Transfection of neural stem cells with Lipofectamine Stem Transfection Reagent in StemPro medium

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NSC media, passaging reagents, and complexation medium

Starting with undifferentiated human primary neural stem cells (NSCs) or pluripotent stem cell (PSC)–derived NSCs, expanded in a defined culture system such as Gibco StemPro NSC Serum-Free Medium (SFM) on Gibco Geltrex matrix, is ideal for efficient transfection.

Passaging

• Maintain NSCs in the format of your choice, such as 6-well plates, 60 cm dishes, T-25 flasks, or T-75 flasks coated with Geltrex matrix, in StemPro SFM. Propagating NSCs in T-25 flasks and transfecting in 24-well plates are convenient formats used in this protocol.
• Passage NSCs every 3 to 5 days at 90–100% confluence.
• Use Gibco StemPro Accutase Cell Dissociation Reagent to generate a single-cell suspension of NSCs for both expansion and seeding for transfection.

Precoating 24-well plates with Geltrex matrix for transfection

1. Prepare a 1:100 dilution of Geltrex matrix in cold Gibco DMEM/F-12 with Gibco GlutaMAX Supplement (Cat. No. 10565).
2. Add 300 μL of diluted Geltrex matrix to each well of a 24-well plate and incubate at 37°C for ≥1 hour, before use.

• Tip: Geltrex matrix–coated plates can be prepared ahead of time and stored for up to 2 weeks at 4°C. Equilibrate at room temperature for 1 hour before plating cells.

Seeding cells for transfections

1. When NSC cultures are ~90–100% confluent, remove the StemPro NSC SFM.
2. Wash NSCs once with 10 mL of DPBS without calcium and magnesium; aspirate the medium and discard.
3. Add 1 mL of room-temperature StemPro Accutase reagent to each T-25 flask, swirl to evenly coat the NSCs, and incubate for 2–5 minutes at room temperature.
   Important: To maximize transfection efficiency, seeding a single-cell suspension of NSCs prepared with StemPro Accutase reagent is recommended.
4. Observe cells on an inverted microscope to confirm that NSCs are detached; firmly tap the flask to aid in the detachment of NSCs, as necessary.
5. Add 9 mL of StemPro NSC SFM to inactivate the StemPro Accutase reagent.
6. Gently triturate and rinse the flask to generate a single-cell suspension, and transfer the cell suspension into a 15 mL conical tube.
7. Centrifuge the NSC cell suspension at 200 x g for 4 minutes.
8. Aspirate the supernatant and resuspend the pellet to a single-cell suspension in 3 mL of StemPro NSC SFM.
9. Perform a total viable cell count with the Invitrogen Countess II Automated Cell Counter or another method.
10. Dilute with additional StemPro NSC SFM to a final concentration of 150,000 cells/mL.
11. Aspirate the Geltrex matrix from the wells of a precoated 24-well plate.
    Important: Proliferating NSC cultures need room to expand during transfection, so plate the recommended starting number of cells (step 12) to achieve 30–60% confluence on the day of transfection.
12. Add 0.5 mL of the NSC suspension in StemPro NSC SFM to plate 75,000 cells/well in the precoated 24-well plate.
13. Return the plate to the incubator and culture at 37°C with 5% CO₂, overnight.
    Important: You do not need to change the medium on the day of transfection.

DNA transfection protocol

Perform the following steps, which have been optimized for using Invitrogen Lipofectamine Stem Transfection Reagent with NSCs:

Analysis of transfection efficiency

Observe NSCs transfected with a GFP reporter construct at 24 and 48 hours posttransfection by fluorescence microscopy or flow cytometry for endpoint analysis (Figure 1).

Figure 1. Posttransfection analysis of NSCs. (A) Fluorescence image demonstrating 59% transfection efficiency, and (B) bright-field image. NSCs are shown 24 hours after transfection with 500 ng of a 6 kb EF1α-GFP plasmid and 1 μL of Lipofectamine Stem reagent in StemPro NSC SFM on Geltrex matrix.

mRNA transfection protocol

Perform the following steps, which have been optimized for using Lipofectamine Stem reagent with NSCs:

Analysis of transfection efficiency

Observe PSCs transfected with a fluorescent mRNA at 24 and 48 hours posttransfection by fluorescence microscopy or flow cytometry for endpoint analysis (Figure 2).

Figure 2. Posttransfection analysis of NSCs. (A) Fluorescence image demonstrating 70% transfection efficiency, and (B) bright-field image. iPSC-derived NSCs (NCRM1) are shown 36 hours after transfection with 250 ng of GFP mRNA and 1 μL of Lipofectamine Stem reagent in StemPro NSC SFM on Geltrex matrix.

Ribonucleoprotein (RNP) transfection protocol

RNP complex components:

• Invitrogen GeneArt Platinum Cas9 Nuclease (Cat. No. B25641)
• gRNA (see “Designing and generating gRNA by in vitro transcription”)

Perform the following steps, which have been optimized for using Lipofectamine Stem reagent with NSCs:

Analysis of transfection efficiency

Observe PSCs transfected with a GFP reporter construct at 24 and 48 hours posttransfection by fluorescence microscopy or flow cytometry, and analyze double-stranded break (DSB) formation using the Invitrogen GeneArt Genomic Cleavage Detection Kit or a similar assay (Figure 3).

Figure 3. Posttransfection analysis of NSCs. (A) Fluorescence image demonstrating 60% transfection efficiency, and (B) bright-field image. iPSC-derived NSCs (NCRM1) are shown 24 hours posttransfection with 500 ng of GeneArt Platinum Cas9 Nuclease, 125 ng of gRNA, 50 ng of GFP mRNA, and 1 μL of Lipofectamine Stem reagent in StemPro NSC SFM on Geltrex matrix. (C) Genomic cleavage detection analysis of iPSC-derived NSCs 48 hours posttransfection, demonstrating 56% indel formation within the EMX1 locus.

Designing and generating gRNA by in vitro transcription

In addition to RNP transfection efficiency, the efficiency of DSB/indel formation at a given locus can depend on gRNA design. Use the Invitrogen GeneArt CRISPR Search and Design Tool, available at thermofisher.com/crisprdesign, to search our database of >600,000 predesigned gRNA sequences specific to every gene in the human genome. These predesigned gRNAs are optimized for gene knockout and typically target the first 3 transcribed exons per gene.

Clone and generate your own gRNA using the Invitrogen GeneArt Precision gRNA Synthesis Kit (Cat. No. A29377). gRNA concentration can be quantified on the Invitrogen Qubit 3 Fluorometer (Cat. No. Q33216) coupled with the Invitrogen Qubit RNA BR Assay Kit (Cat. No. Q10210).