An investigation into the role of chemokines in haemopoietic stem cell quiescence

Sinclair, Amy (2015) An investigation into the role of chemokines in haemopoietic stem cell quiescence. PhD thesis, University of Glasgow.

Abstract

Haemopoietic stem cells (HSC) maintain lifelong haemopoiesis through the monitoring and production of cells from multiple haemopoietic cell lineages. A key property of HSC is their ability to maintain quiescence. Quiescence refers to a state of inactivity in which the cell is not dividing and remains dormant. It is this property of the HSC that is thought to maintain genomic integrity and to allow the HSC to sustain haemopoiesis over the period of a lifetime. However, the regulation of quiescence in this context is not well understood. Numerous studies have aimed to understand the molecular mechanisms underlying HSC quiescence using high-throughput approaches. A previous microarray study by our group aimed to understand the transcriptional differences between quiescent and proliferating human HSC. Data from this microarray showed that the most up regulated group of genes in quiescent compared to proliferating human HSC were chemokine ligands, specifically within the CXC family. Although this was a novel finding at the time, the biological function of these chemokine genes was not studied until the current work presented here. In this thesis, we aimed to extend foregoing research and importantly, investigate the role of CXC chemokines in HSC properties, using both human and mouse systems.

First, we validated the results from the microarray study using gene expression analyses to show that chemokine ligands CXCL1 and CXCL2 were significantly up regulated in quiescent HSC (CD34+CD38-) in comparison to more proliferative progenitors (CD34+CD38+). Focusing on CXCL1, we showed positive expression of the ligand protein in human stem/progenitor cells using immunofluorescence and western blotting on human primary CD34+ cells. In addition, we identified positive expression of receptor CXCR2 by gene and protein analyses on CD34+ cells, indicating the presence of an autocrine chemokine signalling loop. To determine the biological function of CXCL1/CXCR2 signalling in human HSC, we used shRNA to reduce CXCL1 expression and a commercially available inhibitor (SB-225002) to block CXCR2 receptor signalling. Experiments on cell lines expressing CXCL1 and CXCR2 (HT 1080) showed that reduction of CXCL1 and over-expression reduced or increased cell viability and proliferation respectively. Experiments on human primary CD34+ cells revealed that reduction of CXCL1 induced apoptosis and reduced colony formation. Similarly, inhibition of CXCR2 signalling in CD34+ cells using SB-225002 induced apoptosis and reduced colony formation in a dose dependent manner. However, due to human sample availability and technical challenges, experiments need repeated in order for a valid conclusion to be made and statistical analysis could not be carried out for some primary experiments. In addition, further experimental work is required to conclusively prove that human stem/progenitors express CXCL1 and CXCR2 as different techniques showed varying results. In summary, we provide some evidence that CXCL1 and CXCR2 is expressed by human HSC and may be an important survival pathway in normal human HSC which requires further experimental data to provide valid conclusions.

In order to gain a deeper understanding of the biological function of chemokine signalling in HSC biology, we used an in vivo murine system. First, we examined mRNA transcripts of CXC chemokines in mouse HSC populations. We screened a small selected group of CXC chemokines using primitive mouse HSC and single cell quantitative PCR using the Fluidigm™ platform. Gene expression analyses identified that Cxcr2 and Cxcl4 mRNA transcripts were detected including in the most rare, primitive HSC fraction. To elucidate the mechanism of action, we used a transgenic reporter and knock out mouse models for both genes of interest. Analysis of a Cxcr2 null mice model (Cxcr2-/-) validated previous research in which animals lacking Cxcr2 show disrupted haemopoiesis with an expansion of myeloid cells in the haemopoietic organs. Interestingly, within the current work, analysis of steady state haemopoiesis revealed an expansion of the most primitive HSC in the BM of animals lacking Cxcr2 and enhanced mobilisation demonstrated by an increase in the stem/progenitor activity in the spleen and PB. HSC functional analyses using BM reconstitution assays with wildtype (WT) or Cxcr2-/- HSC showed that there was a trend towards a reduction in engraftment in animals transplanted with HSC lacking Cxcr2. However, this result was not statistically significant due to high sample variability and due to time constraints and the length of this assay, this was not repeated. The data suggests that Cxcr2 expressing HSC may be important for stem cell maintenance via a cell autonomous mechanism however experiments are required to be repeated to draw valid conclusions.

Cxcl4-Cre transgenic mice containing a RFP construct under the control of the Rosa26 promoter (Cxcl4-Cre) showed RFP expression in HSC and progeny. RFP expression in HSC populations was in accordance with Cxcl4 mRNA transcripts therefore suggesting RFP expression was correlated with endogenous Cxcl4 expression. Interestingly, flow cytometry analysis identified that not all (~50%) HSC showed positive expression for RFP. Flow cytometry sorting of positive and negative populations revealed that cells with enhanced colony formation potential reside within the RFP (Cxcl4) positive fraction. To extend this data, we aimed to knock out and reduce Cxcl4 expression and examine the phenotype. Targeted deletion of Cxcl4 in vitro using a Cxcl4 shRNA vector demonstrated that Cxcl4 reduction in vitro diminished colony formation in primary and secondary replating assays. Since data for human CXCL4 mRNA were not conclusive from the original microarray, we reassessed the relevance of CXCL4 in the human system. Gene expression analyses showed that CXCL4 transcripts were indeed detected and furthermore, up regulated in primitive HSC (CD34+CD38-CD90+) compared with proliferative progenitors (CD34+CD38+). Collectively, the data indicates that CXCL4 may play an important role in mouse and human HSC biology, however further experimental work is required to address this.

In summary, the data presented in this thesis demonstrate that several chemokines including CXCL1, CXCL4 and receptor CXCR2 may have key roles in HSC survival and maintenance, both in the mouse and human systems. However, increased biological replicates and further experiments are required to draw valid conclusions. Enhanced understanding of the regulation of stem cell properties is critical for improving our ability to manipulate normal stem cells in vitro and in vivo. Furthermore, understanding normal stem cell regulation is fundamental for the research of diseases such as leukaemia in which leukaemic stem cells are less sensitive to drug treatment.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: chemokines, HSC
• Subjects: Q Science > QH Natural history > QH426 Genetics; Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cancer Sciences > Paul O'Gorman Leukemia Research Centre
• Supervisor's Name: Holyoake, Professor Tessa and Graham, Professor Gerard
• Date of Award: 2015
• Depositing User: Dr Amy Sinclair
• Unique ID: glathesis:2015-4956
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Apr 2014 07:34
• Last Modified: 11 Apr 2014 07:35
• URI: https://theses.gla.ac.uk/id/eprint/4956