Routine clonal expansion of mesenchymal stem cells derived from amniotic fluid for perinatal applications

Abstract

Stem cells (SCs) isolated from amniotic fluid (AF) are a promising source for autologous perinatal cell therapy. The aim of this study was to develop a routine isolation, selection, and expansion protocol of clonal SC lines from redundant clinical amniocentesis samples. Amniotic fluids were collected between 15 and 22 weeks of gestation, and SCs were isolated by CD117-based and mechanical selection protocols. SCs were characterized by mesenchymal SC marker expression and differentiation protocols. Cells were manipulated with a lentiviral vector system expressing the β-galactosidase reporter gene and were injected into immunodeficient newborn mouse pups. Qualitative assessment was performed to detect the infused cells after 1 week. A total of 78 clonal AF SC populations were successfully isolated by mechanical selection from 21 consecutive amniocentesis samples. They were positive for mesenchymal SC cluster of differentiation markers and could be differentiated into the different lineages. SCs were stably labeled using β-galactosidase and were detected in the lungs and hearts of the neonatal mice. We demonstrate that mesenchymal SCs can be routinely isolated and clonally expanded from mid-gestation human AF using mechanical isolation. They can easily be transduced and be tested for perinatal treatment in animal models.