Spleen as a site for hematopoiesis

Abstract

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 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.