Bianca Nowlan1,2,3, Kathryn Futrega3,4,5, Elizabeth Deborah Williams1,2,3, Michael Robert Doran6,7,8,9,10,11,12. 1. School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia. 2. Australian Prostate Cancer Research Centre - Queensland (APCCRC-Q) and Queensland Bladder Cancer Initiative (QBCI), Brisbane, Queensland, Australia. 3. Translational Research Institute, 37 Kent Street, Brisbane, Queensland, 4102, Australia. 4. Centre for Biomedical Technologies (CBT) and School of Mechanical, Medical, and Process Engineering (MMPE), Queensland University of Technology (QUT), Brisbane, Australia. 5. Skeletal Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 30, 30 Convent Dr MSC 4320, Bethesda, MD, 20892-4320, USA. 6. School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia. michael.doran@qut.edu.au. 7. Australian Prostate Cancer Research Centre - Queensland (APCCRC-Q) and Queensland Bladder Cancer Initiative (QBCI), Brisbane, Queensland, Australia. michael.doran@qut.edu.au. 8. Translational Research Institute, 37 Kent Street, Brisbane, Queensland, 4102, Australia. michael.doran@qut.edu.au. 9. Centre for Biomedical Technologies (CBT) and School of Mechanical, Medical, and Process Engineering (MMPE), Queensland University of Technology (QUT), Brisbane, Australia. michael.doran@qut.edu.au. 10. Skeletal Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Building 30, 30 Convent Dr MSC 4320, Bethesda, MD, 20892-4320, USA. michael.doran@qut.edu.au. 11. Mater Research Institute - University of Queensland, Brisbane, Australia. michael.doran@qut.edu.au. 12. Australian National Centre for the Public Awareness of Science, Australian National University, Canberra, Australia. michael.doran@qut.edu.au.
Abstract
BACKGROUND: Direct bone marrow injection of cells into murine marrow cavities is used in a range of cell characterization assays and to develop disease models. While human bone marrow-derived stromal cells (hBMSC, also known as mesenchymal stem cells (MSC)) are frequently described in therapeutic applications, or disease modeling, their behavior following direct injection into murine bone marrow is poorly characterized. Herein, we characterized hBMSC engraftment and persistence within the bone marrow of NOD-scid interleukin (IL)-2γ-/- (NSG) mice with or without prior 2 Gy total-body γ-irradiation of recipient mice. METHODS: One day after conditioning NSG mice with sublethal irradiation, 5 × 105 luciferase (Luc) and green fluorescent protein (GFP)-expressing hBMSC (hBMSC-Luc/GFP) were injected into the right femurs of animals. hBMSC-Luc/GFP were tracked in live animals using IVIS imaging, and histology was used to further characterize hBMSC location and behavior in tissues. RESULTS: hBMSC-Luc/GFP number within injected marrow cavities declined rapidly over 4 weeks, but prior irradiation of animals delayed this decline. At 4 weeks, hBMSC-Luc/GFP colonized injected marrow cavities and distal marrow cavities at rates of 2.5 ± 2.2% and 1.7 ± 1.9% of total marrow nucleated cells, respectively in both irradiated and non-irradiated mice. In distal marrow cavities, hBMSC were not uniformly distributed and appeared to be co-localized in clusters, with the majority found in the endosteal region. CONCLUSIONS: While significant numbers of hBMSC-Luc/GFP could be deposited into the mouse bone marrow via direct bone marrow injection, IVIS imaging indicated that the number of hBMSC-Luc/GFP in that bone marrow cavity declined with time. Irradiation of mice prior to transplant only delayed the rate of hBMSC-Luc/GFP population decline in injected femurs. Clusters of hBMSC-Luc/GFP were observed in the histology of distal marrow cavities, suggesting that some transplanted cells actively homed to distal marrow cavities. Individual cell clusters may have arisen from discrete clones that homed to the marrow, and then underwent modest proliferation. The transient high-density population of hBMSC within the injected femur, or the longer-term low-density population of hBMSC in distal marrow cavities, offers useful models for studying disease or regenerative processes. Experimental designs should consider how relative hBMSC distribution and local hBMSC densities evolve over time.
BACKGROUND: Direct bone marrow injection of cells into murine marrow cavities is used in a range of cell characterization assays and to develop disease models. While human bone marrow-derived stromal cells (hBMSC, also known as mesenchymal stem cells (MSC)) are frequently described in therapeutic applications, or disease modeling, their behavior following direct injection into murine bone marrow is poorly characterized. Herein, we characterized hBMSC engraftment and persistence within the bone marrow of NOD-scid interleukin (IL)-2γ-/- (NSG) mice with or without prior 2 Gy total-body γ-irradiation of recipient mice. METHODS: One day after conditioning NSG mice with sublethal irradiation, 5 × 105 luciferase (Luc) and green fluorescent protein (GFP)-expressing hBMSC (hBMSC-Luc/GFP) were injected into the right femurs of animals. hBMSC-Luc/GFP were tracked in live animals using IVIS imaging, and histology was used to further characterize hBMSC location and behavior in tissues. RESULTS:hBMSC-Luc/GFP number within injected marrow cavities declined rapidly over 4 weeks, but prior irradiation of animals delayed this decline. At 4 weeks, hBMSC-Luc/GFP colonized injected marrow cavities and distal marrow cavities at rates of 2.5 ± 2.2% and 1.7 ± 1.9% of total marrow nucleated cells, respectively in both irradiated and non-irradiated mice. In distal marrow cavities, hBMSC were not uniformly distributed and appeared to be co-localized in clusters, with the majority found in the endosteal region. CONCLUSIONS: While significant numbers of hBMSC-Luc/GFP could be deposited into the mouse bone marrow via direct bone marrow injection, IVIS imaging indicated that the number of hBMSC-Luc/GFP in that bone marrow cavity declined with time. Irradiation of mice prior to transplant only delayed the rate of hBMSC-Luc/GFP population decline in injected femurs. Clusters of hBMSC-Luc/GFP were observed in the histology of distal marrow cavities, suggesting that some transplanted cells actively homed to distal marrow cavities. Individual cell clusters may have arisen from discrete clones that homed to the marrow, and then underwent modest proliferation. The transient high-density population of hBMSC within the injected femur, or the longer-term low-density population of hBMSC in distal marrow cavities, offers useful models for studying disease or regenerative processes. Experimental designs should consider how relative hBMSC distribution and local hBMSC densities evolve over time.
Entities:
Keywords:
Bone marrow; Cell competition; Human bone marrow-derived stromal cells; Intrafemoral injection; Sub-lethal irradiation; Xenograft
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