Investigation of haematopoietic stem cell homing post-transplantation using an in vitro model of the bone marrow/vasculature interface

Almalki, Sultan

Abstract

Homing of haematopoietic stem cells (HSCs) to the bone marrow (BM) niches after transplantation (HSCT) is controlled by chemotactic signalling released from the BM microenvironment. This process is incompletely understood, but some factors are known, such as stromal cell-derived factor 1 alpha (SDF-1 α). Studies have shown that HSCs naturally mobilise to sites of ischaemic injury and since the BM microenvironment is hypoxic, it has been theorised that the mandatory conditioning chemotherapy for HSCT might play a role in stem cell homing, by producing an "injury" in the hypoxic BM. This injury leads to the release of chemotactic factors which might induce HSCs to traffic to the BM niches. This combination of chemotherapy and hypoxia in the BM microenvironment is often underestimated in vitro; however, adding the hypoxia element might provide a novel candidate that previously has been overlooked.

A novel model of HSC homing has been developed, using HL-60 cells as HSC equivalent, which traffic through a vasculature endothelium (HMEC-1) on a hanging transwell insert, to reach the BM (HS-5) in hypoxia and compared to normoxic conditions. Transmigrated HL-60 cells were monitored under the influence of SDF-1 or conditioned medium (C.M.) from HS-5 after treatment with clinically relevant doses and low intensity conditioning therapy (melphalan, carmustine, etoposide and cytarabine) and targeted therapies (ibrutinib and imatinib) in hypoxic and normoxic environments.

Herein, peptide-based chemokine signalling factors released by HS-5 post-chemotherapy exposure were explored using proteomics analysis. HS-5 C.M. were analysed after treatment with a combination of conditioning therapy (BEAM) in hypoxia and normoxia to identify the chemokines accounting for the functional effects of the cell secretome. For the first time, this approach led to the recognition of distinct functions associated with HS-5 secretome according to the treatment conditions. In hypoxia, 25 proteins were upregulated, predominantly related to stress and response to hypoxia. For BEAM treatment plus hypoxia 31 proteins were upregulated, evidencing a secretome linked to stress, hypoxia, angiogenesis, localisation, cell migration and vascular permeability. These findings suggest that conditioning therapy might be strongly involved in manipulating vascular endothelium and facilitating HSCs trafficking.

In addition, some candidate proteins were obtained as a recombinant and tested in the transmigration model. MMP-1, MMP-3 and AMOT were investigated for their ability to act as inducers for HL-60 transmigration. Both MMP-1 and MMP-3 were shown to affect the barrier integrity by loosening the endothelial membrane; MMP-1 was shown to have an extra role in inducing HL-60 trafficking actively. Conversely, AMOT was shown to affect the endothelium integrity without inducing HL-60 trafficking. However, both MMPs and AMOT were shown to have more potency in combination with SDF-1α.

Finally, several proteins were upregulated after exposing HS-5 to conditioning therapy and hypoxia, suggesting that HSC homing is a complex process that involves several factors aside from SDF-1α. These factors might be effective inducers for HSCs homing to the BM microenvironment for a limited time, which can be utilised to infuse the donated graft to have better engraftment.

Citation

Almalki, S. Investigation of haematopoietic stem cell homing post-transplantation using an in vitro model of the bone marrow/vasculature interface. (Thesis). University of the West of England. Retrieved from https://uwe-repository.worktribe.com/output/6000940

• Thesis Type: Thesis
• Deposit Date: May 29, 2020
• Publicly Available Date: Jun 28, 2021
• Public URL: https://uwe-repository.worktribe.com/output/6000940
• Award Date: Jun 28, 2021