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Page 2 of 9 Xu et al. Vessel Plus 2023;7:33 https://dx.doi.org/10.20517/2574-1209.2023.98

Conclusions: Although there was a trend in the improvement in infarct size, a single-dose administration of neither
IM nor IV injection of HBMSC-EV resulted in significant improvement in post-MI cardiac function. A major
limitation of this study is the lack of trials determining the optimal dose of HBMSC-EV needed in this model.
However, the current study demonstrates that future studies are required to either optimize administration or
bioengineer HBMSC-EV with cardiac-homing properties.

Keywords: Human bone mesenchymal stem cell-derived extracellular vesicles, biodistribution, intramyocardial
injection, intravenous injection, myocardial infarction

INTRODUCTION
Extracellular vesicles (EV) are secreted by almost every cell type and are lipid bilayer membranes that encase
a variety of compounds such as proteins, lipids, nucleic acids, etc. In stem cells, EVs are thought to be
responsible for the beneficial paracrine effects. Stem cell-derived EVs have a wide range of therapeutic
functions in preclinical models of cardiovascular disease - EVs have been shown to protect several forms of
myocardial ischemia in both small and large animal models .
[1-4]

Furthermore, intramyocardial (IM) injection has been shown to be the most reliable form of EV delivery,
while intravenous (IV) injection is not [1,2,4-7] . A systemic review found that when EVs were intravenously
injected, there was little to no evidence of EV localization to the heart, with primarily hepatic and splenic
[8]
uptake . We recently confirmed these findings in a model of murine acute MI, and only found cardiac
uptake of EVs after IM injection and not with IV injection .
[7]
However, no long-term functional studies have been done to compare the effects of intramyocardial vs. IV
EV injection side-by-side in an acute MI model. These two different delivery methods are important to
compare since IM injection has more reliable cardiac benefits in preclinical studies, but requires a
thoracotomy to deliver. IV injection, though with less reliable cardiac benefits, is a non-invasive delivery
method that is more translatable to clinical practice. In this study, we delivered human bone mesenchymal
stem cell-derived EVs (HBMSC-EV) via either IM or IV injection after murine acute MI and examined
cardiac function 28 days post-injection. Additionally, as our previous findings confirmed hepatic HBMSC-
EV uptake after IV injection, we evaluated for evidence of changes in liver inflammation, fibrosis, growth,
and oxidative status 28 days post-injection since mesenchymal stem cell-derived EVs have been shown to
[9]
have beneficial effects in preclinical liver disease models . We also wanted to determine if there were any
detectable long-term changes in the liver after HBMSC-EV uptake.

METHODS
HBMSC-EV isolation
HBMSC (Lonza, PT-2501) were cultured to passage 7 with the Mesenchymal Stem Cell Growth Medium
(MSCGM) BulletKit (Lonza, PT-3001), per manufacturer’s instructions . At 80%-90% confluency, the old
[7]
MSCGM was removed from the HBMSC and replaced with fresh MSCGM. The HBMSC were placed in a
humidified hypoxia chamber (Billups‐Rothenberg, MIC‐101) containing 95% N and 5% CO for 24 h at
2
2
37 °C. The HBMSC were then removed from the hypoxia chamber and the media was collected. The media
was centrifuged at 2,000× g to remove the cell debris, and then ultracentrifuged (WX Ultra Centrifuge with
Sorvall AH‐629 rotor) twice at 100,000× g for 70 min to isolate the HBMSC‐EV pellet and to wash the
HBMSC-EV with Dulbecco's Phosphate Buffered Saline (PBS). The HBMSC-EV were re-suspended in PBS
with 1% dimethylsulfoxide (DMSO) and stored at -80 °C . Hypoxia pre-conditioned HBMSC-EVs were
[7]
[10]
isolated, given previous studies suggesting greater proteomic promise in cardiovascular disease . Using this