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Xu et al. Vessel Plus 2023;7:33 https://dx.doi.org/10.20517/2574-1209.2023.98 Page 3 of 9

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protocol, approximately 5-7 million HBMSC were needed to produce the injection dosage, 2 × 10 HBMSC-
EV particles.

HBMSC-EV characterization
As previously described, the HBMSC-EV were characterized through electron microscopy, nanoparticle
[7]
tracking analysis, and immunoblotting . The HBMSC-EV were visualized with electron microscopy (FEI
Morgagni 268) after fixation and contrasted in 4% uranyl acetate. The size, number, and distribution of the
HBMSC-EV were quantified with the Nanosight NS500 (Malvern Instruments). 10 µg of HBMSC-EV
protein was loaded in a Bis-Tris gel and run using MOPS-SDS running buffer. The following markers were
evaluated through immunoblotting: CD81 (Cell Signaling #52892S, 1:1,000), CD9 (Cell Signaling #13403S,
1:1,000), Alix (Cell Signaling #92880S, 1:1,000), GAPDH (Cell Signaling #97166S, 1:1,000), heat shock
protein 70 (HSP70) (Cell Signaling #4872T, 1:1,000), and albumin (Cell Signaling #4929S, 1:1,000) .
[11]

Animals
Female and male FVB/NCrl mice (8-10 weeks old, Charles River Stock No. 207) were acclimatized and
housed at the Coro Building Barrier facility. Experiments were carried out in accordance with the approved
protocol via the Institutional Animal Care and Use Committee (Protocol 1844667/CMTT# 5017‐22).

Echocardiogram
Echocardiogram (Vevo 2100, FUJIFILM VisualSonic Inc.) was performed preoperatively, and
postoperatively on postoperative days 3, 7, 14, 21, and 28. The mice were anesthetized with 2% isoflurane
and monitored to ensure normothermia and heart rate between 400-600 beats per minute. Left heart systolic
function was evaluated by obtaining two-dimensional parasternal long-axis views, and proximal, mid-
papillary and distal short-axis views. Through Simpson’s method on Vevo Lab 5.6.0, left ventricular ejection
fraction (LVEF) and fractional shortening (FS) were quantified.

Surgical procedure: left anterior descending (LAD) coronary artery ligation
Anesthesia was induced with 3% isoflurane and ketamine (100 mg/kg). Buprenorphine SR (1 mg/kg) was
administered in the dorsal fat pad. The mice were intubated and ventilated (MiniVent Type 845, Harvard
Apparatus) and maintained at 2% isoflurane. A left thoracotomy in the 3rd interspace was made to expose
the heart. The LAD artery was ligated with an 8-0 nylon suture 2-3 mm below the left atrial appendage with
resulting blanching and dyskinesia .
[12]

Immediately after ligation, the mice received one of the following injections [Figure 1]:

1. IM injection control (IM-C) (n = 8): 10 µL PBS with 1% DMSO.

2. IM injection HBMSC-EV (IM-EV) (n = 9): 2 × 10 HBMSC-EV particles in 10 µL PBS with 1% DMSO.
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3. Tail vein injection control (IV-C) (n = 8): 10 µL PBS with 1% DMSO + 190 µL PBS.

4. Tail vein injection HBMSC-EV (IV-EV) (n = 8): 2 × 10 HBMSC-EV particles in 10 µL PBS with 1%
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DMSO + 190 µL PBS.

The dosage of 2 × 10 HBMSC-EV particles was chosen based on previous biodistribution studies that
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visualized positive organ uptake at this dosage 2 h after injection . The intramyocardial injection was done
[7]
with a Neuros Syringe (Hamilton, 1183U32) in two locations (5 µL per location) - immediately below the