Superior transfection efficiency without toxicity in even the most difficult stem cell assays

Achieve up to 80% transfection efficiency in PSCs and NSCs and up to 45% in MSCs

  • Versatile: co-delivers DNA (up to 11 kb), RNA, and Cas9 protein complexes
  • Gentle on cells: delivers low toxicity and continues cell proliferation without inducing differentiation
  • Flexible: transfects adherent and suspension cells, offering a simple alternative to electroporation

Figure 1. Time-lapse phase-contrast (left) and fluorescence (right) movies of human ES cells (H9) grown in Essential 8 medium on vitronectin and transfected with 1 µL Lipofectamine Stem reagent complexed with a GFP-expression construct (500 ng total plasmid DNA). Cultures were observed over 48 hours from start of transfection.

Featured application note: Versatility and performance of Lipofectamine Stem Transfection Reagent

Lipofectamine Stem Cell Reagent application note

The goal of this study was to illustrate the versatility of Invitrogen Lipofectamine Stem Transfection Reagent, which was developed specifically for stem cells, to co-deliver multiple payloads in addition to large plasmid constructs with high transfection efficiency. Lipofectamine Stem reagent can be used to transfect a wide range of stem cell types with superior efficiency while supporting continued proliferation without inducing differentiation.

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Superior transfection in human embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), neural stem cells (NSC), and mesenchymal stem cells (MSC)

Stem cell transfection is a key technique for many researchers, but the sensitivity of stem cells has made them difficult to transfect efficiently using reagent-based protocols. Designed for dramatically improved transfection efficiency, Invitrogen Lipofectamine Stem Transfection Reagent offers a simple, robust and reproducible method for delivering DNA, RNA and RNP such as Cas9/gRNA complexes into a wide range of stem cells, including pluripotent stem cells, neural stem cells and mesenchymal stem cells (Figure 2). This newest addition to the Lipofectamine transfection reagent family helps ensure high-efficiency transfection while maintaining maximum cell viability and growth in an undifferentiated state.


Versatility for the delivery of DNA, RNA and/or Cas9 protein complexes into stem cells

Research applications such as gene editing, gene expression, and directed differentiation depend on the effective, efficient delivery of DNA, RNA and/or Cas9 protein complexes into stem cells. Experiments involving large constructs have typically been carried out using electroporation, as reagent-based transfection yielded poor results. Lipofectamine Stem reagent offers expanded capabilities for stem cell transfection, making it possible to deliver DNA plasmids in sizes up to 11 kb. For gene editing, Lipofectamine Stem reagent allows the co-transfection of Cas9 protein complexed with guide RNAs along with single-stranded DNA for homology directed repair. In addition, Lipofectamine Stem reagent can be used for the transfection of mRNA for gene expression or directed differentiation studies using stem cells. All of these attributes make this versatile reagent an ideal addition to your stem cell research tool kit and a readily accessible alternative to electroporation.

A. DNA delivery

B. mRNA delivery

C. mRNA & Cas9 protein delivery

Figure 2. Lipofectamine Stem Reagent outperforms FuGENE HD Reagent in pluripotent stem cell transfection, delivering both small and large DNA plasmids, mRNA, and Cas9 protein complexes. (A) Human ES (H9) cells or Human Episomal iPSCs were transfected with Lipofectamine Stem Reagent or FuGENE HD Regent according to the accompanying protocol and either a 5 kb or 11 kb DNA plasmid expressing GFP and examined for GFP expression 24 hours posttransfection. In the bottom panel NCRM-iPS cells transfected using Lipofectamine Stem Reagent with a ~10.5 kb plasmid expressing Cas9 and GFP followed by detection via immunocytochemistry. (B) Human ES (H9) cells were transfected with Lipofectamine Stem Reagent or FuGENE HD Reagent according to the protocol with an mRNA expressing GFP. (C, left) Lipofectamine Stem Reagent was used to transfect cells with Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). (C, right) Genome modification was analyzed using the T7Endo 1 assay.

A. DNA delivery

B. mRNA & Cas9 protein delivery

Figure 3. Lipofectamine Stem Reagent outperforms FuGENE HD Reagent in neural stem cell transfection, delivering both small and large DNA plasmids, mRNA, and Cas9 protein complexes. (A, top) Human iPSC-derived neural stem cells were transfected with a 5 kb DNA plasmid expressing GFP using Lipofectamine Stem Reagent or FuGENE HD Reagent according to the accompanying protocol and examined for GFP expression 24 hours post-transfection. (A, bottom) Human iPSC-derived neural stem cells were transfected with an 11 kb DNA plasmid expressing GFP using Lipofectamine Stem according to the protocol and examined for GFP expression 24 hours post-transfection. (B, left) Human iPSC-derived neural stem cells were transfected in suspension using Lipofectamine Stem Reagent with 250 ng Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). GFP was observed after 24 hours. (B, right) Genome modification was analyzed using the T7Endo 1 assay.

A. DNA delivery

B. mRNA delivery

Figure 4. Lipofectamine Stem Reagent outperforms FuGENE HD Reagent in mesenchymal stem cell transfection, delivering plasmid DNA and mRNA. (A, top) StemPro Human Adipose-Derived Stem Cells were transfected with a 5 kb DNA plasmid expressing GFP using Lipofectamine Stem Reagent or FuGENE HD Reagent according to the accompanying protocol and examined for GFP expression 24 hours post-transfection. (A, bottom) StemPro BM Mesenchymal Stem Cells were transfected with a 5 kb DNA plasmid expressing GFP using Lipofectamine Stem Reagent or FuGENE HD Reagent according to the accompanying protocol and examined for GFP expression 24 hours post-transfection. (B) StemPro BM Mesenchymal Stem Cells were transfected with mRNA expressing GFP using Lipofectamine Stem Reagent and examined for GFP expression 48 hours post-transfection..

A. DNA delivery

B. mRNA delivery

C. mRNA & Cas9 protein delivery

Figure 2. Lipofectamine Stem Reagent outperforms FuGENE HD Reagent in pluripotent stem cell transfection, delivering both small and large DNA plasmids, mRNA, and Cas9 protein complexes. (A) Human ES (H9) cells or Human Episomal iPSCs were transfected with Lipofectamine Stem Reagent or FuGENE HD Regent according to the accompanying protocol and either a 5 kb or 11 kb DNA plasmid expressing GFP and examined for GFP expression 24 hours posttransfection. In the bottom panel NCRM-iPS cells transfected using Lipofectamine Stem Reagent with a ~10.5 kb plasmid expressing Cas9 and GFP followed by detection via immunocytochemistry. (B) Human ES (H9) cells were transfected with Lipofectamine Stem Reagent or FuGENE HD Reagent according to the protocol with an mRNA expressing GFP. (C, left) Lipofectamine Stem Reagent was used to transfect cells with Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). (C, right) Genome modification was analyzed using the T7Endo 1 assay.

A. DNA delivery

B. mRNA & Cas9 protein delivery

Figure 3. Lipofectamine Stem Reagent outperforms FuGENE HD Reagent in neural stem cell transfection, delivering both small and large DNA plasmids, mRNA, and Cas9 protein complexes. (A, top) Human iPSC-derived neural stem cells were transfected with a 5 kb DNA plasmid expressing GFP using Lipofectamine Stem Reagent or FuGENE HD Reagent according to the accompanying protocol and examined for GFP expression 24 hours post-transfection. (A, bottom) Human iPSC-derived neural stem cells were transfected with an 11 kb DNA plasmid expressing GFP using Lipofectamine Stem according to the protocol and examined for GFP expression 24 hours post-transfection. (B, left) Human iPSC-derived neural stem cells were transfected in suspension using Lipofectamine Stem Reagent with 250 ng Cas9 mRNA (modified)/ gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified) or Cas9 protein/gRNA: Emx-1 crRNA (Exon2)-tracrRNA oligo/ GFP mRNA (modified). GFP was observed after 24 hours. (B, right) Genome modification was analyzed using the T7Endo 1 assay.

A. DNA delivery

B. mRNA delivery

Figure 4. Lipofectamine Stem Reagent outperforms FuGENE HD Reagent in mesenchymal stem cell transfection, delivering plasmid DNA and mRNA. (A, top) StemPro Human Adipose-Derived Stem Cells were transfected with a 5 kb DNA plasmid expressing GFP using Lipofectamine Stem Reagent or FuGENE HD Reagent according to the accompanying protocol and examined for GFP expression 24 hours post-transfection. (A, bottom) StemPro BM Mesenchymal Stem Cells were transfected with a 5 kb DNA plasmid expressing GFP using Lipofectamine Stem Reagent or FuGENE HD Reagent according to the accompanying protocol and examined for GFP expression 24 hours post-transfection. (B) StemPro BM Mesenchymal Stem Cells were transfected with mRNA expressing GFP using Lipofectamine Stem Reagent and examined for GFP expression 48 hours post-transfection..

Lipofectamine Stem Reagent supports a healthy culture without inducing differentiation

When transfecting stem cells, it is important that culture conditions support their continued proliferation in an undifferentiated state. While introduction of exogenous constructs can have intended and unintended consequences, Lipofectamine Stem Transfection Reagent does not adversely affect proliferation (Figure 5).

Figure 5. Cells continue to proliferate at a rate similar to un-transfected. By plotting confluency measurements of transfected and un-transfected iPSCs, a similar rate of growth and time to confluence is observed.

Tips for maximizing efficiency in adherent and suspension PSCs

There are many choices of culture systems for pluripotent stem cells including feeder-based and feeder-free systems, which include using a variety of matrices. Lipofectamine Stem reagent can be used in conjunction with a variety of culture systems. Below are some tips to maximize transfection efficiency in your culture system of choice:

  1. For culture conditions where PSCs are passaged as clusters of cells and grown as large colonies on feeders or on Geltrex matrix, transfection in suspension at the time of passaging and re-plating can maximize access of the reagent to all the cells and increase transfection efficiency. This option can be used with monolayer culture systems as well to ensure even, high transfection efficiency or as an alternative to electroporation.
  2. When transfecting adherent cultures growing on Geltrex or Matrigel matrices, it is important to plate small clusters or single pluripotent stem cells at an optimal density of 30–60% to allow them room to continue to expand during a transfection time of 24 to 48 hours.
  3. When transfecting adherent cultures, growing cells on vitronectin or laminin—which allow the cells to spread out in more of a monolayer—can maximize transfection.
  4. While Lipofectamine Stem reagent is compatible with most culture systems, components in some stem cell media can interfere with liposomal-based transfection. In those cases, using Opti-MEM medium for an initial transfection period of 1–4 hours followed by the addition of your medium of choice can minimize this effect while still supporting continued expansion. Refer to the   Lipofectamine Stem Transfection Reagent user guide for specific details.

Stem cell–specific protocols

In addition to the protocol developed for the   Lipofectamine Stem Transfection Reagent user guide we’ve created stem cell–specific protocols that will help guide you to high efficiency with low toxicity in individual stem cell types (ESC, NSC or MSC) and with your media of choice. The protocols cover growing cells, culture conditions, and passaging through to successful transfection results.

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