Dec 18, 2019
LIPID-MEDIATED TRANSFECTION OF iPSCs (Basic Protocol 2)
- Michael S. Fernandopulle1,
- Ryan Prestil1,
- Christopher Grunseich1,
- Chao Wang2,
- Li Gan2,
- Michael E. Ward1
- 1National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland;
- 2Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California
- Neurodegeneration Method Development CommunityTech. support email: [email protected]
External link: https://doi.org/10.1002/cpcb.51
Protocol Citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. LIPID-MEDIATED TRANSFECTION OF iPSCs (Basic Protocol 2). protocols.io https://dx.doi.org/10.17504/protocols.io.5deg23e
Manuscript citation:
Fernandopulle, M. S., Prestil, R., Grunseich, C., Wang, C., Gan, L., & Ward, M. E. (2018). Transcription-factor mediated differentiation of human iPSCs into neurons. Current Protocols in Cell Biology, e51. doi:https://doi.org/10.1002/cpcb.51
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 11, 2019
Last Modified: December 18, 2019
Protocol Integer ID: 25734
Keywords: i3LMN, i3Neurons, iPSC, iPSC-derived neurons, transcription factor-mediated differentiation, transfection of ipsc, delivery of dna plasmid, transfection reagent, other lipid reagent, mediated transfection, based transfection reagent, general transfection, lipofectamine stem, lipid, transfection, dna plasmid, several lipid, translated ribonucleoprotein, transcribed rna, rna, insertion of the transgene cassette, e8 medium on matrigel, dna, transgene cassette, transgene, ipsc
Abstract
This protocol describes the lipid-mediated transfection of iPSCs maintained in E8 medium on Matrigel. While several lipid-based transfection reagents are commercially available, Lipofectamine Stem is used here because it is specifically optimized for delivery of DNA plasmids into hiPSCs. If preferred, similar results may also be achieved by electroporation or nucleofection, and other lipid reagents are available for in vitro-transcribed RNA or in vitro-translated ribonucleoproteins (RNPs). Furthermore, while this protocol provides the steps for a general transfection, specific details are provided below regarding insertion of the transgene cassettes relevant for neural differentiation (See Basic Protocols 5 and 7).
Attachments
Guidelines
Passaging with Accutase immediately before transfection improves efficiency by generating a single-cell suspension that increases exposure to the Lipofectamine reagent; however, if Accutase passaging for certain iPSC lines results in low viability, transfection may also be performed on EDTA-passaged cells or on adherent cells at low confluency
(20 % to 30 %). Transfection efficiency may be monitored by including a fluorescent protein reporter under a promoter that is active in human stem cells (e.g., CAG, PGK, EF-1α containing introns; not CMV) and viewing the cells 1 day after transfection. This reporter does not need to be integrated, as transient expression should persist for 3 to 4 days after transfection. Finally, increased cell death is typical for 1 to 2 days after transfection, and can result in the accumulation of debris, so the culture medium should be changed daily, and cells may also be washed with PBS after aspiration of spent medium to further reduce debris carryover. The transfected iPSCs should be passaged for expansion, enrichment, and/or clonal selection (Basic Protocol 3 or 4) after the cells have reached approximately 80 % confluency, which commonly occurs 2 to 4 days after transfection.
Materials
- Lipofectamine Stem (Invitrogen, cat. no. STEM00001) or other lipid-based transfection reagent
Lipofectamine™ Stem Transfection ReagentInvitrogen - Thermo FisherCatalog #STEM00001
- Opti-MEM I Reduced Serum Medium (Gibco, cat. no. 31985062)
Opti-MEM™ I Reduced Serum MediumGibco - Thermo Fisher ScientificCatalog #31985062
- DNA plasmid(s) (e.g., CRISPR-Cas9 and guide RNA, TALENs, and/or DNA insert with appropriate homology arms; DNA obtained from an endotoxin free maxi-prep kit)
- Additional reagents and equipment for general iPSC culture (Basic Protocol 1) and counting cells (Phelan & May, 2015)
Troubleshooting
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Grow a sufficient number of iPSCs for transfection and prepare cells as for an Accutase split (see Basic Protocol 1).
Note
One or two wells of a 6-well dish at 80 % confluency should provide more than enough cells for one transfection.
Count the cells (Phelan & May, 2015), transfer 1.5 × 106 cells to a 15-ml conical tube, and centrifuge 00:05:00 at 300 x g , Room temperature .
Aspirate the supernatant and resuspend in 2 mL of E8 medium supplemented with 10 micromolar (µM) Y-27632 ROCK inhibitor.
Note
If iPSCs are normally maintained in a Flex medium, it is best to transition to regular E8 medium on the day of transfection to improve efficiency.
Pipet the medium and cells into 1 well of a 6-well dish pre-coated with Matrigel and return plate to the incubator. Gently shake the plate front-to-back and side-to-side.
Allow the cells to adhere in the incubator for 01:00:00 to 02:00:00 before adding the transfection
solution.
For each transfection, add 100 µL of Opti-MEM and 3 µg of total DNA to one 1.5-ml microcentrifuge tube and vortex for 00:00:02 to 00:00:03 . In a second tube, combine 100 µL of Opti-MEM and 10 µL of Lipofectamine Stem reagent, and vortex for 00:00:02 to 00:00:03 .
Note
For TALEN-mediated insertion to the AAVS1 or CLYBL locus, such as for the hNGN2 (Addgene, cat. no. 105840) and hNIL (Addgene. cat. no. 105841) differentiation cassettes, use a 2:1:1 ratio of 1.5 µg donor construct with 0.75 µg of each of the site-specific TALENs.
For AAVS1: 0.75 µg of pTALdNC-AAVS1_T2 (Addgene, cat. no. 80496) and 0.75 µg of pTALdNC-AAVS1_T1 (Addgene, cat. no. 80495) per transfection.
For CLYBL: 0.75 µg of pZT-C13-R1 (Addgene, cat. no. 62197) and 0.75 µg of pZT-C13-L1 (Addgene, cat. no. 62196) per transfection.
Combine the contents of the two tubes and vortex again for 00:00:02 to 00:00:03 . Incubate this
mixture for 00:10:00 at Room temperature .
Retrieve the cells plated in steps 3 to 4, and, using a P200 pipet tip and repeat pipettor, add 200 µL of the complete transfection solution from step 6 dropwise, evenly across the surface of the well. Return the cells to the incubator overnight.
24:00:00 after transfection, aspirate transfection medium and replace with fresh E8.
If applicable, evaluate transfection efficiency by fluorescence microscopy.
Note
All cells transfected with the hNGN2 (Addgene, cat. no. 105840 or 110492) and hNIL constructs (Addgene, cat. no. 105841 or 105842) will transiently express mCherry for 3 to 4 days, while only those cells with transgene insertion will maintain stable expression of mCherry for longer periods of time.
Change medium daily with normal culture medium, and wash with PBS if necessary to remove debris. Once the cells have reached 80 % confluency, they may be passaged to a new dish for expansion or used for enrichment or clonal selection.