Ramachandra, Durrgah Latchumi; (2017) The Haematopoietic potential of Human Amniotic Fluid Stem Cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

There is a constant demand for haematopoietic stem cells (HSC) for clinical applications. Amniotic fluid stem (AFS) cells serve as a potential autologous cell source for therapy. Previously murine and sheep AFS have shown to have significant haematopoietic activity after transplantation in immune deficient mice. The haematopoietic potential of Human AFS have never been established in vivo, and its use has been limited by the presence of debris and low cell number at sample collection. My thesis explored the (1) isolation of human amniotic fluid (AF), (2) haematopoietic potential of human AF (CD117/c-Kit+; AFSC) by reconstituting the haematopoietic system of NOD-SCID/IL2rγnull (NSG) mice in vivo and (3) expansion of haematopoietic human AFSC in vitro. Human AF samples (2nd and 3rd trimester, n=110) were collected for the study under an ethically approved project from women undergoing amniocentesis for prenatal diagnosis of congenital disease, or amniodrainage procedures for fetal abnormality. I have employed several strategies to eliminate the large amount of cellular debris from the collected human AF and provide a more homogeneous cell population. Percoll density centrifugation demonstrated a reduction in cell debris and enrichment of the CD117+ population. The haematopoietic potential of human AFSC was explored in vivo. Human AF (2nd and 3rd trimester) and cord blood (CB; control) were selected for CD117 and CD34 respectively. Sorted cells (104 in 200μl PBS) were injected intravenously into sub-lethally irradiated NSG mice (~n=6/group). Human AFSC engrafted the haematopoietic system of NSG mice at levels similar to those achieved with CB-HSC post-primary and secondary transplantation. Importantly, multi-lineage haematopoietic reconstitution was observed at 16 weeks post-primary and secondary transplantation. Moreover, the possibility of expanding haematopoietic progenitors from human AF in vitro was demonstrated with the use of a cytokine-based media and the generation of haematopoietic progenitors by AF derived-induced pluripotent stem cell (AF-iPS) lines. In conclusion, I showed that human AF could be isolated, have long-term multi-lineage haematopoietic potential that is similar to the current “gold-standard” stem cell source for haematopoietic transplantation as well as demonstrates haematopoietic expansion. These findings make human AFSC to be an alternative novel fetal cell source for pre- and post-natal cell or cell-based gene therapy for the treatment of haematological disorders in the future.

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