Multipotent mesenchymal stem cells from human placenta: critical parameters for isolation and maintenance of stemness after isolation.

OBJECTIVE This study was undertaken to isolate and characterize multipotent mesenchymal stem cells from term human placenta (placenta-derived mesenchymal stem cells, PD-MSCs). STUDY DESIGN Sequential enzymatic digestion was used to isolate PD-MSCs in which trypsin removes the trophoblast layer, followed by collagenase treatment of remaining placental tissue. Karyotype, phenotype, growth kinetics, and differentiability of PD-MSC isolates from collagenase digests were analyzed. RESULTS PD-MSC isolation was successful in 14 of 17 cases. Karyotyping of PD-MSC isolates from deliveries with a male fetus revealed that these cells are of maternal origin. Flow cytometry and immunocytochemistry confirmed the mesenchymal stem cell phenotype. Proliferation rates of PD-MSCs remained constantly high up to passage 20. These cells could be differentiated toward mesodermal lineage in vitro up to passage 20. Nonconfluent culture was critical to maintain the MSC stemness during long-term culture. CONCLUSION Term placenta constitutes a rich, very reliable source of maternal mesenchymal stem cells that remain differentiable, even at high passage numbers.