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Transfecting Stem Cells
Why, Where and How?
Mirus Bio LLC
Miguel Dominguez, M.S.
Technical Services Scientist
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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
Outline
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What Are Stem Cells?
Development, Growth and Repair
Self-renewal Pluripotent
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Self-renewal
• Unlimited proliferation (regeneration)
• Formation of same cell or new cell
• symmetric vs. asymmetric division
Self-renewal
Symmetric
division
Asymmetric
division
A. Stem Cell
B. Progenitor Cell
C. Differentiated Cell
http://en.wikipedia.org/wiki/Stem_cell
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Pluripotency
Plurimus (very many) + Potens (having power)
Differentiation to any cell type
Tissue and organ formation
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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
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Embryonic Stem Cells
• Derived from embryos fertilized in vitro
• Self-renew and pluripotent
• Ethical and legal hurdles
Types of Stem Cells
http://en.wikipedia.org/wiki/Stem_cell
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Adult/Somatic Stem Cells
• Undifferentiated cells within tissue/organ
• Self-renew and multipotent (not pluripotent)
Types of Stem Cells
http://stemcells.nih.gov/info/basics/basics4
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Induced Pluripotent Stem Cells (iPSCs)
• Somatic cells reprogrammed to pluripotent state
• Self-renew and pluripotent
• Unlimited quantities
Types of Stem Cells
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Basic biology
• Development and differentiation
• Regeneration and repair
Disease modeling
• Specific genetic backgrounds
• Drug target identification
Treatments
• Cell based therapies
• New drug development
What Capabilities Can Stem Sells Provide?
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How Do We Get There?
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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
Why Transfection?
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Why Transfection? E
ffic
iency
Safety
MMLV-derived retrovirus
Lentivirus Excisable lentivirus RNA
Transposon
Episomal vector
Small molecule
Protein
Adapted from Nature Review
Genetics (2011) 12: 231-242
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Where is Transfection Applicable?
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Where is Transfection Applicable?
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Reprogramming via plasmid DNA
• Integrative
• PiggyBac transposons
• Linear DNA with loxP sites
Transfection for Reprogramming
K Woltjen et al. Nature 458, 766-770 (2009)
K Kaji et al. Nature 458, 771-775 (2009)
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Reprogramming via plasmid DNA
• Non-integrative
• DNA minicircles
• Episomal vectors
• Biotherapeutics
Transfection for Reprogramming
Okita et al. Science 2009
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Reprogramming via plasmid DNA
• Non-integrative
• DNA minicircles
• Episomal vectors
• Biotherapeutics
Transfection for Reprogramming
Jia et al. Nature Methods 7: 197-199 (2010)
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Transfection for Reprogramming
Angel and Yanik. PLoS ONE (2010) 5: e11756.
RNA mediated
• Non-integrative
• Faster, higher efficiencies
• Modified transcripts
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RNA mediated
• Non-integrative
• Faster, higher efficiencies
• Modified transcripts
Transfection for Reprogramming
Warren et al. Cell Stem Cell (2010) 7: 618-630
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Transfection for Reprogramming
Nucleic Acids Research (2011) 39: e142p
Human Dendritic Cells Primary Keratinocytes
RNA mediated
• Non-integrative
• Faster, higher efficiencies
• Modified transcripts
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Where is Transfection Applicable?
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Transfection of iPSCs
• Gene targeting strategies
• Nuclease mediated homologous recombination
Transfection of Stem Cells
Nature Rev Genetics (2010) 11: 636-646
Nature Rev Mol Cell Bio (2012) Online 11-21-12
Zinc Finger Nucleases (ZFN)
Transcription Activator-like Effector Nucleases (TALENS)
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Transfection of iPSCs
• Gene targeting strategies
• Nuclease mediated homologous recombination
Transfection of Stem Cells
Sigma-Aldrich CompoZr® Knockout Zinc Finger Nucleases (ZFN)
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Transfection of iPSCs
• Gene targeting strategies
• Nuclease mediated homologous recombination
Transfection of Stem Cells
Electroporation of iCell® Cardiomyocytes
Data courtesy of
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Where is Transfection Applicable?
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Transfection of Stem Cell Derivatives
Transfection of iCell® Cardiomyocytes
Data courtesy of
Transfection of iPSC Derivatives
• Cardiomyocytes, endothelial, neurons etc.
• Purify stem cell derivatives
• Toxicity screening
• Inducible systems
• Drug target identification
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Transfection of iPSC Derivatives
• Cardiomyocytes, hepatocytes, neurons etc.
• Purify stem cell derivatives
• Toxicity screening
• Inducible systems
• Drug target identification
Transfection of Stem Cell Derivatives
40k cells/well
80k cells/well
cAMP Pathway Induction Measured Via Luciferase Reporter Plasmid
Data courtesy of
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Transfection of iPSC derived cell types
• Cardiomyocytes, hepatocytes, neurons etc.
• Purify stem cell derivatives
• Toxicity screening
• Inducible systems
• Drug target identification
Transfection of Stem Cell Derivatives
Data courtesy of
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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
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TransIT®-2020 for DNA delivery
Mirus for Stem Cell Applications
The TransIT®-2020 Transfection Reagent was used to transfect 0.5 x 106 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.
Fluorescent Merged Flow Cytometery
Data courtesy of
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TransIT®-LT1 for DNA delivery
Mirus for Stem Cell Applications
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.
Phase Contrast Fluorescent Merged
Data courtesy of Natasa Savic, MSc, Santoro Lab, University of Zürich
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TransIT®-mRNA for RNA delivery
Mirus for Stem Cell Applications
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.
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TransIT-TKO for RNAi
Mirus for Stem Cell Applications
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.
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Ingenio® for Electroporation
Mirus for Stem Cell Applications
The Ingenio® Electroporation Kit was used to transfect 2 x 106 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 106 cells/well. Cells were visualized 24 hours post-transfection and imaged under 4X objective with an Olympus IX71® Inverted Microscope.
Fluorescent Merged Flow Cytometery
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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®
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Strike Balance – High Efficiency and Low Tox
What About Toxicity?
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.
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What About Toxicity?
Mirus TransIT® Transfection Reagents Minimize
Stress Response in Transfected HeLa Cells. Stress-
related 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 stress-
related gene expression changes were lower when cells
were transfected with TransIT®-LT1 or TransIT®-2020
than when cells were transfected with Lipofectamine®
2000.
Minimize Stress Response
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What About Toxicity?
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.
Affect Fewer Pathways
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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
Transfection and Stem Cell Applications
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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
Mirus for Transfection
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