A. Lange-Consiglio

B. Corradetti

C. Perrini

D. Bizzaro

E. Esposti

F. Cremonesi

G. Camussi

L. Pascucci

M.C. Deregibus

M.G. Marini

AIR Universita degli studi di Milano

European Cells and Materials Vol. 31. Suppl. 1, 2016 (page 187)                                                            ISSN 1473-2262 
 
                                                http://www.ecmjournal.org 
 
Microvesicles from amniotic cells as potential novel therapeutics in regenerative 
medicine: first in vitro result in equine stressed tendon and endometrial cells 
A Lange-Consiglio1, C Perrini1, P Esposti1, MC Deregibus2, G Camussi2, L Pascucci3, MG Marini4, 
B Corradetti4, D Bizzaro4, F Cremonesi1 
 1 Reproduction Unit, Large Animal Hospital, Università degli Studi di Milano, Italy. 2 Università di 
Torino, Italy. 3 Università di Perugia, Italy. 4 Università Politecnica delle Marche, Italy  
 
INTRODUCTION: The unfavorable 
microenvironment of injured or degenerating 
tissues may result in the death or apoptosis of a 
large proportion of implanted MSCs in the short 
period immediately post-transplantation. Recovery 
of in vivo spontaneous equine tendon lesions by 
administration of horse amniotic mesenchymal 
cells conditioned medium (AMC-CM) suggests a 
paracrine mechanisms in the regeneration process 
[1]. It has recently been demonstrated that 
microvesicles (MVs) released from cells are an 
integral component of the paracrine cell-to-cell 
communication during tissue regeneration [2]. 
Aims of this study were to investigate the presence 
and type of MVs secreted by equine amniotic 
mesenchymal cells (AMCs), their incorporation in 
equine tendon and endometrial cells and their 
effect on these cell lines stressed in vitro by 
lipopolysaccharide (LPS). 
METHODS: MVs were obtained by 
ultracentrifugation at 100.000g for 1h at 4°C of the 
media obtained culturing AMCs isolated from 
three different amnion. MVs size was evaluated by 
Nanosight technology and transmission electron 
microscopy (TEM). Tendon and endometrial cells 
were obtained from collagenase digestion for 17h 
and 3h respectively and cultured in HG-DMEM 
with 10% fetal calf serum. To study the ability of 
tendon and endometrial cells to incorporate MVs, 
a dose-response curve was performed adding 10-
20-30-40-50x106 MVs/ml labeled with PKH-26 for 
24h, 48h and 72h. The uptake of MVs was 
evaluated by an Olympus BX51 microscope 
equipped with software for image acquisition. A 
dose/response curve of LPS investigated by 
apoptotic and MTT tests showed that 100ng/ml at 
48h on tendon cells and 10ng at 24h on 
endometrial cells were the doses and the times 
most effective in inducing cellular stress. RT-
qPCR expression of pro-inflammatory genes such 
as tumor necrosis factor-α (TNF-α), 
metallopeptidase (MMP) 1 and 13, and of an anti-
inflammatory gene such as transforming growth  
factor-ß (TGF-ß) was evaluated in the in vitro LPS 
stressed cells by Mann-Whitney U-test. 
RESULTS: Nanosight showed that AMCs secrete 
MVs in the range of 100-200 nm. TEM revealed 
budding of AMCs membrane, proving that these 
MVs fall within the shedding vesicles category. 
The MVs uptake was gradual over time (Fig. 1). 
The same semi-quantitative fluorescence uptake 
signal was obtained when 50x106 MVs were 
incorporated at 24h, or 40x106 MVs at 48h and 
30x106 MVs at 72h, suggesting that an inverse 
correlation between concentration and time was 
found in MVs uptake equally by tendon and 
endometrial cells. MVs induced a significant 
(P<0.05) down-regulation of TNF-α, MMP1 and 
MMP13 expression in both cellular lines after in 
vitro LPS stress, and up-regulation of TGF-ß 
expression. 
 
Fig. 1: Representative micrographs of 
internalization at 24h (A) and 72h (B) of MVs 
labeled with PKH-26 by tendon cells.  
DISCUSSION & CONCLUSIONS: Our data 
suggest that MVs can be incorporated in tendon 
and endometrial cells and have a role in 
modulating inflammatory genes in vitro. 
REFERENCES: 1A. Lange-Consiglio, D. Rossi, 
S. Tassan, et al (2013). Stem Cell and 
Development 22:3015-24. 2G. Camussi, M.C. 
Deregibus, S. Bruno et al (2010). Kidney Int 
78:838-48. 
ACKNOWLEDGEMENTS: Grants from 
Università degli Studi di Milano, Italy and 
Università Politecnica delle Marche, Ancona, Italy 
are acknowledged. 


Microvesicles from amniotic cells as potential novel therapeutics in regenerative medicine: first in vitro result in equine stressed tendon and endometrial cells

https://air.unimi.it/retrieve/handle/2434/437379/752964/Vol031Supp01a187.pdf

Microvesicles from amniotic cells as potential novel therapeutics in regenerative medicine: first in vitro result in equine stressed tendon and endometrial cells

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