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Background Mesenchymal stromal cells are multipotent cells considered to be of great promise for use in regenerative medicine. However, the cell dose may be a critical factor in many clinical conditions and the yield resulting from the ex vivo expansion of mesenchymal stromal cells derived from bone marrow may be insufficient. Thus, alternative sources of mesenchymal stromal cells need to be explored. In this study, mesenchymal stromal cells were successfully isolated from second trimester amniotic fluid and analyzed for chromosomal stability to validate their safety for potential utilization as a cell therapy product.Design and Methods Mesenchymal stromal cells were expanded up to the sixth passage starting from amniotic fluid using different culture conditions to optimize large-scale production.Results The highest number of mesenchymal stromal cells derived from amniotic fluid was reached at a low plating density; in these conditions the expansion of mesenchymal stromal cells from amniotic fluid was significantly greater than that of adult bone marrow-derived mesenchymal stromal cells. Mesenchymal stromal cells from amniotic fluid represent a relatively homogeneous population of immature cells with immunosuppressive properties and extensive proliferative potential. Despite their high proliferative capacity in culture, we did not observe any karyotypic abnormalities or transformation potential in vitro nor any tumorigenic effect in vivo.Conclusions Fetal mesenchymal stromal cells can be extensively expanded from amniotic fluid, showing no karyotypic abnormalities or transformation potential in vitro and no tumorigenic effect in vivo. They represent a relatively homogeneous population of immature mesenchymal stromal cells with long telomeres, immunosuppressive properties and extensive proliferative potential. Our results indicate that amniotic fluid represents a rich source of mesenchymal stromal cells suitable for banking to be used when large amounts of cells are required.

Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.

Objective: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. Methods: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients, respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. Results: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (≥98%) for CD73, CD105, CD106, CD29, CD13, CD44 and negative (≤1%) for CD34, CD45, CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients, respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. Conclusions: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units. © 2006 International Society for Experimental Hematology.