining of different lineage markers.

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Figure 4.A0.3 iPSC characterization by teratoma formation. (A ) 4‐ to 6‐week‐old athymus nude mice were anesthetized with avertin and injected with ∼1 × 106 iPS cells at the dorsal neck region. After 3 to 4 weeks, the tumors were then harvested and fixed in zinc formalin solution before further paraffin embedding and H&E staining. (B ) Teratomas contain tissues from different lineages. Cartilage, neural tissue, skeleton muscle, and epidermis tissue were shown.

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Literature Cited

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