Isolation and Characterization of Mesenchymal Stem Cells from Amniotic Fluid and Chorionic Villi

Abstract

s / Placenta 32 (2011) S326–S340 S338 ISOLATION AND CHARACTERIZATION OF MESENCHYMAL STEM CELLS FROM AMNIOTIC FLUID AND CHORIONIC VILLI E.A. Roselli , R. Colognato , E. Antonini , E. Sanna , S. De Toffol , M. Manganini , F. Maggi , F.R. Grati , G. Simoni a,b a Research & Development, Cytogenetics and Molecular Biology, TOMA Advanced Biomedical Assays S.p.A., Busto Arsizio, Varese, Italy; b Biocell Center S.p.A., Busto Arsizio, Varese, Italy This study was design to assess amniotic-(AF-MSC) and chorionic villiderived mesenchymal stem cells (CV-MSC) characteristics, in order to verify their possible applications for regenerative medicine. Starting from 3 mL of amniotic fluid and approximately 5 mg of chorionic villi, the samples were analyzed for biological endpoints: cell viability, proliferation rate, doubling time, immunophenotype, and differentiation potential. Genome stability, by karyotype analysis, genome-wide array-CGH and microsatellite analysis, were also explored. AF and CV samples showed the presence of cells with features of staminality and differentiation potential towards osteogenic, adipogenic and condrogenic phenotypes. Karyotype and microsatellite stabilities were assessed until the 15th and 27th culture passages, respectively. The frequency of the chromosomes aberrations at the different culture passages was not significantly different from the basal frequency found in primary cultures. Preliminary data obtained from array CGH analysis comparing DNA from early to late passages did not show any copy number variations of DNA segments, indicating that the in vitro culture did not induce any modification of the genome stability. Immunophenotyping of cultured MSC revealed two distinct expression patterns related to the two different prenatal sample sources. Our findings indicate that it is possible to isolate and extensively expand MSC from AF and CV and that the in vitro growth culture does not interfere with the DNA-repair systems. Under these circumstances, AF-and CV-MSC could be suitable for therapeutic proposes. Moreover the use of cell bank technology, on native samples, might represent a life-long available autologous cell source for perinatal or adult regenerative medicine. doi:10.1016/j.placenta.2011.07.070 ABILITY OF POLYURETHANE FOAMS TO SUPPORT HUMAN PLACENTADERIVED MESENCHYMAL CELL ADHESION AND OSTEOGENIC DIFFERENTIATION D. Rossi , S. Bertoldi , S. Fare , M. Denegri , O. Parolini , M. Tanzi b a Centro di Ricerca E. Menni, Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy; b Biomaterials Laboratory, Bioengineering Department, Politecnico di Milano, Milano, Italy Application of engineered constructs composed of cells cultured on porous scaffolds is an appealing alternative to current therapies for bone tissue reconstruction. Mesenchymal cells hold great promise in this context, with human placenta representing an easily accessible pluripotent cell source which is exempt from ethical debate. We evaluated the osteogenic differentiation capacity of human amniotic (hAMSCs) and chorionic (hCMSCs) mesenchymal stromal cells cultured on polyurethane foams (PUFs) with or without a a-tricalcium phosphate (TCP PUF) coating. PUF matrix was synthesized by reacting a polyether-polyol mixture with MDI prepolymer, using Fe-acetyl-acetonate as the catalyst and water (2% w/wpolyol) as the expanding agent. The matrix was then coated by immersion in TCP suspension under magnetic stirring. hAMSCs and hCMSCs isolated from two human term placentas were cultured on the polyurethane foams or in glass chamber slides (control), in either osteoinductive or control medium, for up to 20 days. Cell morphology was investigated by SEM while scaffold colonization and cell differentiation were evaluated by routine and specialized histological stains. PUF characterization allowed evaluation of foam density (0.127 0.003g/ cm3), porosity (w90%) and average pore size (268mm). SEM revealed good cell colonization and adhesion onto both PUF and TCP PUF for all tested cell types. Cell adhesion onto the porous surface of PUF was confirmed by histological analysis, which also revealed calcium deposition around cells seeded on TCP PUF, even when cultured without osteoinductive medium. Therefore, the tested polyurethane foams seem to be a valid scaffold for supporting adhesion and differentiation of placenta-derived mesenchymal cells. doi:10.1016/j.placenta.2011.07.071 MESENCHYMAL TRANSITION OF AMNION EPITHELIAL CELLS R. Roy, M. Nitschke, A. Brodarac, S.K. Kang, C. Stamm Berlin-Brandenburg School for Regenerative Therapies, Germany Amnion epithelial cells (AEC) are a readily available cell source for potential use in regenerative medicine. Subpopulations of AEC may express early stem cell markers such as SSEA-3/4, TRA1-60, TRA1-81, Oct3/4, Nanog, SOX-2 and display stem cell behaviour, but usually AEC behave like mature epithelial cells. We sought to induce epithelial-to-mesenchymal transition (EMT) in AEC to improve their capacity for cardiovascular regeneration. Human AEC were derived from full-term placenta and expanded in customized media. To induce EMT, transforming growth factor-b (TGFb) was added. EMT was then assessed by analysis of cell morphology, immunocytology, in vitro scratch wound assay, Transwell migration assay and FACS analysis. Followingexpansion in customizedmedia,AECexpressed stemcellmarkers but lost Oct-4 expression at higher passages and behaved like mature epithelial cells. 25ng/ml TGFb added to the medium for 5-6 days induced striking changes in AEC morphology. Cells acquired an elongated, fibroblastoid shape, equivalent to thatofmesenchymal stemcells. TGF-b induced AEC showed up-regulation of N-cadherin, irregular migration pattern that lead to accelerated wound closure and only stimulated AEC migrated through an 8mm transwell membrane. FACS analysis showed an increase in CD 90 expression and a decrease in CD14 and HLA-DR expression. Under the influence of TGF-b, mature AEC undergo epithelial-to-mesenchymal transition and acquire a mesenchymal stem cell-like phenotype. EMTgreatly enhances cell mobility andmay thus help optimize AEC for use in cardiovascular cell therapy. doi:10.1016/j.placenta.2011.07.072 EXTRACELLULAR MATRIX: FROM THE PLACENTAL DEVELOPMENT AT TISSUE ENGINEERING S. San Martin , J. Villenas , M. Parraga , M. Alaminos , A. Campos c a Centre for Reproductive Biology Studies, School of Medicine, Universidad de Valparaiso, Chile; b CREAS, Valparaiso, Chile; c Department of Histology, University of Granada, Spain During the morphogenesis and development of tissues, a coordinated process of proliferation and differentiation of cells are required. In this context, adequate relationships with the extracellular matrix (ECM) components are essential for normal development. The ECM comprises a variety of versatile proteins arranged in a cell surface-associated network and its interaction with cells provide the basic principles of material sciences that could be applied to explain the interaction of the cells in artificial construct for tissue engineering. Some examples are present that showed the ECM is necessary for placenta in human and animal models during the pregnancy, as well artificial tissues construction develop for tissue engineering. Placental tissues of human and rodent were obtained and process for morphological analysis. Primary cell cultures of stromal fibroblasts were generated from normal oral mucosa biopsies. A substitute of the stroma was constructed using fibrin. Extracellular matrix proteins were evaluated by immunohistochemistry.