Haematopoietic stem cells (HSCs) are routinely used to treat haematological
disorders, as they can engraft into the bone marrow of immuno-compromised
recipients where they undergo self-renewal and multilineage differentiation to
provide long-term reconstitution of the blood system. Identification of novel factors
able to regulate or expand HSCs would have a significant impact in a clinical setting.
Mouse embryonic stem (ES) cells can be used as a model system to investigate
haematopoietic regulation, since these pluripotent cells are amenable to large-scale
culture and have the capacity to differentiate into a variety of cell types in vitro,
including cells of haematopoietic lineages. Mature blood cells can be generated
relatively easily from ES cells; however, HSCs are generated at relatively low
frequencies and there has been only limited success in the contribution of these cells
to the adult haematopoietic system in vivo. Previous work demonstrated that the
frequency of haematopoietic progenitors was significantly increased when ES cells
were co-cultured with primary El0.5 aorta-gonad-mesonephros (AGM) tissue
explants, a region which is able to give rise to HSCs in vivo. Therefore, the AGM
region is a potent source of haematopoietic inductive signals both in vivo and for ES
cells in vitro.This project aimed to determine which subregion(s) of the AGM were
responsible for the haematopoietic enhancing effects that primary AGM explants had
on differentiating ES cells. To this end, a novel co-culture system has been
established to test the enhancing effects of a panel of clonal stromal cell lines derived
from different subregions of the midgestational AGM. It was found that three clonal
stromal cell lines derived from the dorsal aorta and surrounding mesenchyme (AM)
subregion of the AGM were able to significantly enhance the frequency of ES cell
derived multipotent haematopoietic progenitors, as measured by in vitro colony
assays and flow cytometry. By contrast, two stromal cell lines derived from the urogenital ridges (UG) of the AGM did not enhance haematopoietic differentiation of
ES cells. Interestingly, the enhancing capacity of the AM-derived stroma was
comparable with that of the bone marrow derived OP9 stromal cell line, which has
been widely used in the literature to promote haematopoietic differentiation of ES
cells. Further investigation revealed that the enhancing capacity is not retained by
extracellular matrices isolated from the AM stromal cell layers and the effects were
dependent on direct ES cell-stromal cell contact. Co-culture of an ES cell line
carrying a mesoderm specific Brachyury-eGFP reporter gene demonstrated that the
stromal lines mediated their effects post- Brachyury (mesoderm) induction in the ES
cells. In addition, co-culture of sorted ES cell populations confirmed that
Brachyury*, but not Brachyury', cells gave rise to haematopoietic progenitors in AM
stromal co-culture, supporting the notion that ES cell differentiation recapitulated the
in vivo pattern of lineage specification. Transplantation of co-cultured ES cells into
irradiated adult NOD/SCID mouse recipients led to low levels of donor cell
engraftment in the spleen and bone marrow, which expanded upon serial
transplantation; but full repopulation of the recipient haematopoietic system was not
confirmed. Adult bone marrow cells were found to achieve repopulation more
readily in the NOD/SCID animal model when transplanted intra-splenically, as
compared to intra-venous injection. This suggests that transplantation of ES-derived
haematopoietic cells directly into the haematopoietic niche, by intra-splenic or intrafemoral injection, could facilitate repopulation
