Investigating the haematopoietic and regenerative capacity of the spleen

Abstract

Haematopoietic stem cells (HSC) have distinct differentiation outputs of myeloid and lymphoid cell progeny and platelets. Over the lifespan of an individual, a skewing towards myeloid differentiation by HSC can be observed with ageing. This dominant myelopoiesis is attributed to a combination of both increased HSC differentiation into myeloid progeny and higher frequencies of these ‘myeloid-biased’ HSC as a result of increased self-renewal. As well as ‘myeloid bias’, myeloproliferative disorders are also common in aged individuals. If these are life-threatening, they are commonly treated by HSC transplantation (HSCT) after myeloablation, a procedure that resets haematopoiesis and cell repopulation in the affected individual.

Although widely successful, HSCT is associated with high rates of mortality. The individual is most compromised during the time between pre-transplant conditioning or myeloablation, and haematopoietic recovery following transplantation of HSC. This period when patients are highly immunocompromised remains an important target for therapeutic improvement. The success of HSCT is known to be correlated with the number of available niche spaces for occupation by migrating donor HSC. It is also clear that the spleen is a primary site for HSC localisation following HSCT and as a result can contribute to recovery. The power of the spleen to provide niches for HSC is also supported by evidence that it has remarkable regenerative capacity following irradiation and can support haematopoiesis, hosting its own small but distinct population of HSC. Splenic HSC are also primed for myelopoiesis, and retain this myeloid bias even after circulation and lodgement in bone marrow haematopoietic niches. The spleen could therefore be an important contributor to the myeloid bias of HSC associated with an ageing phenotype. The importance of the spleen in the phenotype of myeloid bias with ageing, and as a target for improvement of recovery following HSCT, has not yet been investigated fully.

This thesis aims to examine 1) the potential effect of the spleen on the development of myeloid bias observed in aged HSCs, as well as 2) its potential to act as an extramedullary niche to augment haematopoietic recovery following HSCT.

The first aim employs two animal models to assess whether haematopoietic niches in spleen contribute to increased myeloid bias in HSC. Flow cytometric analyses indicate CD150hi expression can be used to inspect shifts in the number or proportion of HSC with an aged, myeloid-biased phenotype. The two models investigate the role of spleen in the myeloid-biased phenotype by comparing splenectomised animals with normal animals. The first model involves steady-state splenectomised aged female mice at 21 months of age (equivalent to 56-69 years in humans) and compares these with age-matched control mice. This study found no significant difference in the number or proportion of myeloid-biased HSC. The second model examines the importance of extramedullary haematopoiesis in spleen, which occurs during pregnancy, on the development of the myeloid-biased phenotype. Mice were splenectomised and then exposed to multiple rounds of pregnancy, then the number and proportion of CD150hi HSC present in bone marrow was compared with normal age-matched exbreeder mice. Again, no differences were found in this model. Together, the data indicate that spleen does not play any significant role in the shift to myeloid bias observed amongst bone marrow HSC during haematopoiesis in ageing or following extended periods of extramedullary haematopoiesis. One possible explanation is that the influence of the spleen on numbers and proportion of myeloid-biased HSC may have been difficult to detect under conditions of steady-state haematopoiesis. Changes may only be detectable during pregnancy or during stress-induced extramedullary haematopoiesis. Changes may also have been masked by other intrinsic and extrinsic factors that contribute to HSC ageing.

The second aim investigates whether cellular therapy could be used to augment the regenerative capacity of spleen following irradiation and so hasten recovery of HSC niches in spleen. This could allow earlier haematopoietic recovery and reduce the period of immunosuppression. Myeloablative damage to spleen was evident immediately following lethal irradiation (9.5Gy total, delivered in a split dose). This was indicated by loss of several cellular and structural compartments involving both white pulp and red pulp regions. This damage resolved between 4- and 8-weeks post-irradiation through intravenous infusion of bone marrow-derived HSPC. The first cellular therapy tested whether co-infusion with enzymatically dissociated neonatal spleen cells could influence recovery of the spleen. This study demonstrated enhanced development of stromal and endothelial cells, which provide the supporting microenvironments and niches for haematopoietic recovery. However, co-infusion of neonatal spleen cells also delayed the formation of distinct regions of white and red pulp in spleen compared with control irradiated spleen given only an infusion of bone marrow HSPC. In a second study, irradiated animals given HSPC were co-infused with cells from the cloned splenic stromal cell line, STX3. This was previously developed by our lab and is capable of supporting haematopoiesis with production of myeloid cells in vitro. In contrast to animals given neonatal spleen cells as an infusion, the transfer of STX3 cells did not noticeably influence splenic regeneration. This study demonstrates how the regeneration of spleen following irradiation can be augmented using cellular therapies, specifically neonatal spleen cell transfer in tandem with HSPC infusion at the time of irradiation.

This thesis provides insight into the potential role of the spleen in HSC ageing, and in its suitability as an extramedullary haematopoietic niche, which can be targeted for faster recovery following myeloablative conditioning during HSCT. Taken together, these studies posit the spleen as a haematopoietic niche that can be targeted to enhance or augment haematopoiesis.

Date of Award	28 Nov 2024
Original language	English
Supervisor	Jonathan Tan (Supervisor) & Helen O'Neill (Supervisor)