Table 1.

Characteristics of controlled preclinical studies of human MSC-derived EVs to treat or prevent GVHD in mice

Study	Model	n* I / C	MSC source	Mouse GVHD induction and confirmation	EV Isolation	EV Characterization	Intervention	Dose of EV (per animal unless stated)
[23]	Treatment	30 / 32	hBM	8 Gy TBI and tail vein injection of spleen cells from allogeneic mice GVHD confirmation: ≥10% loss of body weight	UCa	EV size: 90–400 nm Mean protein: 4.48 mg/ml Particles: 4.23 × 109 /ml EM: Yes Western blot: CD63, CD81	Treatment: once on day +5 Route: tail vein	Controls: saline MSC-EV group: EVs derived from 2 × 106 MSC per kg of body weight
[24]	Treatment	8 / 8	hBM	TBI and tail vein injection of BM and spleen cells of allogeneic mice GVHD confirmation: clinical score ≥ 0.6 and molecular donor chimerism on day 20	UCb	EV size: <100 nm Mean protein:: NR Particles: NR EM: Yes Western blot: CD63, CD9, CD81	Treatment: Weekly × 6 weeks starting day +22 Route: tail vein	Controls: saline or 100 μg fibroblast EVs MSC-EV group: 100 μg
[25]	Prevention	20 / 20	hESC	TBI 100 cGy and tail vein injection of human PBMC. Human engraftment confirmation: % human cells in blood by flow cytometry	0.22 μ filter then TFF	EV size: NR Mean protein:: 1.9 mg/mL Particles: 1.9 × 1011 /mL EM: NR Western blot: NR	Prophylaxis: every 3 days starting day +1 until death or day +34 Route: intraperitoneal	Control: saline MSC-EV group: 1 or 10 μg
[26]	Prevention	14 / 15	hUCB	TBI 7.5 Gy and tail vein injection of BM and splenocytes allogeneic mice Allogenic engraftment confirmation: NR	UCc	EV size: 30–100 nm Mean protein: protein concentration by bicinchoninic acid. Protein concentration NR. Particles: NR EM: Yes Western blot: CD63, CD9, CD81	Prophylaxis: day 0 and day +7 Route: tail vein	Control: no prophylaxis MSC-EV group: 200 μg

*n = total number of mice included in control and treatment groups and reported as part of primary survival outcome data

I/C intervention / control groups

UC Ultracentrifugation, TFF Tangential flow, NR Not reported

^aisolation involved centrifugation at 2000 g × 30 min, 0.22 μfiltration, Total Exosome Isolation Reagent (Invitrogen/Thermo Fisher Scientific), followed by centrifugation at 100,000 g × 1 h

^bisolation involved centrifugation at 200 g × 10 min, 2000 g × 20 min, 10,000 g × 30 min, 110,000 g × 7 h then 0.22 μfiltration and repeating all steps again

^cisolation involved centrifugation at 2000 g × 30 min followed by centrifugation at 100,000 g × 2 h and repeated