Shaw, SS; (2013) The potential of amniotic fluid stem cells in prenatal gene and cell therapy. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Amniotic fluid stem (AFS) cells can be expanded without feeder layers and can differentiate into mesenchymal and haematopoietic lineages. Long term engraftment has been difficult to achieve after prenatal stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained from amniotic fluid (AF) with minimal risk. My thesis aims to define the potential of human, sheep and mouse AFS cells as an autologous stem cell source for prenatal cell/gene therapy. Using pregnant sheep, I explored using AF mesenchymal stem cells (AFMSCs) and CD34+ cells for autologous in utero therapy. AF was collected under ultrasound-guided amniocentesis in early gestation. Those cells were transduced with enhanced green-fluorescent-protein (GFP) using lentivirus vector. After expansion, transduced AFMSCs were injected into peritoneal cavity of each donor fetal sheep. Widespread transgenic GFP expression was detected in fetal tissue. For looking into haematopoietic potential, I transplanted autologous fresh and frozen CD34+AFS, and bone marrow cells into immunocompromised mice. Sheep CD34+AFS cells formed colonies, and were positive for CD45, but negative for CD14/CD31/CD4/ CD58. Flow cytometric analysis at 3 months showed GFP positive cells in all haematopoietic organs. To prove congenic transplantation is better than allogeneic, I collected AF from YFP+/C57BL/6 mice at E13. CKit+/Lin- cells were injected into peritoneal cavity of every mouse fetus. Peripheral blood engraftment was significantly higher in mice transplanted with congenic, versus allogeneic cells, as was liver and spleen engraftment. Finally, looking ahead to clinical translation, human AF cells could be cultured, transduced, sorted and expanded in vitro by using conditional medium in adherent plates. Xenogeneic transplantation of human Ckit+ AFS cells into fetal mice also showed minimal engraftment in peripheral blood. In conclusion, AF derived stem cells are an important source of autologous cells that could have prenatal therapeutic value in cell or cell-based gene therapy in the future.

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