ISOLATION AND CHARACTERIZATION OF AMNION-DERIVED INHIBITORY FACTORS ON LYMPHOCYTE PROLIFERATION
Creators: name: ROSSI, DANIELE
Publisher: name: Universit� degli Studi di Milano
Year: 2013

description: Background
Cells derived from the amniotic membranes of human term placenta are
receiving particular attention because of their characteristics of stemness,
multipotency and their immunologycal features, supporting a variety of
possible clinical applications in the field of cell transplantation and
regenerative medicine. We have previously demonstrated that cells isolated
from the mesenchymal region of the human amniotic membrane (human
amniotic mesenchymal tissue cells, hAMTC) possess immunoregulatory
properties, such as the ability to inhibit lymphocyte proliferation and
cytokine production and to suppress generation and maturation of
monocyte-derived dendritic cells, as also reported for MSC from other
sources. All of the results described above were obtained when hAMTC
were cultured either in direct cell contact or in a transwell system, thereby
suggesting the involvement of soluble inhibitory factor(s) secreted by these
cells. However, the precise factors and mechanisms responsible for the
immunoregulatory roles of hAMTC remain unknown.
Aims
Therefore, in this study, we aimed to identify the soluble factors released by
hAMTC which are responsible for the anti-proliferative effects of these cells
on lymphocytes, and also to gain insight into the mechanisms underlying
their actions through in vitro studies.
Materials and Methods
Conditioned medium (CM) was prepared by culture of isolated hAMTC
(CM-hAMTC) and fragments of the whole human amniotic membrane (CMhAM)
for 5 days in UltraCulture medium. Chemical and physical properties,
such as the thermostability, chemical nature and molecular weight, of the
factors associated with the anti-proliferative effects of hAMTC were
evaluated. Blocking of specific synthetic pathways involved in the
production of immunomodulatory factors, such as nitric oxide, kynurenine
and prostaglandins, was achieved by adding specific inhibitors during CM
production. Finally, the involvement of certain cytokines in the
antiproliferative effects of the CM was evaluated by adding neutralizing
antibodies to the CM during lymphocyte proliferation tests.
Results
In this study, we have demonstrated that:
? the inhibitory factors are temperature-stable, have a small molecular weight, and are likely to be non-proteinaceous;
? only inhibition of the cyclooxygenase pathway partially reversed the antiproliferative
effects of hAMTC, suggesting that prostaglandins may be
key effector molecules in this phenomenon;
? factors which have been previously documented to play a role in the
inhibitory effects of MSCs from other sources (HGF, TGF-?, NO and
IDO) were not shown to be involved;
? the anti-proliferative effects of the amniotic membrane are intrinsic to
this tissue and its derived cells, since these effects are manifested in the
absence of stimulating culture conditions, as opposed to MSC derived
from the bone marrow, which possess anti-proliferative ability only when
cultured in the presence of activating stimuli.
Conclusions
We have demonstrated that both isolated cells or the amniotic membrane in
toto are capable of releasing factors that present with immunomodulatory
capacity, thereby providing new insights toward the identification of some of
these factors and toward the characterization of other factors which remain
to be identified.