Transfecting Stem Cells Why, Where and How?

Transfecting Stem Cells Why, Where and How? Mirus Bio LLC Miguel Dominguez, M.S. Technical Services Scientist

Outline What What are stem cells? What capabilities can stem cells provide? Why, Where and How Why transfection? Where is transfection applicable? How is transfection employed? Solutions from The Transfection Experts Mirus solutions for DNA and RNA delivery Toxicity considerations

What Are Stem Cells? Development, Growth and Repair Self-renewal Pluripotent

Self-renewal Self-renewal Unlimited proliferation (regeneration) Formation of same cell or new cell symmetric vs. asymmetric division Symmetric division Asymmetric division A. Stem Cell B. Progenitor Cell C. Differentiated Cell http://en.wikipedia.org/wiki/stem_cell

Pluripotency Plurimus (very many) + Potens (having power) Differentiation to any cell type Tissue and organ formation

Types of Stem Cells Embryonic Stem Cells (ESCs) Derived from embryos fertilized in vitro Self-renew and pluripotent Ethical and legal hurdles Adult or Somatic Stem Cells Undifferentiated cells within tissue/organ Self-renew and multipotent (not pluripotent) Induced Pluripotent Stem Cells (ipscs) Somatic cells reprogrammed to pluripotent state Self-renew and pluripotent Unlimited quantities

Types of Stem Cells Embryonic Stem Cells Derived from embryos fertilized in vitro Self-renew and pluripotent Ethical and legal hurdles http://en.wikipedia.org/wiki/stem_cell

Types of Stem Cells Adult/Somatic Stem Cells Undifferentiated cells within tissue/organ Self-renew and multipotent (not pluripotent) http://stemcells.nih.gov/info/basics/basics4

Types of Stem Cells Induced Pluripotent Stem Cells (ipscs) Somatic cells reprogrammed to pluripotent state Self-renew and pluripotent Unlimited quantities

What Capabilities Can Stem Sells Provide? Basic biology Development and differentiation Regeneration and repair Disease modeling Specific genetic backgrounds Drug target identification Treatments Cell based therapies New drug development

How Do We Get There?

Why Transfection? Virus transduction High efficiencies Genomic integration Oncogenic effects Immune response Unsuitable for biotherapeutics Protein transfection Non-integrative Low efficiencies Large amounts of protein necessary Small molecule Non-integrative Inefficient

Efficiency Why Transfection? MMLV-derived retrovirus Lentivirus Excisable lentivirus RNA Transposon Episomal vector Small molecule Safety Protein Adapted from Nature Review Genetics (2011) 12: 231-242

Where is Transfection Applicable?

Where is Transfection Applicable?

Transfection for Reprogramming Reprogramming via plasmid DNA Integrative PiggyBac transposons Linear DNA with loxp sites K Woltjen et al. Nature 458, 766-770 (2009) K Kaji et al. Nature 458, 771-775 (2009)

Transfection for Reprogramming Reprogramming via plasmid DNA Non-integrative DNA minicircles Episomal vectors Biotherapeutics Okita et al. Science 2009

Transfection for Reprogramming Reprogramming via plasmid DNA Non-integrative DNA minicircles Episomal vectors Biotherapeutics Jia et al. Nature Methods 7: 197-199 (2010)

Transfection for Reprogramming RNA mediated Non-integrative Faster, higher efficiencies Modified transcripts Angel and Yanik. PLoS ONE (2010) 5: e11756.

Transfection for Reprogramming RNA mediated Non-integrative Faster, higher efficiencies Modified transcripts Warren et al. Cell Stem Cell (2010) 7: 618-630

Transfection for Reprogramming RNA mediated Non-integrative Faster, higher efficiencies Modified transcripts Primary Keratinocytes Human Dendritic Cells Nucleic Acids Research (2011) 39: e142p

Where is Transfection Applicable?

Transfection of Stem Cells Transfection of ipscs Gene targeting strategies Nuclease mediated homologous recombination Zinc Finger Nucleases (ZFN) Nature Rev Genetics (2010) 11: 636-646 Transcription Activator-like Effector Nucleases (TALENS) Nature Rev Mol Cell Bio (2012) Online 11-21-12

Transfection of Stem Cells Transfection of ipscs Gene targeting strategies Nuclease mediated homologous recombination Sigma-Aldrich CompoZr Knockout Zinc Finger Nucleases (ZFN)

Transfection of Stem Cells Transfection of ipscs Gene targeting strategies Nuclease mediated homologous recombination Electroporation of icell Cardiomyocytes Data courtesy of

Where is Transfection Applicable?

Transfection of Stem Cell Derivatives Transfection of ipsc Derivatives Cardiomyocytes, endothelial, neurons etc. Purify stem cell derivatives Toxicity screening Inducible systems Drug target identification Transfection of icell Cardiomyocytes Data courtesy of

Transfection of Stem Cell Derivatives Transfection of ipsc Derivatives camp Pathway Induction Measured Via Luciferase Reporter Plasmid Cardiomyocytes, hepatocytes, neurons etc. Purify stem cell derivatives Toxicity screening Inducible systems Drug target identification Data courtesy of 40k cells/well 80k cells/well

Transfection of Stem Cell Derivatives Transfection of ipsc derived cell types Data courtesy of Cardiomyocytes, hepatocytes, neurons etc. Purify stem cell derivatives Toxicity screening Inducible systems Drug target identification

Mirus for Stem Cell Applications TransIT -Transfection Reagents Low toxicity High efficiency DNA, RNA and sirna Ingenio Electroporation Kit Universal solution Compatible with conventional electroporators

Mirus for Stem Cell Applications TransIT -2020 for DNA delivery Fluorescent Merged Flow Cytometery The TransIT -2020 Transfection Reagent was used to transfect 0.5 x 10 6 ips cells with a ZsGreen expressing plasmid (Clontech). Transfections were performed in 6-well plates using 7.5 µl of TransIT-2020 Transfection Reagent to deliver 2.5 µg of DNA (3:1, reagent: DNA). Cells were visualized 24 hours post-transfection and imaged at 4X objective with an Olympus IX71 Inverted Microscope. Data courtesy of

Mirus for Stem Cell Applications TransIT -LT1 for DNA delivery Phase Contrast Fluorescent Merged Data courtesy of Natasa Savic, MSc, Santoro Lab, University of Zürich Mouse embryonic stem cells (mescs) were seeded at 250,000 cells per well of a 6-well plate and transfected 2 hours after plaiting with 6 ul of TransIT -LT1 Transfection Reagent and 2.5 ug of a GFP expressing plasmid. Efficiency was visualized at approx. 60%. Images were taken using a Leica DMI 6000B inverted microscope 48 hours post-transfection.

Mirus for Stem Cell Applications TransIT -mrna for RNA delivery The TransIT -mrna Transfection Kit was used to transfect BJ human neonatal foreskin fibroblasts (A) and MRC-5 human lung fibroblasts (B) with a pseudouridine and 5mC modified based GFP mrna (Trilink Biotechnologies, Inc.). Transfections were performed in 12-well plates using 1-3 μl of TransIT-mRNA Transfection Reagent and mrna Boost Reagent to deliver 1 μg of RNA (1:1:1, 2:2:1 and 3:3:1; reagent: boost: RNA ratio). Cells were assayed 18 hours post-transfection on a BD LSR II Flow Cytometer. Cell viability was measured using propidium iodide stain.

Mirus for Stem Cell Applications TransIT-TKO for RNAi Panels A and B show the effect of GAPDH-targeted sirna on GAPDH (targeted) and HPRT1 (non-targeted) mrna expression, respectively. icell Cardiomyocytes were cultured for 7 days in a 12-well cell culture plate before transfection with either control (scrambled) or GAPDH sirna using TransIT-TKO (3-5 μl/well). 72 hours post-transfection the GAPDH and HPRT1 (non-targeted) mrna levels were measured relative to 18s rrna levels and normalized to the mrna levels obtained following transfection of the control sirna in each experiment.

Mirus for Stem Cell Applications Ingenio for Electroporation Fluorescent Merged Flow Cytometery The Ingenio Electroporation Kit was used to transfect 2 x 10 6 ips cells on the Amaxa Nucleofector II Device. Cells were electroporated with 8 µg ZsGreen expressing plasmid (Clontech) in 100 µl and plated in 6-well plates at 0.33 x 10 6 cells/well. Cells were visualized 24 hours post-transfection and imaged under 4X objective with an Olympus IX71 Inverted Microscope.

Why Mirus? What do your cells look like? What is your reagent doing? HeLa cells 8 and 24 hours post-transfection with TransIT and Lipofectamine

What About Toxicity? Strike Balance High Efficiency and Low Tox TransIT Broad Spectrum Reagents Balance High Efficiency Delivery with Low Toxicity. HeLa cells were transfected with luciferase encoding plasmid DNA using either TransIT -LT1, TransIT -2020 or Lipofectamine 2000 for 24 hours. Transfection was measured by luciferase activity using a conventional assay. Cytotoxicity was assessed by quantifying the LDH released from the cytosol of damaged cells compared to cells alone.

What About Toxicity? Minimize Stress Response Mirus TransIT Transfection Reagents Minimize Stress Response in Transfected HeLa Cells. Stressrelated gene expression changes were determined by RT-qPCR from total RNA samples harvested from HeLa cells that were transfected with TransIT -LT1, TransIT -2020 or Lipofectamine 2000 at 8 and 24 hours. Eighty-four genes were analyzed using the Human Stress Response 96 StellARray (Lonza). At both time points, the number and magnitude of stressrelated gene expression changes were lower when cells were transfected with TransIT -LT1 or TransIT -2020 than when cells were transfected with Lipofectamine 2000.

What About Toxicity? Affect Fewer Pathways Transfections with TransIT -LT1 or TransIT -2020 Affect Fewer Core Pathways than Transfections with Lipofectamine 2000. Canonical pathway enrichment analysis shows the primary biological processes impacted in HeLa cells that were transfected with TransIT -LT1, TransIT -2020 or Lipofectamine 2000 at 24 hours. The negative log of the p values is the probability of obtaining these genes associated with the given pathways by random chance. The bar line at the bottom of each graph is derived from the ratio of the number of genes on our list associated with a given pathway divided by the total number of genes that make up that pathway. Pathway analysis was performed using Pathway Analysis (Ingenuity Systems). Representative data from two independent tests is shown.

Transfection and Stem Cell Applications Stem cell research focus towards Less integrative and scar-free applications Higher efficiencies Transfection provides Safer alternatives to viral transduction Efficiencies suitable for reprogramming and differentiation

Mirus for Transfection TransIT Transfection Reagents for DNA, RNA and sirna delivery Ingenio Electroporation Kit for a compatible and cost-effective alternative Validated solutions for stem cell research Less toxic imperative for stem cell research Free samples to prove it to yourself

Where to find Mirus Visit The Transfection Experts @ Booth 423 Transfection Fundamentals Rm 111, 6:45pm Win an ipad mini every day!