Esther Nolte-’t Hoen, Utrecht University, The Netherlands

Basic science

cancer

(cardio)vascular

hypoxia

reproduction/pregnancy

tissue repair and remodelling

body fluids / biomarkers

therapeutics

pathogen-derived EV

immune system

nervous system

isolation

characterization

RNA

biogenesis

Late breaking

Clinical

ISEV 2014: Basic science topics

ISEV 2013

cancer

(cardio)vascular

hypoxia

reproduction/pregnancy

tissue repair and remodelling

body fluids / biomarkers

therapeutics

pathogen-derived EV

immune system

nervous system

isolation

characterization

RNA

biogenesis

Late breaking

ISEV 2014: Basic science topics

Clinical

Flow cytometry

Single vesicle analysis

novel characterizationmethods

standardization inmeasurement

EV isolation and characterization

cancer

(cardio)vascular

hypoxia

reproduction/pregnancy

tissue repair and remodelling

body fluids / biomarkers

therapeutics

pathogen-derived EV

immune system

nervous system

isolation

characterization

RNA

biogenesis

Late breaking

EV isolation

Buzz words 2014:

• Size exclusion chromatography

• Comparative analysis

- Purpose: Separate EV from contaminants

- Fluids:- Prostate fluid

- Blood plasma

- Milk

- Cell culture supernatant

- Big advantage: low labor intensity

- Future:- Different packing material to remove different contaminants?

- Loss of EV? How to assess this?

Buzz words 2014:

• Size exclusion chromatography

• Comparative analysis

Sucrose / iodixanol

Gradient separationUltracentrifugation

? hrs

Matrix-based isolation

SE chromatography

Differences in:

- Particle yield

- Enrichment of certain EV

populations

- Contamination with non-EV

- EV functionality

Future:

- Adjust method of choice to research

question?

- Serial usage for optimal purification

and subpopulation isolation?

EV isolation

Standardization

Topics 2014:

-Inter- and intra-individual variability

-Pre-analytical variables:

- Freeze/thaw (blood, milk)

- Shear stress in needles (blood)

- Handling time and temperature before/during EV

isolation (blood, milk)

- Timely removal of interfering protein complexes

(e.g. THP in urine and casein/whey in milk)

-Quality Control (e.g. protein/lipid ratios)

- Standardization of measurements

(?)

EV characterization

Buzz words 2014:

• At the single vesicle level

• Comparative analysis

- (fluorescent mode) NTA

- t-Resistive Pulse Sensing (qNano)

- Flow Cytometry

- (cryo) EM

- Asymmetric flow-field-flow fractionation

- Small Angle X-ray Scatter

- Dynamic Light Scattering

- Atomic Force Microscopy

- On-chip size/conc measurement

- ….

Compare:

EV characteristics (e.g. size)

EV concentration

EV characterization

Buzz words 2014:

• At the single vesicle level

• Comparative analysis

- (fluorescent mode) NTA

- t-Resistive Pulse Sensing (qNano)

- Flow Cytometry- (cryo) EM

- Asymmetric flow-field-flow fractionation

- Small Angle X-ray Scatter

- DLS

- New high-end cytometers & labeling strategies to use

conventional cytometers

- Comparison of different flow cytometers

- Crucial settings for correct measurements

- EV sorting

High-throughput analysis of

EV contentsEV numbers

EV cargo selection and release

Release:

- Live cell fluorescence imaging (e.g. pH sensitive optical reporter)

- Substances inducing EV release

Biogenesis:

- C-Elegans and Drosophila as model organisms (e.g. RNAi screens)

- EV from plasma membrane versus MVB & different types of MVB

EV contents:

-Subpopulations analysis: Lipidomics / proteomics / transcriptiomics

-Overlap RNA species in EV from mammalian cells, parasites, fungi, bacteria!

-Mechanisms for RNA sorting

EV function

STEVE GSCHMEISSNER/SCIENCE PHOTO LIBRARY

Key player ISEV 2014:

Mesenchymal Stem Cell

Physiological role Tissue repair Other therapeutic effects

Osteogenicdifferentiation of MSC

Ischemia cancer

angiogenesis Irradiation damage GvHD

Skin burns

nephrotoxicity

Muscle injury

Remarkable new insights

Release of organelles via EV:

Proteasomes (T cell lines)

Mitochondria (from MSC to macrophages)

1

Remarkable new insights

Facts:

- Pub-crawl driven research

- Consumption of high numbers of EV

- No pro-inflamm effects

Release of organelles via EV1

Yeast-derived EV in beer

2

Thank you all!