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Defining regulators of hematopoietic stem cell homing and engraftment

Final Report Summary - HEMHOME (Defining regulators of hematopoietic stem cell homing and engraftment)

Project: 255451
Final report: period covered: 01/07/2010 – 31/01/2013

Background and objective:
The objective of this project is to identify new molecules that mediate homing and adhesion of hematopoietic stem and progenitor cells (HSPCs) to their niche in the bone marrow. The project is based on human HSPCs isolated from cord blood as a widely accessible but underused source of cells for transplantation. Firm attachment of HSPCs, defined as CD34+, to mesenchymal stroma cells (MSCs) gave the ground to our in vitro model of niche interactions. MSCs are present in the bone marrow and have been suggested to be a crucial component of the HSC specific niche. When plated onto a layer of human MSCs, the vast majority of human CD34+ cells stay adherent despite the gravitational force upon inversion of the plate. We use these highly adhesive properties of CD34+ cells as a baseline to screen for modifiers (shRNAs) that interfere with adhesion. ShRNAs retrieved in this manner may target genes that are important mediators of adhesion. Previous proof of concept experiments for the experimental setup had been performed before start of the project.

Results and conclusions:
A lentiviral library of pooled shRNAs targeting adhesion molecules (366 genes, 1778 shRNA) was used to transduce CD34+ cells. The cells were assayed for adhesion; viable CD34+ cells were sorted from initial, adherent and non-adherent fractions and subsequently, integrated shRNAs were identified and quantified by deep sequencing (a collaboration with Clinical Oncology Dept. in Lund). Altogether, 7 independent screens were performed. Genes, for whom shRNAs were enriched in the non-adherent fraction i.e. down-regulation of which decreases adhesion to MSC, are considered as candidates for further validation. Results from 7 replicates of adhesion assay confirmed a role of beta1 integrin and alpha6 integrin in HSPC niche interactions (Potocnik 2000, Qian 2007). Indeed, 2 independent shRNAs for each of these genes scored among top positions in the screens.
In order to fine-tune the screen results, we constructed a sub-library of shRNAs which scored high throughout 7 replicates. Consequently, a screen with sub-library narrowed down to 200 shRNA allowed us to obtain more consistent results and more reliable candidate shRNAs. Additionally, we performed microarrays on initial, adherent and non-adherent fractions to compare gene expression in CD34+ cells in relation to their adhesive properties. The majority of genes targeted by our candidate shRNAs was found to have low to medium expression levels in the initial fraction without significant change upon adhesion assay. Our observations were compared with database available online (biogps, hematopoietic atlas) and published data. Another important aspect when choosing a candidate gene to follow-up is the efficiency of gene expression knockdown by a particular shRNA. Therefore, we consulted data accessible online (Sigma-Aldrich) and information available form The RNAi Consortium. Such a thorough preparatory analysis of candidate shRNAs and genes should allow identification of hits with a role in CD34+ adhesion to MSC while eliminating noise inherent to screening strategy.
Next, candidate shRNAs were assessed individually: CD34+ cells are transduced with a single shRNA and its ability to modify adhesion is tested. We obtained a high validation rate for top subscreen shRNAs as 16 out of 20 scored positive in adhesion assay.
After validation of an shRNA in adhesion assay, we searched for at least 1 more shRNA for a targeted gene that would reproduce the effect in adhesion assay and therefore, confirm the specificity of the observed gene KD phenotype. Among the novel candidate genes previously not implicated in HSCP adhesion, we first focused on the GTPase cytohesin1 (CYTH1). Initially, CYTH1 has been identified as a regulator of beta2 integrin inside-out regulation in immune cells (Kolanus 2007). We confirmed CYTH1 expression in total CD34+ cells from cord blood and bone marrow as well as in more purified progenitor/stem fractions CD34+CD38-CD90+. Validation assays showed that knockdown (KD) of CYTH1 from four independent shRNAs significantly increased the fraction of non-adherent cells in the stroma adhesion assay. Moreover, CYTH1 KD specifically inhibited adherence of CD34+ cells to the integrin ligands: glycoproteins fibronectin and ICAM1, implicating CYTH1 as a crucial mediator of cell adhesion in human HSPC. In vivo, shRNA-mediated KD of CYTH1 significantly decreased both short-term and long-term engraftment of CD34+ cells in NSG mice, indicating a role for CYTH1 in mediating homing and engraftment of HSPCs. Elaborated analysis of CYTH1 KD influence on CD34+ cells homing is a result of a new collaboration with Dominique Bonnet’s group from London Cancer Research. Preliminary results from homing assays using intravital microscopy show increased mobility of CD34+CD38- cells with CYTH1 KD coupled with a tendency to localize at a further distance from the bone surface and endothelium compared to control cells.

Potential impact and use:
Hematopoietic stem cells (HSCs) have been used in the clinic for several decades in life-saving treatment of hematological disorders and malignant diseases through continuously improved bone marrow transplantation (BMT) protocols. However, a significant proportion of patients who would benefit from bone marrow transplantation cannot receive treatment due to insufficient numbers of stem cells. Methods to increase the efficiency of BMT may therefor have a profound and immediate impact on clinical medicine. Even when large numbers of cells are available for BMT there is a need for myeloablative preconditioning to get efficient engraftment of the transplanted cells. Myeloablation is necessary to kill resident cells and free niche space for the incoming cells. When treating cancer with BMT, irradiation or cytotoxic agents are part of the treatment to kill cancer cells but when considering HSC transplantation for non-malignant disorders and in conjunction with gene therapy the severe side-effects of myeloablative preconditioning is a significant constraint. A better understanding of the mechanisms regulating migration and adhesion of HSCs will be crucial to find new approaches enabling efficient engraftment in non-conditioned hosts.
We successfully developed an shRNA-based screen for mediators of adhesion between human cord blood derived CD34+ cells and MSCs. Deep sequencing is a convenient method to deconvolute screen results in a laborsaving and efficient manner. Our screens detected known regulators of HSPC adhesion demonstrating feasibility of the approach. Currently, cytohesin1 (CYTH1) previously not associated with HSPC adhesion, is investigated for its role in HSPC-niche interactions. Its successful validation in niche interactions in vitro and in vivo will rank cytohesin1 highly interesting target to modulate the engraftment process in a clinical setting. In collaboration with Lena Svensson’s group (Leukocyte migration, Immunology Dept., Lund) we are currently using interference reflection microscopy (IRM) to understand in more details how lack of CYTH1 alters the adhesive properties of HSCPs. Link between cytohesin1 and integrin activation status is investigated with total internal reflection microscopy (TIRF). Moreover, migration of CD34+ cells with CYTH1 KD in random or under shear stress is analyzed with time-lapse microscopy.

Socio-economic impact:
Scientific research and socio-economic development are tightly coupled. Regenerative medicine, which includes all stem cell research, is a critical element in the future for the biotech and life science industry. The global market for stem cell research has grown extensively and involves not only potential cures for serious diseases but it also means more jobs, income and economic growth. Commercializing research results with industrial applications have the potential to give rise to economic growth.

Potocnik et al., Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity 12 (2000) 653-663

Qian et al., Contribution of #6 integrins to hematopoietic stem and progenitor cell homing to bone marrow and collaboration with alpha6 integrins. Blood 107 (2007) 3503-3510

Kolanus, Guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction. Immunological Reviews 218 (2007) 102-113