Lim, Hong Kiat (Don)
Description
Hematopoiesis occurs throughout the lifespan of an organism and
involves the formation of blood cells from hematopoietic stem
cell (HSC) which are self-renewing multipotent progenitors. The
hematopoietic niche environment comprises nonhematopoietic cells,
extracellular matrix components and soluble regulatory factors
which contribute to the quiescence, dormancy, self-renewal and
differentiation of HSC. While multiple HSC niches have been
described in bone...[Show more] marrow, as endosteal, vascular and
perivascular, niches which support hematopoiesis in other sites
like spleen remain to be elucidated. Previous studies in this lab
have described unique splenic stroma cell lines 5G3 and 3B5 which
can support hematopoiesis and reflect HSC niches. In addition,
previously obtained transcriptome data has shown that 5G3 and 3B5
stroma express many genes in parallel with perivascular cells
described in bone marrow. This information forms the basis of the
current study.
In this thesis, both 5G3 and 3B5 stroma have been shown to share
a mesenchymal lineage origin with perivascular cells in bone
marrow, including mesenchymal stem cells and C-XC motif ligand 12
(CXCL12)-abundant reticular cells. 5G3 and 3B5 express many cell
surface markers in common with these cells including CD105, CD29,
VCAM1, Sca-1, CD51, CD140a and Thy1.2. In addition, the concept
of niches for hematopoiesis in spleen has been advanced. Splenic
stromal cells with the phenotype of Sca-1+gp38+Thy1.2+CD29+CD51+
were found to be important for in vitro hematopoiesis. Their
phenotype reflects cells of mesenchymal lineage, consistent with
our primary hypothesis that HSC niches involve at least
perivascular reticular cells resembling the stromal line models
of 5G3 and 3B5. Stromal cells expressing gp38 or Thy1.2 appear to
be associated with some HSC in spleen identified through section
staining, although this is restricted to neonatal spleen in the
case of gp38+ stromal cells.
Restricted hematopoiesis giving rise to L-DC and myeloid cells
was replicated in vivo following grafting of splenic stromal cell
lines including 5G3 and 3B5 under the kidney capsule. While L-DC
production was clearly shown to occur within 3B5 grafts, 5G3 was
difficult to engraft and then formed niches which appeared to
support hematopoietic cells with a transformed phenotype.
Specific signaling pathway inhibitors added into in vitro stromal
co-cultures involving lineage-depleted bone marrow over 5G3
stroma, was also used to identify the important role of SCF but
not CXCL12 in supporting in vitro hematopoiesis. In similar
experiments, the DAPT inhibitor of Notch signalling was used to
identify a role for Notch signalling in the development of L-DC
from MPP added into co-cultures over 5G3 stroma in vitro.
This study has improved our current understanding of HSC niches
in spleen and identified some of the molecular regulators of
haematopoiesis. Information obtained in this thesis verifies the
existence of HSC niches in spleen and will be important for
development therapies involving spleen as an extramedullary niche
for hematopoiesis. The amplification of existing splenic niches
or the ability to generate ectopic niches could be used to
support enhancement of hematopoietic output, in immunocompromised
patients or following HSC transplantation.
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