Transfecting Stem Cells Why, Where and How? - Mirus Bio€¦ · iCell® Cardiomyocytes were...

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Transfecting Stem Cells

Why, Where and How?

Mirus Bio LLC

Miguel Dominguez, M.S.

Technical Services Scientist


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


What Are Stem Cells?

Development, Growth and Repair

Self-renewal Pluripotent


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


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


• Unlimited quantities


Embryonic Stem Cells

• Derived from embryos fertilized in vitro


• Ethical and legal hurdles

Types of Stem Cells

http://en.wikipedia.org/wiki/Stem_cell


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


Induced Pluripotent Stem Cells (iPSCs)

• Somatic cells reprogrammed to pluripotent state


• Unlimited quantities

Types of Stem Cells


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?


How Do We Get There?


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?


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


Where is Transfection Applicable?




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)



• Non-integrative

• DNA minicircles

• Episomal vectors

• Biotherapeutics


Okita et al. Science 2009



• Non-integrative

• DNA minicircles

• Episomal vectors

• Biotherapeutics


Jia et al. Nature Methods 7: 197-199 (2010)



Angel and Yanik. PLoS ONE (2010) 5: e11756.

RNA mediated

• Non-integrative

• Faster, higher efficiencies

• Modified transcripts


RNA mediated

• Non-integrative




Warren et al. Cell Stem Cell (2010) 7: 618-630



Nucleic Acids Research (2011) 39: e142p

Human Dendritic Cells Primary Keratinocytes

RNA mediated

• Non-integrative






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)






Sigma-Aldrich CompoZr® Knockout Zinc Finger Nucleases (ZFN)






Electroporation of iCell® Cardiomyocytes

Data courtesy of




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



Transfection of iPSC Derivatives

• Cardiomyocytes, hepatocytes, neurons etc.






40k cells/well

80k cells/well

cAMP Pathway Induction Measured Via Luciferase Reporter Plasmid

Data courtesy of


Transfection of iPSC derived cell types

• Cardiomyocytes, hepatocytes, neurons etc.






Data courtesy of


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


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


TransIT®-LT1 for DNA delivery


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


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.


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.


Ingenio® for Electroporation


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


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®


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.



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



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


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


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


Visit The Transfection Experts @ Booth 423

Transfection Fundamentals

Rm 111, 6:45pm

Win an iPad mini every day!

Where to find Mirus

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Transfecting Stem Cells Why, Where and How? - Mirus Bio€¦ · iCell® Cardiomyocytes were...

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