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The ability of stem cells to self-renew and differentiate into various specialized cell types promises to contribute greatly to future applications in regenerative medicine and the development of novel therapeutic treatments. Technologies enabling the genetic manipulation of stem cells support preclinical and clinical research to develop new gene correction and tissue replacement therapies. The ability to subsequently isolate and expand lines of stem cells under feeder-free culture conditions, using systems like StemFlex and Essential 8 media, further enable the movement of stem cell research from the bench to the clinic.
Transfecting stem cells without inhibiting cell viability and cell growth have been shown to be difficult, as the delivery method itself must have little to no effect on the properties of stem cells (e.g., maintenance of pluripotency posttransfection) for downstream assay results to be reliable. Research applications such as gene editing, gene expression, and directed differentiation all require the ability to deliver a variety of constructs into stem cells. Advances in gene editing using the CRISPR-Cas9 system also require the ability to deliver large plasmid constructs or a combination of DNA, RNA and/or Cas9 ribonucleoprotein (RNP) complexes.
3.2 Choosing a transfection method and technologies
Different transfection methods and technologies have their own advantages and disadvantages that must be considered. However, nonviral transfection methods are the most widely used methods for delivering foreign molecules into cells (Figure 3.1). The main features to consider when transfecting cells include transfection efficiency, toxicity, versatility, and cost. The products below are recommended, keeping these key considerations in mind (Table 3.1).
Figure 3.1. Recommended transfection methods by cell type.
Table 3.1. Transfection selection guide for stem cells—recommended payload by product and transfection efficiency by cell type (more blocks represent higher efficiency).
3.3 Transfecting DNA
Plasmid DNA remains the most common transgene construct for transfection. Until recently, it was difficult to get high levels of DNA delivery using chemical transfection reagents in stem cells. However, with the recent introduction of Invitrogen Lipofectamine Stem Transfection Reagent, efficiency of 60% or higher is easily achieved.
Lipofectamine Stem Transfection Reagent is optimized for efficient DNA delivery into stem cells (human ESCs and iPSCs), with high efficiency (Figure 3.2). This reagent has also been validated to achieve high-efficiency delivery of large plasmid DNA (>10 kb) into PSCs, while supporting their continued proliferation in an undifferentiated state (Figure 3.3).
Applications like gene editing frequently require large plasmids encoding gene-editing constructs or template DNA, and often require electroporation to transfect cells. Lipofectamine Stem reagent provides a complementary alternative to electroporation to introduce a wide range of plasmid DNA into stem cells and is gentler on cells. Lipofectamine Stem reagent is also compatible with a variety of cell culture media, including feeder-free culture systems such as Essential 8 Medium with vitronectin and StemFlex Medium with Geltrex matrix(Figure 3.4). In addition, human PSCs can be transfected in suspension during re-plating as a convenient alternative to electroporation.
A. Human pluripotent stem cells (Gibco iPSCs)
B. Human embryonic stem cells (H9 ESCs)
Figure 3.2. High-efficiency DNA transfection with Lipofectamine Stem reagent in human stem cells (A) iPSCs and (B) ESCs.
Transfects large DNA constructs
Lipofectamine Stem reagent achieves high-efficiency delivery of large plasmid DNA (>10 kb) into human PSCs, while supporting their continued proliferation in an undifferentiated state in defined and complex feeder-free culture systems.
Figure 3.3. Delivery of large DNA constructs with significantly higher efficiency than leading supplier’s reagent.(A) H9 ESCs transfected with 11 kb DNA plasmid using a leading DNA delivery reagent in mTeSR1 Medium. (B) H9 ESCs transfected with 11 kb DNA plasmid using Lipofectamine Stem reagent in mTeSR1 Medium. (C) H9 ESCs transfected with 11 kb DNA plasmid using Lipofectamine Stem reagent with StemFlex Medium.
Figure 3.4. Lipofectamine Stem reagent is compatible with a variety of media systems. Results show human PSCs 38–44 hours posttransfection. Human iPSCs plated in 24-well plates were transfected with 1 or 2 µL Lipofectamine Stem reagent in (A)StemFlex Medium on Geltrex matrix, (B)Essential 8 Medium on vitronectin, and (C) mTeSR1 Medium on Geltrex matrix.
Gentle on cells—keeps stem cells viable and healthy
Transfecting stem cells without inhibiting cell viability and cell growth can be challenging due to the sensitivity of these cells. Transfection requires a balancing act between introducing foreign nucleic acids into a cell, and not killing the cell in the process. Lipofectamine Stem reagent delivers low amounts of nucleic acid with high translational Figure 3.5. Lipofectamine Stem reagent maintains healthy, proliferating stem cells during transfection. (A) Human iPSCs cultured in feede-rfree Essential 8 Medium were transfected with Lipofectamine Stem reagent or not transfected (control). Cells remain healthy and viable with normal morphology and continued to proliferate with continuous transfection, reaching confluency by 48 hours. (B) Percent area confluency of transfected and untransfected cells. efficiency, allowing stem cells to stay healthy and continue proliferating without inducing differentiation. Pluripotent stem cells continue to proliferate with continuous transfection, reaching near-confluency by 48 hours (Figure 3.5).
Figure 3.5. Lipofectamine Stem reagent maintains healthy, proliferating stem cells during transfection. (A) Human iPSCs cultured in feeder-free Essential 8 Medium were transfected with Lipofectamine Stem reagent or not transfected (control). Cells remain healthy and viable with normal morphology and continued to proliferate with continuous transfection, reaching confluency by 48 hours. (B) Percent area confluency of transfected and untransfected cells.
After transfection with Lipofectamine Stem reagent, PSCs can be serially passaged and expanded, while maintaining pluripotency. Wells of control and transfected Gibco iPSCs were passaged in parallel with Versene Solution for 48 hours after transfection with Lipofectamine Stem reagent to deliver a GFP DNA plasmid, replated in a 6-well format, and allowed to expand in StemFlex Medium for 4 additional days. They were then passaged again, allowed to expand for 3 more days, and fixed after reaching >75% confluency. Cultures exhibited uniform cell morphology and homogeneous expression of Oct4 after transfection with Lipofectamine Stem reagent and continued culture in StemFlex Medium(Figure 3.6).
Figure 3.6. PSCs maintain markers of pluripotency after transfection with Lipofectamine Stem reagent and growth in StemFlex Medium. (A)Gibco Episomal iPSCs transfected with circular plasmid DNA and subsequently expanded for 2 passages. (B)Gibco Episomal iPSCs not transfected. Note: Gibco iPSCs retain residual GFP expression two passages after transfection of a circular plasmid DNA (A, left panel), while continuing to express Oct4 protein (A, middle panel) in the nuclei, similar to untransfected Gibco iPSCs grown in parallel in StemFlex Medium(B, middle panel).
3.4 Transfection with mRNA
Messenger RNA (mRNA) tends to transfect more efficiently than DNA, making it a great alternative for transfection when working with difficult-to-transfect stem cells. Transfection of mRNA only requires entry into the cell cytoplasm—without the need for translocation to the nucleus for transcription. Since nuclear entry is not necessary with mRNA, it eliminates any risk of integration into the host genome, which can cause insertional mutagenesis and activation of oncogenes; and transfection efficiency is generally higher. Additional benefits include a transient transfection and faster time to protein expression than with DNA. Transfection with mRNA is a useful platform for manipulating cell genotype and phenotypes by gene editing and transcription factor–directed differentiation. Having control over the timing, dosage, and stoichiometry of transgene delivery provides a precise way to drive foreign protein production in stem cells.
As shown in Figure 3.7, Lipofectamine Stem Transfection Reagent demonstrates outstanding transfection efficiency in stem cells with low amounts of mRNA.
Figure 3.7. High-efficiency transfection of human stem cells with mRNA using Lipofectamine Stem reagent. (A) Experimental conditions for H9 ESCs. (B) The cells were examined for GFP expression 24 and 48 hours posttransfection.
3.5 Transfection with RNP complexes
Research applications such as gene editing require the ability to deliver a variety of substrates into cells. Lipofectamine Stem reagent can be used to efficiently co-deliver RNP complexes of Cas9 protein with a guide RNA (gRNA) to support high-efficiency insertion/deletion (indel) formation of your target gene (Figure 3.8). Single-stranded DNA constructs can also be mixed with Cas9– mRNA, Cas9–gRNA, or Cas9 protein–gRNA to promote homology-directed repair (HDR) and introduce targeted genomic sequences.
Figure 3.8. Transfection with Lipofectamine Stem reagent supports high-efficiency gene editing in stem cells. (A) Experimental conditions for human iPSCs. (B) (Left) Human iPSCs were cotransfected with Cas9 mRNA, gRNA, and GFP mRNA (not shown) or Cas9 RNP targeting the EMX1 gene and GFP mRNA. (Right) Genomic cleavage detection of the EMX1 locus in iPSCs was analyzed 48 hours after transfection.
For certain media systems, such as StemFlex Medium, some of the media components may be inhibitory to lipid-based transfection. For these media systems, the following workflow can be used to accomplish high-efficiency lipid-based delivery using the Lipofectamine Stem reagent(Figure 3.9).
For detailed instructions, go to thermofisher.com/lipofectaminestem
As shown in Figure 3.10 and Table 3.2, using the previous workflow (Figure 3.9), high-efficiency delivery of Cas9 RNP complex is attained using Lipofectamine Stem reagent with >40% cleavage or indel efficiency being observed using the Invitrogen GeneArt Genomic Cleavage Detection (GCD) Kit. PSCs are also shown to attain high maintenance of pluripotency as assessed via immunocytochemistry of PSCs stained for Oct4, an intracellular marker of pluripotency.
Figure 3.10. GeneArt Genomic Cleavage Detection Kit analysis of cleavage efficiency. The assay was performed 72 hours following transfection of Gibco Human Episomal iPSCs with Cas9 RNP complex via Lipofectamine Stem reagent.
At times, lipid-based reagent solutions are not a viable option. Therefore, we recommend electroporation using the Invitrogen Neon Transfection System in these instances. StemFlex Medium is a versatile medium, which also supports PSCs in electroporation-based workflows, providing robust recovery of PSCs following electroporation (Figure 3.11 and 3.14), improved cleavage efficiency relative to electroporation-based workflows (Figure 3.12), and high maintenance of pluripotency of stem cells following electroporation and recovery (Figure 3.13).
Figure 3.13. Maintenance of pluripotency of iPSCs cultured in StemFlex Medium after electroporation and recovery. Cultures transfected with Cas9–gRNA complexes targeting the HPRT gene were assessed by (A) qualitative immunocytochemistry of Oct4 and TRA-1-60 expression and (B) quantitative assessment of Nanog expression via flow cytometric analysis.
Figure 3.14. Cell growth and viability following electroporation of Cas9 plasmid and donor plasmid into ESI-017 cells.
- In addition to vitronectin, PSCs can be successfully grown and transfected in Essential 8 and StemFlex media on rhLaminin-521, as well as Geltrex matrix.
- PSCs passaged with Versene solution should be triturated into small clumps of 3–5 cells. Colonies broken into too large clusters (>10 cells) will only transfect well around the outer edges. Alternatively, use TrypLE Select Enzyme as recommended for use with the StemFlex Medium system.
- Lipofectamine Stem reagent complexes should be made in Gibco Opti-MEM I Reduced Serum Medium and can be added directly to stem cells in culture media growing with or without serum. Note: an overlay method is required for StemFlex Medium–cultured PSCs.
- While the optimal timing of transfection (24–72 hours) of Cas9 RNP for maximal indel formation may need to be determined for each gene of interest, it is not necessary to remove transfection complexes. An equal volume of additional Essential 8 Medium should be added to the wells the following day without interfering with the transfection, if stem cell cultures need to be transfected for 48 hours. For StemFlex cultures, removal of complexes 24 hours post-delivery is recommended.
- The amount of Lipofectamine Stem reagent required for optimal transfection depends upon the amount of stem cells plated and the amount of DNA used (see recommended amounts below).
- Proliferating stem cell cultures need room to expand during transfection, so plate the recommended starting cell numbers to achieve 30–60% confluency on the day of transfection.
- Using lower amounts of DNA, mRNA, or RNP complexes can yield improved expression while minimizing risks of cytotoxicity from excess foreign constructs and erroneous cleavage events.
- In instances where poor lipid-based transfection is observed, the Neon Transfection System may be used as an alternative method.
For Research Use Only. Not for use in diagnostic procedures.