Molecular and Proteomic Characterization of Human Mesenchymal Stem Cells Derived from Amniotic Fluid: Comparison to Bone Marrow Mesenchymal Stem Cells

Abstract

Human mesenchymal stem cells (hMSCs) constitute a population of multipotent adherent cells able to give rise to multiple mesenchymal lineages such as osteoblasts, adipocytes, or chondrocytes. So far, the most common source of MSCs has been the bone marrow (BM); however BM-MSC harvesting and processing exhibits major drawbacks and limitations. Thus, identification and characterization of alternative sources of MSCs are of great importance. In the present study, we isolated and expanded fetal MSCs from second-trimester amniotic fluid (AF). We documented that these cells are of embryonic origin, can differentiate under appropriate conditions into cell types derived from all three germ layers, and express the pluripotency marker Oct-4, the human Nanog protein, and the stage-specific embryonic antigen-4 (SSEA-4). Furthermore, we systematically tested the immunophenotype of cultured MSCs by flow cytometry analysis using a wide variety of markers. Direct comparison of this phenotype to the one derived from cultured BM-MSCs demonstrated that cultured MSCs from both sources exhibit similar expression patterns. Using the two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approach, we have generated for the first time the protein map of cultured AF-MSCs by identifying 261 proteins, and we compared it directly to that of cultured BM-MSCs. The functional pattern of the identified proteins from both sources was similar. However, cultured AF-MSCs displayed a number of unique proteins related to proliferation and primitive phenotype, which may confer to the distinct features of the two types. Considering the easy access to this new cell source and the yield of expanded MSCs for stem cell research, AF may provide an excellent source of MSCs both for basic research and for potential therapeutic applications.

Exploring the Human Term Placenta as a Novel Source for Stem Cells and their Application in the Clinic

“…Thus, they may be a superior MSC source for clinical trials than the traditional source which is adult bone marrow. This section will focus on the isolation of MSC from amniotic fluid (Mareschi et al, 2009, Nadri and Soleimani, 2007, Roubelakis et al, 2007, the amniotic membrane (Soncini et al, 2007, Portmann-Lanz et al, 2006, Diaz-Prado et al, 2010, the chorion (chorion laeve and chorionic villi) (Soncini et al, 2007, Portmann-Lanz et al, 2006, Igura et al, 2004, Poloni et al, 2008, the decidua (Macias et al, 2010, Aghajanova et al, 2010, umbilical cord/Wharton's jelly (Mitchell et al, 2003, Reinisch et al, 2007, Petsa et al, 2009) and umbilical cord blood (Mareschi et al, 2001, Bieback et al, 2004, Romanov et al, 2003. This section will also discuss the isolation of haematopoietic stem cells from the umbilical cord blood and the application of these cells.…”

Células-tronco do líquido amniótico

“…31,32 Enquanto as AF-MSCs atingem o nú-mero de 3 milhões de células em nove dias, as MSCs da medula óssea expandem-se para 400 mil células, em condições semelhantes de cultura e plaqueamento. 33 O fenótipo das AF-MSCs é bastante semelhante às células mesenquimais de outros tecidos, como aquelas originadas da medula óssea e do cordão umbilical. Expressam os antígenos de superfície da membrana celular CD29, CD44, CD90 e CD105.…”

“…Quanto ao CD73, alguns relatos demonstram expressão nessas células, enquanto outros mostram expressão somente nas células derivadas da medula óssea e não deri-vadas de líquido amniótico. 11,[31][32][33][34][35][36][37][38] Esse mesmo padrão imunofenotípico, do tipo mesenquimal, é mantido pelas AF-MSCs após isolamento, expansão, e descongelamento pós-criopreservação por cinco meses. 39 …”

Expressão da P-gp, MPR1 e LRP em células-tronco mesenquimais humanas derivadas do líquido amniótico e medula óssea

“…Seguindo este raciocínio Roubelakis, ao comparar CTM de líquido amniótico e medula óssea demonstrou que, embora ambas demonstrassem expressão gênica e proteômica em comum, apenas as células de LA apresentam transcritos e proteínas relacionados à atividade proliferativa e fenótipo primitivo, ou seja, mesmo possuindo características mesenquimais em comum, as células-tronco possuem propriedades dependentes do tecido de origem (Wolpert, 2000;Roubelakis et al,2007).…”

“…Sua maioria é de natureza epitelioide, provavelmente células da pele, tratos respiratórios e gastrointestinal fetal ou da membrana amniótica, e uma pequena porcentagem é de células-tronco mesenquimais, que podem proceder dos tecidos fibroconectivos fetais (Slack, 2008;Dobreva et al, 2010;Pappa & Anagnou, 2011 (Tsai et al, 2007;Roubelakis et al, 2007).…”