857: Third trimester amniotic fluid as source of mesenchymal stem cells for regenerative applications

Mesenchymal stem cells (MSCs) found in the amniotic fluid (AF) have potential applications in cellular therapy and regenerative medicine. Banking has been possible since 2007 in Europe and 2009 in the US, but limited to 2nd trimester fluid. Our thesis proposes that 3rd trimester MSC are similar in viability, multipotency and proliferation potential to the ‘younger’ stem cells.Also data is limited regarding the maintenance of biological properties of the 3rd trimester stem cells after cryopreservation. Research and Laboratory protocol: This IRB approved study was to collect residual fluid from 3d trimester amniocentesis (indication included FLM testing and TTTS). Fluid was sent to Children's Hospital in Boston. Additional sites in Europe (Biocell-TOMA) collected 3rd trimester fluids, which were analyzed separately. In Boston, AF was spun and the cell pellet resuspended, plated and incubated. First yield (harvest) and sometimes second harvests were obtained. In the Biocell-TOMA experiments, AF cells were either cryopreserved immediately or resuspended in growth media and plated. AF cultures were subcultured at first passage in MSCs selective media and then expanded. Phenotype of the expanded cells was analyzed by flow-cytometry. Multipotency was tested by growing cells in tissue-specific culture conditions. To date 42/60 samples collected in Boston yielded results in 1st or 2nd passage. 15 European AF samples were collected, 5 were cryopreserved and 10 were plated. FACS analysis of cultured AF-MSCs showed expression of CD29, CD44, CD117, CD105, CD73 and CD90, but not CD45, CD34, CD14, CD19 and HLA-DR surface markers. Experiments on cryopreserved samples are ongoing to verify the maintenance of these properties following thawing procedure. Conclusion: Our findings indicate that it is possible to isolate, cryopreserve and expand AF-MSC at late stages of gestation. They are comparable to younger AF-MSC. In the second phase of the study we will further characterize 3d trimester AF-MSCs and improve collection techniques.