Generation of HLA-Specific Humanised Mice Using Bone Marrow-Derived Haematopoietic Stem Cells from Cadaveric Organ Donors
Department of Surgery, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom
Meeting: 2013 American Transplant Congress
Abstract number: C1175
Introduction
‘Humanised’ mice reconstituted with a functional immune compartment are an invaluable tool in the study of the immune response and can be generated using haematopoietic stem cells (HSCs) from a variety of sources. Bone marrow (BM) from cadaveric organ donors represents a potentially abundant source of HSCs of pre-specified HLA type. In addition, cells and tissues can be harvested from syngeneic and allogeneic cadaveric donors with which to challenge the immune system in the resulting humanised mice. We therefore examined the ability of BM-derived HSCs from cadaveric organ donors to generate a humanised mouse model to investigate human responses to alloantigens.
Methods
Bone marrow was aspirated from the lumbar vertebrae and iliac bones of human cadaveric donors after other organs were retrieved for transplantation and the mononuclear fraction was separated using Ficoll gradient and cryopreserved at -180°C in 10% DMSO+90% FCS. After thawing, live CD34+ HSCs were isolated using magnetic beads and adoptively transferred into sub-lethally irradiated immmunodeficient NOD/SCID/IL2rΓ-/- mice (NSG; 1-5×105 cells/animal). Engraftment with human CD45+ cells was assessed by flow cytometric analysis of peripheral blood samples at weekly intervals. Skin, splenocytes and mesenteric blood vessels from the same donors were also cryopreserved at -180°C.
Results
Bone marrow was successfully aspirated (22-76 ml/donor) from cadaveric donors that proceeded to donation after either brain death or circulatory death (age range 35–81 years). The CD34+ fraction of bone marrow aspirates was 1% (range 0.9-1.4%) and post-thaw viability of the CD34+ fraction was >80%. NSG mice (n=9) were successfully reconstituted with CD45+ cells. Engraftment levels in the peripheral blood reached 94.5% at 30 weeks, consisting of 63.3% B cells (CD19+), 29.1% T cells (CD3+), 22.45 CD4 T cells and 7.8% CD8 T cells.
Conclusion
BM-derived HSCs survive circulatory arrest for several hours and maintain their engraftment potential in immunodeficient mice. This model enables the generation of HLA-specific humanised mice, using a readily available source of HSCs, to investigate human immune responses to alloantigens.
To cite this abstract in AMA style:
Elliott K, Conlon T, Negus M, Rouhani F, Vallier L, Bolton E, Bradley J, Pettigrew G, Saeb-Parsy K. Generation of HLA-Specific Humanised Mice Using Bone Marrow-Derived Haematopoietic Stem Cells from Cadaveric Organ Donors [abstract]. Am J Transplant. 2013; 13 (suppl 5). https://atcmeetingabstracts.com/abstract/generation-of-hla-specific-humanised-mice-using-bone-marrow-derived-haematopoietic-stem-cells-from-cadaveric-organ-donors/. Accessed November 22, 2024.« Back to 2013 American Transplant Congress