Novel ES cell differentiation system enables the generation of low-level repopulating haematopoietic stem cells with lymphoid and myeloid potential
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Date
28/06/2014Item status
Restricted AccessEmbargo end date
31/12/2100Author
Fanning, Niamh Catherine
Metadata
Abstract
The potential of embryonic stem (ES) cells to generate any developmental or adult
cell type holds much promise for regenerative medicine and in vitro modelling of
development and disease. Haematopoietic stem cells (HSCs) regenerate all lineages
of the blood throughout adult life and are essential for the treatment of a vast number
of haematalogic disorders. Current sources of HSCs for clinical use and research,
including adult bone marrow, peripheral blood stem cells and umbilical cord blood,
are limited by the number of HSCs they contain and by the availability of a suitable
donor. A system that generates a reliable source of HSCs from ES cells would
therefore be an ideal alternative. While much progress has been made in the
generation of downstream lineages of the haematopoietic system, progress in the
derivation of HSCs capable of long-term self-renewal and multilineage reconstitution
from ES cells has been limited. Understanding of the developmental steps leading to
HSC emergence in the embryo has been advancing in recent years. In particular,
precursors of HSCs (preHSCs) have been isolated from the mouse embryo,
characterised and matured into HSCs ex vivo using the specialised conditions of
aggregate culture systems (Taoudi et al 2008, Rybtsov et al 2011). We hypothesised
that application of the aggregate culture system in the differentiation of ES cells
could provide a missing link in the in vitro generation of HSCs. Here I have
developed a novel ES cell differentiation system that employs the specialised
conditions of the aggregate culture system, after an initial stage of mesoderm
differentiation. I show that this system creates an environment for efficient
haematopoietic and endothelial progenitor formation and generates cells of a preHSC
type I (VE-Cadherin+CD45-CD41lo) and preHSC type II (VE-Cadhein+CD45+)
surface phenotype. Notably, the system gives rise to cells that achieve low-levels of
haematopoietic repopulation in sublethally irradiated NSG mice. The low-level
repopulating cells persist for over 4 months in animals and show both myeloid and
lymphoid potential. I identify genes that are expressed in cells of a preHSC II surface
marker-phenotype from the E11.5 dorsal aorta, but not in cells of this phenotype
from the E11.5 Yolk sac or differentiated ES cells. I also show that enforced
expression of Notch downstream target Hes1 in Flk1+ mesoderm during ES cell
differentiation does not improve levels of ES-derived repopulation.