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Achieve up to 80% transfection efficiency in pluripotent stem cells (PSCs) and neural stem cells (NSCs), and up to 45% in mesenchymal stem cells (MSCs).
Figure 1. Time-lapse fluorescence (left) and phase-contrast (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.
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.
Download Lipofectamine Stem Transfection Reagent application note
Although stem cell transfection is a a key technique for many researchers, 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 Lipofectamine transfection reagent family helps ensure high-efficiency transfection while maintaining maximum cell viability and growth in an undifferentiated state.
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.
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.
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.
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.
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.
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.
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.
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. Transfected cells continue to proliferate at a rate similar to untransfected cells. By plotting confluency measurements of transfected and untransfected iPSCs, a similar rate of growth and time to confluence is observed.
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:
In addition to the Lipofectamine Stem Transfection Reagent user guide, please see stem cell-specific protocols that will help guide you to high efficiency transfection with low toxicity in individual stem cell types (ESC, NSC, or MSC) using your media of choice. These protocols cover growing cells, culture conditions, and passaging through to successful transfection results.
Our Lipofectamine reagent-specific protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Search this library to find protocols and citations curated to fit your experimental needs. Filter by cell type, payload, and product of choice to easily unlock your answers.
Search our extensive collection of Transfection FAQs to find the answers related to Lipofectamine Stem Transfection Reagent.
For Research Use Only. Not for use in diagnostic procedures.