70: Multipotent mesenchymal stromal cells from human placenta: Critical criteria for isolation and maintenance of ‘stemness' after isolation

ObjectiveMultipotent mesenchymal stromal cells (MSCs) provide new opportunities for regenerative medicine because of their ability to differentiate into mesodermal lineage cells. Evidence has emerged that stroma of chorionic villi of human term placenta represents a potentially highly valuable source of human MSCs for autologous cell therapy. For clinical application of chorionic villi-derived MSCs (MSCs), reliable and reproducible protocols for isolation will be necessary. A central point concerns the maintenance of 'stemness' after isolation.Study DesignWe report a new protocol for isolation of CV-MSCs from term placental tissue, which rests on trypsin digest to remove the epithelial trophoblast cell layer, followed by collagenase digest. The CV-MSC isolates were analyzed for growth kinetics and expression marker profiles at passages 1, 10, and 20. Phenotypic profiling was performed by flow cytometry and immunocytochemistry. Specific differentiation was induced in commercial differentiation media.ResultsCV-MSC isolation was successful in 11 out of 12 human cases. The CV-MSC isolates grew out as adherent, fast proliferating colonies. Growth rates remained constantly high up to passage 20. CV-MSCs fulfilled all criteria defining MSCs, such as expression of CD73, CD90, CD105 and CD166, but absence of hematopoietic markers CD34 and CD45. Early passage CV-MSCs expressed CD133, a marker for immaturity. Interestingly, CV-MSCs were positive for von Willebrand factor and alpha smooth muscle actin. CV-MSCs could be differentiated into osteocytes, chondrocytes, and adipocytes. In non-confluent culture, the 'stemness' marker profile was stable up to passage 20, while subcultures beyond 70% confluence lost their MSC markers.ConclusionHuman placenta emerged as robust, very reliable source of truly multipotent cells. Non-confluent culture of CV-MSCs was found critical to maintain their 'stemness' during expansion. Differentiation capacity of late passage CV-MSCs, as well as their differentiation into muscle-, endothelial-, and trophoblast-like cells remain to be tested. ObjectiveMultipotent mesenchymal stromal cells (MSCs) provide new opportunities for regenerative medicine because of their ability to differentiate into mesodermal lineage cells. Evidence has emerged that stroma of chorionic villi of human term placenta represents a potentially highly valuable source of human MSCs for autologous cell therapy. For clinical application of chorionic villi-derived MSCs (MSCs), reliable and reproducible protocols for isolation will be necessary. A central point concerns the maintenance of 'stemness' after isolation. Multipotent mesenchymal stromal cells (MSCs) provide new opportunities for regenerative medicine because of their ability to differentiate into mesodermal lineage cells. Evidence has emerged that stroma of chorionic villi of human term placenta represents a potentially highly valuable source of human MSCs for autologous cell therapy. For clinical application of chorionic villi-derived MSCs (MSCs), reliable and reproducible protocols for isolation will be necessary. A central point concerns the maintenance of 'stemness' after isolation. Study DesignWe report a new protocol for isolation of CV-MSCs from term placental tissue, which rests on trypsin digest to remove the epithelial trophoblast cell layer, followed by collagenase digest. The CV-MSC isolates were analyzed for growth kinetics and expression marker profiles at passages 1, 10, and 20. Phenotypic profiling was performed by flow cytometry and immunocytochemistry. Specific differentiation was induced in commercial differentiation media. We report a new protocol for isolation of CV-MSCs from term placental tissue, which rests on trypsin digest to remove the epithelial trophoblast cell layer, followed by collagenase digest. The CV-MSC isolates were analyzed for growth kinetics and expression marker profiles at passages 1, 10, and 20. Phenotypic profiling was performed by flow cytometry and immunocytochemistry. Specific differentiation was induced in commercial differentiation media. ResultsCV-MSC isolation was successful in 11 out of 12 human cases. The CV-MSC isolates grew out as adherent, fast proliferating colonies. Growth rates remained constantly high up to passage 20. CV-MSCs fulfilled all criteria defining MSCs, such as expression of CD73, CD90, CD105 and CD166, but absence of hematopoietic markers CD34 and CD45. Early passage CV-MSCs expressed CD133, a marker for immaturity. Interestingly, CV-MSCs were positive for von Willebrand factor and alpha smooth muscle actin. CV-MSCs could be differentiated into osteocytes, chondrocytes, and adipocytes. In non-confluent culture, the 'stemness' marker profile was stable up to passage 20, while subcultures beyond 70% confluence lost their MSC markers. CV-MSC isolation was successful in 11 out of 12 human cases. The CV-MSC isolates grew out as adherent, fast proliferating colonies. Growth rates remained constantly high up to passage 20. CV-MSCs fulfilled all criteria defining MSCs, such as expression of CD73, CD90, CD105 and CD166, but absence of hematopoietic markers CD34 and CD45. Early passage CV-MSCs expressed CD133, a marker for immaturity. Interestingly, CV-MSCs were positive for von Willebrand factor and alpha smooth muscle actin. CV-MSCs could be differentiated into osteocytes, chondrocytes, and adipocytes. In non-confluent culture, the 'stemness' marker profile was stable up to passage 20, while subcultures beyond 70% confluence lost their MSC markers. ConclusionHuman placenta emerged as robust, very reliable source of truly multipotent cells. Non-confluent culture of CV-MSCs was found critical to maintain their 'stemness' during expansion. Differentiation capacity of late passage CV-MSCs, as well as their differentiation into muscle-, endothelial-, and trophoblast-like cells remain to be tested. Human placenta emerged as robust, very reliable source of truly multipotent cells. Non-confluent culture of CV-MSCs was found critical to maintain their 'stemness' during expansion. Differentiation capacity of late passage CV-MSCs, as well as their differentiation into muscle-, endothelial-, and trophoblast-like cells remain to be tested.