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Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10 Page 27

vesicles are proposed to mediate their effects and, to date, the reparative and regenerative effects of stem
cell EVs have been demonstrated in a wide range of cell types. While a high potential for their therapeutic
use exists, there is a gap of knowledge surrounding their characterization, mechanisms of action, and how
they may regulate cells of the central nervous system (CNS). We have isolated and recovered high yields
of EVs from large scale cultures of both induced pluripotent stem cells (iPSCs) and mesenchymal stem
cells (MSCs) using tangential flow filtration. Our EV characterization includes both phenotypic (size,
tetraspanin expression) and biochemical assays. EV functionality has also been assessed in vitro utilizing
several cell-based assays related to cellular viability, migration, angiogenesis, and immunomodulation
in both healthy and damaged recipient cells with relevance to the CNS. Our data suggests that EVs from
different sources of stem cells display unique phenotypes, exhibit differential association with various
cytokines, proteins, and long non-coding RNAs, and have the ability to significantly enhance processes
[4]
that are critical for cellular repair . Lastly, utilizing an iPSC-derived neurosphere model, we have observed
a robust uptake of stem cell EVs and have found that these EVs are able to effectively penetrate these 3D
structures. Collectively, these results highlight the “holistic” properties of stem cell EVs by demonstrating
their ability to partially reverse or reduce damage in various cell types.

REFERENCES
1. Sampey GC, Saifuddin M, Schwab A, et al. Exosomes from HIV-1-infected cells stimulate production of pro-inflammatory cytokines
through trans-activating response (TAR) RNA. J Biol Chem 2016;291:1251-66.
2. Pleet ML, Erickson J, DeMarino C, et al. Ebola virus VP40 modulates cell cycle and biogenesis of extracellular vesicles. J Infect Dis
2018;218:S365-87.
3. Pinto DO, DeMarino C, Pleet ML, et al. HTLV-1 extracellular vesicles promote cell-to-cell contact. Front Microbiol 2019;10:2147.
4. Branscome H, Paul S, Khatkar P, et al. Stem cell extracellular vesicles and their potential to contribute to the repair of damaged CNS
cells. J Neuroimmune Pharmacol 2020;15:520-37.

8. Extracellular vesicles from HTLV-1 infected cells regulate viral spread and pathogenesis

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Authors: Sarah Al Sharif , Daniel O. Pinto , Gifty Mensah , Maria Cowen , Pooja Khatkar , James
1
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2
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Erickson , Heather Branscome , Catherine DeMarino , Weidong Zhou , Lance A. Liotta , Fatah Kashanchi 1
E-mail: salshar@gmu.edu
Affiliations:
1 Laboratory of Molecular Virology, School of Systems Biology, George Mason University, Manassas, VA, USA.
2 Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA.
Abstracts: Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T- cell leukemia/
[1,2]
lymphoma (ATLL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) .
Approximately 10 million worldwide are infected with HTLV-1, however, it is most likely that the true
[2]
figure is greater than this number as many cases are not reported . It is endemic in many areas such
[2]
as southern Japan, the Caribbean, and parts of Africa . In recent publications, we showed that HTLV-
1 infected cells release Extracellular Vesicles (EVs) containing viral RNA and proteins (gp61/Tax/HBZ)
[3,4]
and are not infectious . However, they can enhance cell-to-cell contact of uninfected cells, eventually
[4]
aid in spread of virus . We separated distinct EV subpopulations from HTLV-1 infected cell supernatants
by differential ultracentrifugation (DUC) into 2k, 10k, and 100k EVs based upon centrifugation speed.
Proteome profiling of various HTLV-1 EV subpopulations showed that viral/host protein was highly
abundant in 2k subpopulation, compared to other subpopulations. Western Blots revealed that p19
and Tax (i.e., viral proteins), as well as LC3 and p62 (i.e., autophagy proteins) were mainly present in
the 2k and 10k subpopulation. Using an in vitro angiogenesis assay, 2k HTLV-1 EVs were primarily
responsible for tubular deterioration. Different EV subpopulations were incubated with cells involved in
neuroinflammation (i.e., astrocytes, macrophages, and neurons) where the highest level of IL-8 expression