Forraz, Nicolas (2003) Multi-tissue differentiation potential of umbilical cord blood stem cell populations. (PhD thesis), Kingston University, .

Abstract

Scientific investigation of the haemopoietic stem cell has been (and still is) successfully translated to the clinical arena with its major therapeutic application being haemopoietic stem / progenitor cell (HSPC) transplantation to reconstitute haemopoiesis in myelocompromised patients. Although conventional sources include bone marrow (BM) and mobilised peripheral blood, HSPC can also be harvested from umbilical cord blood (CB), with the latter providing an easy access to ontogenically primitive and immunologically "naïve" cell population of particular interest for transplantation. Further optimisation of highly primitive haemopoietic stem cells purification techniques remains, however, necessary to ensure not only short-term haemopoietic reconstitution, but also long-term blood cell formation post-transplant. Because current protocols overly rely on the sialomucin CD34 (a cell surface marker expressed on both primitive and committed HSPC) to predict success of a transplant but also to directly isolate HSPC, the main objective of this work was to develop alternative means for the isolation of a highly primitive pre-haemopoietic stem cell subset from CB. High definition laser-scanning confocal microscopy three-dimensional cell analysis revealed that in both CB and BM HSPC, CD34 interacted with another sialomucin CD164 to co-localise along dense crests correlating with membrane activity. Direct isolation of HSPC was therefore performed by targeting the CD133 antigen, known to be restricted to a more primitive subset that CD34+ counterparts. The synergistic action of thrombopoietin (TPO), flt3-ligand (FL) and c-Kit (K) proved extremely potent at expanding CD133+HSPC in liquid cultures by maintaining a primitive phenotype, limiting apoptosis and inducing self-renewal. TPOFLK also caused modulation of HOX transcription factor family gene expression. Blockade of this intrinsic regulatory pathway by a synthetic peptide (namely "HXP4") notably inhibited CD133+HSPC proliferation and induced them into quiescence. However, despite having interesting 'ex vivo' expansion potential, CD133+HSPC still encompassed cells at various stages of differentiation. A rapid and cost-efficient negative immunomagnetic separation method was hence developed depleting CB from cells expressing haemopoietic markers. This reproducibly isolated a discrete "lineage negative" (LinNeg) stem cell population. This highly primative pre-haemopoietic "LinNeg" stem cell population demostrated multi-tissue differentiation potential and could be stimulated with TPOFLK to simultaneously expand both haemopoietic but also neuroglial progenitors. Taken together, these findings could widen greatly the horizons of CB as cell source for cellular therapies.

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