Isolation, Phenotypic, Molecular and First-Time Proteomic Characterization of Human Mesenchymal Stem Cells (MSCs) Derived from Amniotic Fluid: Comparison to Bone Marrow MSCs.

Human mesenchymal stem cells (hMSCs) constitute a population of multipotent cells, easily expanded in culture and able to give rise to many lineages. These characteristics make MSCs a very attractive tool for developing new strategies for clinical applications based on cell therapy. So far, the most common source of MSCs has been the bone marrow (BM). However, identification and characterization of alternative sources of MSCs is of great importance. One such alternative source is the amniotic fluid (AF), which can be collected during scheduled amniocentesis without any ethical concerns.

To this end, in the present study, we introduced an improved protocol for isolating and clonally expanding fetal MSCs from second trimester amniotic fluid (AF) and we further characterized these cells based on their

• phenotype,

• pluripotency,

• differentiation potential and

• proteomic profile.

The AF samples were obtained during routine amniocentesis and AF-MSCs were enriched by a modified culture protocol. The isolated MSCs expanded rapidly and exhibited differentiation potential into adipocytes and osteoblasts. More importantly, we showed that these cells can differentiate in vitro not only into cell types derived from mesoderm (adipocytes and osteoblasts) and ectoderm (neural cells) but also more interestingly into endoderm (hepatocytes) derived cells. Moreover, we documented that AF-MSCs express Oct-4 transcription factor, a marker of pluripotency, and we studied for the first time its expression over different passages by real time PCR and documented that it remained constant for at least 17 doublings.

An extensive characterization of the phenotypic features of AF-MSCs by using a wide range of surface markers and flow cytometry, indicated that they are positive for all the mesenchymal stem cell markers such as CD90, CD105, CD73 and CD166 and generally exhibit a similar expression pattern to the BM-MSCs.

To characterize these cells in more detail, we established the first proteomic database for human AF-MSCs. Using 2D-gel electrophoresis and matrix-assisted laser desorption ionisation-time of flight-mass (MALDI-TOF) spectrometry approach, we have generated for the first time the protein map of AF MSCs, by identifying 260 proteins and directly compared this protein profile with that of MSCs derived from BM. We further performed a similar analysis for BM-MSCs, identifying 170 different proteins and generating a reference map for these cells. The comparison of the proteomic pattern from both sources was similar. In general, 140 proteins were identified in AF-MSCs related to cell growth/maintenance, metabolism/energy pathways, protein metabolism, apoptosis, signal transduction and communication as well as transcription and transport, that are not present in BM-MSCs.

The approach we initiated, is expected to facilitate systematic functional studies for these multipotent cells. One such approach could be the implementation of the proteomic analysis, during differentiation of AF-MSCs to cells derived from all three germ layers as shown in our study. Data derived from these approaches are expected to clarify the therapeutic potential of the MSCs.