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Du EJ, Muench MO. A Monocytic Barrier to the Humanization of Immunodeficient Mice. Curr Stem Cell Res Ther 2024; 19:959-980. [PMID: 37859310 PMCID: PMC10997744 DOI: 10.2174/011574888x263597231001164351] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2023] [Revised: 08/17/2023] [Accepted: 08/25/2023] [Indexed: 10/21/2023]
Abstract
Mice with severe immunodeficiencies have become very important tools for studying foreign cells in an in vivo environment. Xenotransplants can be used to model cells from many species, although most often, mice are humanized through the transplantation of human cells or tissues to meet the needs of medical research. The development of immunodeficient mice is reviewed leading up to the current state-of-the-art strains, such as the NOD-scid-gamma (NSG) mouse. NSG mice are excellent hosts for human hematopoietic stem cell transplants or immune reconstitution through transfusion of human peripheral blood mononuclear cells. However, barriers to full hematopoietic engraftment still remain; notably, the survival of human cells in the circulation is brief, which limits overall hematological and immune reconstitution. Reports have indicated a critical role for monocytic cells - monocytes, macrophages, and dendritic cells - in the clearance of xenogeneic cells from circulation. Various aspects of the NOD genetic background that affect monocytic cell growth, maturation, and function that are favorable to human cell transplantation are discussed. Important receptors, such as SIRPα, that form a part of the innate immune system and enable the recognition and phagocytosis of foreign cells by monocytic cells are reviewed. The development of humanized mouse models has taken decades of work in creating more immunodeficient mice, genetic modification of these mice to express human genes, and refinement of transplant techniques to optimize engraftment. Future advances may focus on the monocytic cells of the host to find ways for further engraftment and survival of xenogeneic cells.
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Affiliation(s)
- Emily J. Du
- Vitalant Research Institute, 360 Spear Street, Suite 200, San Francisco, CA, 94105, USA
| | - Marcus O. Muench
- Vitalant Research Institute, 360 Spear Street, Suite 200, San Francisco, CA, 94105, USA
- Department of Laboratory Medicine, University of California, San Francisco, CA, 94143, USA
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2
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Calderbank EF, Magnani L, Laurenti E. Intrafemoral Injection of Human Hematopoietic Stem and Progenitor Cells into Immunocompromised Mice. J Vis Exp 2023:10.3791/66315. [PMID: 38145377 PMCID: PMC7615601 DOI: 10.3791/66315] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2023] Open
Abstract
Hematopoietic stem cells (HSCs) are defined by their lifelong ability to produce all blood cell types. This is operationally tested by transplanting cell populations containing HSCs into syngeneic or immunocompromised mice. The size and multilineage composition of the graft is then measured over time, usually by flow cytometry. Classically, a population containing HSCs is injected into the circulation of the animal, after which the HSCs home to the bone marrow, where they lodge and begin blood production. Alternatively, HSCs and/or progenitor cells (HSPCs) can be placed directly in the bone marrow cavity. This paper describes a protocol for intrafemoral injection of human HSPCs into immunodeficient mice. In short, preconditioned mice are anesthetized, and a small hole is drilled through the knee into the femur using a needle. Using a smaller insulin needle, cells are then injected directly into the same conduit created by the first needle. This method of transplantation can be applied in varied experimental designs, using either mouse or human cells as donor cells. It has been most widely used for xenotransplantation, because in this context, it is thought to provide improved engraftment over intravenous injections, therefore improving statistical power and reducing the number of mice to be used.
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Affiliation(s)
- Emily F Calderbank
- Wellcome and Medical Research Council Cambridge Stem Cell Institute, Department of Haematology, University of Cambridge;
| | - Laura Magnani
- Wellcome and Medical Research Council Cambridge Stem Cell Institute, Department of Haematology, University of Cambridge
| | - Elisa Laurenti
- Wellcome and Medical Research Council Cambridge Stem Cell Institute, Department of Haematology, University of Cambridge;
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Shajib MS, Futrega K, Davies AM, Franco RAG, McKenna E, Guillesser B, Klein TJ, Crawford RW, Doran MR. A tumour-spheroid manufacturing and cryopreservation process that yields a highly reproducible product ready for direct use in drug screening assays. J R Soc Interface 2023; 20:20230468. [PMID: 37817581 PMCID: PMC10565407 DOI: 10.1098/rsif.2023.0468] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2023] [Accepted: 09/11/2023] [Indexed: 10/12/2023] Open
Abstract
If it were possible to purchase tumour-spheroids as a standardised product, ready for direct use in assays, this may contribute to greater research reproducibility, potentially reducing costs and accelerating outcomes. Herein, we describe a workflow where uniformly sized cancer tumour-spheroids are mass-produced using microwell culture, cryopreserved with high viability, and then cultured in neutral buoyancy media for drug testing. C4-2B prostate cancer or MCF-7 breast cancer cells amalgamated into uniform tumour-spheroids after 48 h of culture. Tumour-spheroids formed from 100 cells each tolerated the cryopreservation process marginally better than tumour-spheroids formed from 200 or 400 cells. Post-thaw, tumour-spheroid metabolic activity was significantly reduced, suggesting mitochondrial damage. Metabolic function was rescued by thawing the tumour-spheroids into medium supplemented with 10 µM N-Acetyl-l-cysteine (NAC). Following thaw, the neutral buoyancy media, Happy Cell ASM, was used to maintain tumour-spheroids as discrete tissues during drug testing. Fresh and cryopreserved C4-2B or MCF-7 tumour-spheroids responded similarly to titrations of Docetaxel. This protocol will contribute to a future where tumour-spheroids may be available for purchase as reliable and reproducible products, allowing laboratories to efficiently replicate and build on published research, in many cases, making tumour-spheroids simply another cell culture reagent.
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Affiliation(s)
- Md. Shafiullah Shajib
- School of Biomedical Science, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
- Translational Research Institute, Brisbane, Queensland, Australia
| | - Kathryn Futrega
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
- Translational Research Institute, Brisbane, Queensland, Australia
- Department of Health and Human Services, National Institute of Dental and Craniofacial Research (NIDCR), National Institutes of Health (NIH), Bethesda, MD, USA
| | - Anthony M. Davies
- Translational Research Institute, Brisbane, Queensland, Australia
- Vale Life Sciences, Brisbane, Australia
| | - Rose Ann G. Franco
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
- Translational Research Institute, Brisbane, Queensland, Australia
| | - Eamonn McKenna
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
- Translational Research Institute, Brisbane, Queensland, Australia
| | - Bianca Guillesser
- School of Biomedical Science, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
- Translational Research Institute, Brisbane, Queensland, Australia
| | - Travis J. Klein
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
| | - Ross W. Crawford
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
| | - Michael R. Doran
- School of Biomedical Science, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology (QUT), Brisbane, Queensland, Australia
- Translational Research Institute, Brisbane, Queensland, Australia
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4
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Garrigós MM, de Oliveira FA, Nucci MP, Nucci LP, Alves ADH, Dias OFM, Gamarra LF. How mesenchymal stem cell cotransplantation with hematopoietic stem cells can improve engraftment in animal models. World J Stem Cells 2022; 14:658-679. [PMID: 36157912 PMCID: PMC9453272 DOI: 10.4252/wjsc.v14.i8.658] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/15/2022] [Revised: 04/27/2022] [Accepted: 07/26/2022] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Bone marrow transplantation (BMT) can be applied to both hematopoietic and nonhematopoietic diseases; nonetheless, it still comes with a number of challenges and limitations that contribute to treatment failure. Bearing this in mind, a possible way to increase the success rate of BMT would be cotransplantation of mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) to improve the bone marrow niche and secrete molecules that enhance the hematopoietic engraftment.
AIM To analyze HSC and MSC characteristics and their interactions through cotransplantation in murine models.
METHODS We searched for original articles indexed in PubMed and Scopus during the last decade that used HSC and MSC cotransplantation and in vivo BMT in animal models while evaluating cell engraftment. We excluded in vitro studies or studies that involved graft versus host disease or other hematological diseases and publications in languages other than English. In PubMed, we initially identified 555 articles and after selection, only 12 were chosen. In Scopus, 2010 were identified, and six were left after the screening and eligibility process.
RESULTS Of the 2565 articles found in the databases, only 18 original studies met the eligibility criteria. HSC distribution by source showed similar ratios, with human umbilical cord blood or animal bone marrow being administered mainly with a dose of 1 × 107 cells by intravenous or intrabone routes. However, MSCs had a high prevalence of human donors with a variety of sources (umbilical cord blood, bone marrow, tonsil, adipose tissue or fetal lung), using a lower dose, mainly 106 cells and ranging 104 to 1.5 × 107 cells, utilizing the same routes. MSCs were characterized prior to administration in almost every experiment. The recipient used was mostly immunodeficient mice submitted to low-dose irradiation or chemotherapy. The main technique of engraftment for HSC and MSC cotransplantation evaluation was chimerism, followed by hematopoietic reconstitution and survival analysis. Besides the engraftment, homing and cellularity were also evaluated in some studies.
CONCLUSION The preclinical findings validate the potential of MSCs to enable HSC engraftment in vivo in both xenogeneic and allogeneic hematopoietic cell transplantation animal models, in the absence of toxicity.
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Affiliation(s)
- Murilo Montenegro Garrigós
- Hospital Israelita Albert Einstein, São Paulo 05652-900, São Paulo, Brazil
- Instituto de Química, Universidade de São Paulo, São Paulo 05508-900, São Paulo, Brazil
| | | | - Mariana Penteado Nucci
- Hospital Israelita Albert Einstein, São Paulo 05652-900, São Paulo, Brazil
- LIM44-Hospital das Clínicas, Faculdade Medicina da Universidade de São Paulo, São Paulo 05403-000, Brazil
| | - Leopoldo Penteado Nucci
- Centro Universitário do Planalto Central, Área Especial para Industria nº 02 Setor Leste - Gama-DF, Brasília 72445-020, Distrito Federal, Brazil
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Nowlan B, Williams ED, Doran MR. Direct bone marrow injection of human bone marrow-derived stromal cells into mouse femurs results in greater prostate cancer PC-3 cell proliferation, but not specifically proliferation within the injected femurs. BMC Cancer 2022; 22:554. [PMID: 35581599 PMCID: PMC9112579 DOI: 10.1186/s12885-022-09430-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2021] [Accepted: 03/14/2022] [Indexed: 11/21/2022] Open
Abstract
Background While prostate cancer (PCa) cells most often metastasize to bone in men, species-specific differences between human and mouse bone marrow mean that this pattern is not faithfully replicated in mice. Herein we evaluated the impact of partially humanizing mouse bone marrow with human bone marrow-derived stromal cells (BMSC, also known as "mesenchymal stem cells") on human PCa cell behaviour. Methods BMSC are key cellular constituents of marrow. We used intrafemoral injection to transplant 5 × 105 luciferase (Luc) and green fluorescence protein (GFP) expressing human BMSC (hBMSC-Luc/GFP) into the right femur of non-obese diabetic (NOD)-severe combined immunodeficiency (scid) interleukin (IL)-2γ−/− (NSG) mice. Two weeks later, 2.5 × 106 PC-3 prostate cancer cells expressing DsRed (PC-3-DsRed) were delivered into the mice via intracardiac injection. PC-3-DsRed cells were tracked over time using an In Vivo Imaging System (IVIS) live animal imaging system, X-ray and IVIS imaging performed on harvested organs, and PC-3 cell numbers in femurs quantified using flow cytometry and histology. Results Flow cytometry analysis revealed greater PC-3-DsRed cell numbers within femurs of the mice that received hBMSC-Luc/GFP. However, while there were overall greater PC-3-DsRed cell numbers in these animals, there were not more PC-3-DsRed in the femurs injected with hBMSC-Luc/GFP than in contralateral femurs. A similar proportion of mice in with or without hBMSC-Luc/GFP had bone lessions, but the absolute number of bone lesions was greater in mice that had received hBMSC-Luc/GFP. Conclusion PC-3-DsRed cells preferentially populated bones in mice that had received hBMSC-Luc/GFP, although PC-3-DsRed cells not specifically localize in the bone marrow cavity where hBMSC-Luc/GFP had been transplanted. hBMSC-Luc/GFP appear to modify mouse biology in a manner that supports PC-3-DsRed tumor development, rather than specifically influencing PC-3-DsRed cell homing. This study provides useful insights into the role of humanizing murine bone marrow with hBMSC to study human PCa cell biology. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-09430-6.
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Affiliation(s)
- Bianca Nowlan
- School of Biomedical Science, Faculty of Health, Queensland University of Technology at the Translational Research Institute, Brisbane, Australia.,Australian Prostate Cancer Research Centre-Queensland, Brisbane, Australia
| | - Elizabeth D Williams
- School of Biomedical Science, Faculty of Health, Queensland University of Technology at the Translational Research Institute, Brisbane, Australia.,Australian Prostate Cancer Research Centre-Queensland, Brisbane, Australia
| | - Michael Robert Doran
- School of Biomedical Science, Faculty of Health, Queensland University of Technology at the Translational Research Institute, Brisbane, Australia. .,Australian Prostate Cancer Research Centre-Queensland, Brisbane, Australia. .,Centre for Biomedical Technologies, Queensland University of Technology, Brisbane, Australia. .,Mater Research Institute - University of Queensland, Brisbane, Australia. .,Skeletal Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, USA.
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6
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Felker S, Shrestha A, Bailey J, Pillis DM, Siniard D, Malik P. Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment. JCI Insight 2022; 7:151847. [PMID: 35531956 PMCID: PMC9090236 DOI: 10.1172/jci.insight.151847] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2021] [Accepted: 04/06/2022] [Indexed: 11/24/2022] Open
Abstract
Gene therapy involves a substantial loss of hematopoietic stem and progenitor cells (HSPC) during processing and homing. Intra-BM (i.b.m.) transplantation can reduce homing losses, but prior studies have not yielded promising results. We studied the mechanisms involved in homing and engraftment of i.b.m. transplanted and i.v. transplanted genetically modified (GM) human HSPC. We found that i.b.m. HSPC transplantation improved engraftment of hematopoietic progenitor cells (HPC) but not of long-term repopulating hematopoietic stem cells (HSC). Mechanistically, HPC expressed higher functional levels of CXCR4 than HSC, conferring them a retention and homing advantage when transplanted i.b.m. Removing HPC and transplanting an HSC-enriched population i.b.m. significantly increased long-term engraftment over i.v. transplantation. Transient upregulation of CXCR4 on GM HSC-enriched cells, using a noncytotoxic portion of viral protein R (VPR) fused to CXCR4 delivered as a protein in lentiviral particles, resulted in higher homing and long-term engraftment of GM HSC transplanted either i.v. or i.b.m. compared with standard i.v. transplants. Overall, we show a mechanism for why i.b.m. transplants do not significantly improve long-term engraftment over i.v. transplants. I.b.m. transplantation becomes relevant when an HSC-enriched population is delivered. Alternatively, CXCR4 expression on HSC, when transiently increased using a protein delivery method, improves homing and engraftment specifically of GM HSC.
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Affiliation(s)
- Sydney Felker
- Immunology Graduate Program, Cincinnati Children’s Hospital Medical Center (CCHMC) and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
- Division of Experimental Hematology and Cancer Biology and
| | | | - Jeff Bailey
- Division of Experimental Hematology and Cancer Biology and
| | - Devin M Pillis
- Division of Experimental Hematology and Cancer Biology and
| | - Dylan Siniard
- Division of Experimental Hematology and Cancer Biology and
| | - Punam Malik
- Immunology Graduate Program, Cincinnati Children’s Hospital Medical Center (CCHMC) and the University of Cincinnati College of Medicine, Cincinnati, Ohio, USA
- Division of Experimental Hematology and Cancer Biology and
- Division of Hematology, CCHMC, Cincinnati, Ohio, USA
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7
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Shajib MS, Futrega K, Jacob Klein T, Crawford RW, Doran MR. Collagenase treatment appears to improve cartilage tissue integration but damage to collagen networks is likely permanent. J Tissue Eng 2022; 13:20417314221074207. [PMID: 35096364 PMCID: PMC8793122 DOI: 10.1177/20417314221074207] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2021] [Accepted: 01/03/2022] [Indexed: 11/16/2022] Open
Abstract
When repairing cartilage defects a major challenge is achieving high-quality integration between the repair tissue and adjacent native cartilage. Matrix-rich cartilage is not easily remodeled, motivating several studies to trial enzyme treatment of the tissue interface to facilitate remodeling and integration. Studying and optimizing such processes is tedious, as well as potentially expensive, and thus simpler models are needed to evaluate the merits of enzyme treatment on cartilage tissue integration. Herein, we used engineered cartilage microtissues formed from bone marrow-derived stromal cells (BMSC) or expanded articular chondrocytes (ACh) to study the impact of enzyme treatment on cartilage tissue integration and matrix remodeling. A 5-min treatment with collagenase appeared to improve cartilage microtissue integration, while up to 48 h treatment with hyaluronidase did not. Alcian blue and anti-collagen II staining suggested that collagenase treatment did facilitate near seamless integration of cartilage microtissues. Microtissue sections were stained with Picrosirius red and characterized using polarized light microscopy, revealing that individual microtissues contained a collagen network organized in concentric shells. While collagenase treatment appeared to improve tissue integration, assessment of the collagen fibers with polarized light indicated that enzymatically damaged networks were not remodeled nor restored during subsequent culture. This model and these data paradoxically suggest that collagen network disruption is required to improve cartilage tissue integration, but that the disrupted collagen networks are unlikely to subsequently be restored. Future studies should attempt to limit collagen network disruption to the surface of the cartilage, and we recommend using Picrosirius red staining and polarized light to assess the quality of matrix remodeling and integration.
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Affiliation(s)
- Md. Shafiullah Shajib
- School of Biomedical Science, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology, Brisbane, QLD, Australia
- Translational Research Institute, Brisbane, QLD, Australia
| | - Kathryn Futrega
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology, Brisbane, QLD, Australia
- Translational Research Institute, Brisbane, QLD, Australia
- National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA
| | - Travis Jacob Klein
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology, Brisbane, QLD, Australia
| | - Ross W Crawford
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology, Brisbane, QLD, Australia
| | - Michael Robert Doran
- School of Biomedical Science, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia
- Centre for Biomedical Technologies, School of Mechanical, Medical, and Process Engineering, Faculty of Engineering, Queensland University of Technology, Brisbane, QLD, Australia
- Translational Research Institute, Brisbane, QLD, Australia
- National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA
- Mater Research Institute – University of Queensland, Brisbane, QLD, Australia
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8
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Devaraju N, Rajendiran V, Ravi NS, Mohankumar KM. Genome Engineering of Hematopoietic Stem Cells Using CRISPR/Cas9 System. Methods Mol Biol 2022; 2429:307-331. [PMID: 35507170 DOI: 10.1007/978-1-0716-1979-7_20] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Ex vivo genetic manipulation of autologous hematopoietic stem and progenitor cells (HSPCs) is a viable strategy for the treatment of hematologic and primary immune disorders. Targeted genome editing of HSPCs using the CRISPR-Cas9 system provides an effective platform to edit the desired genomic locus for therapeutic purposes with minimal off-target effects. In this chapter, we describe the detailed methodology for the CRISPR-Cas9 mediated gene knockout, deletion, addition, and correction in human HSPCs by viral and nonviral approaches. We also present a comprehensive protocol for the analysis of genome modified HSPCs toward the erythroid and megakaryocyte lineage in vitro and the long-term multilineage reconstitution capacity in the recently developed NBSGW mouse model that supports human erythropoiesis.
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Affiliation(s)
- Nivedhitha Devaraju
- Centre for Stem Cell Research (a unit of inStem, Bangalore), Christian Medical College Campus, Bagayam, Vellore, Tamil Nadu, India
- Manipal Academy of Higher Education, Mangalore, Karnataka, India
| | - Vignesh Rajendiran
- Centre for Stem Cell Research (a unit of inStem, Bangalore), Christian Medical College Campus, Bagayam, Vellore, Tamil Nadu, India
| | - Nithin Sam Ravi
- Centre for Stem Cell Research (a unit of inStem, Bangalore), Christian Medical College Campus, Bagayam, Vellore, Tamil Nadu, India
| | - Kumarasamypet M Mohankumar
- Centre for Stem Cell Research (a unit of inStem, Bangalore), Christian Medical College Campus, Bagayam, Vellore, Tamil Nadu, India.
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Bedel A, Boutin J, Amintas S, Lamrissi-Garcia I, Rousseau B, Poglio S, Brunet de la Grange P, Moranvillier I, Blouin JM, Richard E, Moreau-Gaudry F, Dabernat S. Spleen route accelerates engraftment of human hematopoietic stem cells. Biochem Biophys Res Commun 2021; 569:23-28. [PMID: 34216994 DOI: 10.1016/j.bbrc.2021.06.054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Revised: 06/11/2021] [Accepted: 06/16/2021] [Indexed: 10/21/2022]
Abstract
Intravenous injections of human hematopoietic stem cells (hHSCs) is routinely used in clinic and for modeling hematopoiesis in mice. However, unspecific dilution in vascular system and non-hematopoietic organs challenges engraftment efficiency. Although spleen is capable of extra medullar hematopoiesis, its ability to support human HSC transplantation has never been evaluated. We demonstrate that intra-splenic injection results in high and sustained engraftment of hHSCs into immune-deficient mice, with higher chimerisms than with intravenous or intra-femoral injections. Our results support that spleen microenvironment provides a niche for HSCs amplification and offers a new route for efficient HSC transplantation.
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Affiliation(s)
- A Bedel
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France; CHU de Bordeaux, Bordeaux, France
| | - J Boutin
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France; CHU de Bordeaux, Bordeaux, France
| | - S Amintas
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France; CHU de Bordeaux, Bordeaux, France
| | - I Lamrissi-Garcia
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France
| | - B Rousseau
- Université de Bordeaux, Bordeaux, France; Animalerie spécialisée, Université de Bordeaux, Bordeaux, France
| | - S Poglio
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France
| | - P Brunet de la Grange
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France; Etablissement Français du Sang-Aquitaine Limousin (EFS-AqLi), Bordeaux, France
| | - I Moranvillier
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France
| | - J M Blouin
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France; CHU de Bordeaux, Bordeaux, France
| | - E Richard
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France; CHU de Bordeaux, Bordeaux, France
| | - F Moreau-Gaudry
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France; CHU de Bordeaux, Bordeaux, France.
| | - S Dabernat
- Université de Bordeaux, Bordeaux, France; INSERM U1035, Bordeaux, France; CHU de Bordeaux, Bordeaux, France
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10
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Nowlan B, Futrega K, Williams ED, Doran MR. Human bone marrow-derived stromal cell behavior when injected directly into the bone marrow of NOD-scid-gamma mice pre-conditioned with sub-lethal irradiation. Stem Cell Res Ther 2021; 12:231. [PMID: 33845908 PMCID: PMC8042930 DOI: 10.1186/s13287-021-02297-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 03/18/2021] [Indexed: 11/10/2022] Open
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.
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Affiliation(s)
- Bianca Nowlan
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia.,Australian Prostate Cancer Research Centre - Queensland (APCCRC-Q) and Queensland Bladder Cancer Initiative (QBCI), Brisbane, Queensland, Australia.,Translational Research Institute, 37 Kent Street, Brisbane, Queensland, 4102, Australia
| | - Kathryn Futrega
- Translational Research Institute, 37 Kent Street, Brisbane, Queensland, 4102, Australia.,Centre for Biomedical Technologies (CBT) and School of Mechanical, Medical, and Process Engineering (MMPE), Queensland University of Technology (QUT), Brisbane, Australia.,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
| | - Elizabeth Deborah Williams
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia.,Australian Prostate Cancer Research Centre - Queensland (APCCRC-Q) and Queensland Bladder Cancer Initiative (QBCI), Brisbane, Queensland, Australia.,Translational Research Institute, 37 Kent Street, Brisbane, Queensland, 4102, Australia
| | - Michael Robert Doran
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology (QUT), Brisbane, Australia. .,Australian Prostate Cancer Research Centre - Queensland (APCCRC-Q) and Queensland Bladder Cancer Initiative (QBCI), Brisbane, Queensland, Australia. .,Translational Research Institute, 37 Kent Street, Brisbane, Queensland, 4102, Australia. .,Centre for Biomedical Technologies (CBT) and School of Mechanical, Medical, and Process Engineering (MMPE), Queensland University of Technology (QUT), Brisbane, Australia. .,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. .,Mater Research Institute - University of Queensland, Brisbane, Australia. .,Australian National Centre for the Public Awareness of Science, Australian National University, Canberra, Australia.
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11
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Monterosso ME, Futrega K, Lott WB, Vela I, Williams ED, Doran MR. Using the Microwell-mesh to culture microtissues in vitro and as a carrier to implant microtissues in vivo into mice. Sci Rep 2021; 11:5118. [PMID: 33664329 PMCID: PMC7933425 DOI: 10.1038/s41598-021-84154-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Accepted: 02/03/2021] [Indexed: 11/09/2022] Open
Abstract
Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of "pieces" of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ-/- (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 μm achieving superior microtissue vascularisation relative to mesh with 36×36 μm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.
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Affiliation(s)
- Melissa E Monterosso
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia.,Translational Research Institute, Brisbane, Australia
| | - Kathryn Futrega
- Translational Research Institute, Brisbane, Australia.,Centre for Biomedical Technologies (CBT), School of Mechanical, Medical, and Process Engineering (MMPE), Science and Engineering Faculty (SEF), Queensland University of Technology, Brisbane, Australia
| | - William B Lott
- Translational Research Institute, Brisbane, Australia.,Centre for Biomedical Technologies (CBT), School of Mechanical, Medical, and Process Engineering (MMPE), Science and Engineering Faculty (SEF), Queensland University of Technology, Brisbane, Australia
| | - Ian Vela
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia.,Translational Research Institute, Brisbane, Australia.,Australian Prostate Cancer Research Centre - Queensland (APCRC-Q) and Queensland Bladder Cancer initiative (QBCI), Brisbane, Australia.,Department of Urology, Princess Alexandra Hospital, Brisbane, Australia
| | - Elizabeth D Williams
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia.,Translational Research Institute, Brisbane, Australia.,Australian Prostate Cancer Research Centre - Queensland (APCRC-Q) and Queensland Bladder Cancer initiative (QBCI), Brisbane, Australia
| | - Michael R Doran
- School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, Australia. .,Translational Research Institute, Brisbane, Australia. .,Centre for Biomedical Technologies (CBT), School of Mechanical, Medical, and Process Engineering (MMPE), Science and Engineering Faculty (SEF), Queensland University of Technology, Brisbane, Australia. .,Australian Prostate Cancer Research Centre - Queensland (APCRC-Q) and Queensland Bladder Cancer initiative (QBCI), Brisbane, Australia. .,Mater Research Institute - University of Queensland (UQ), Translational Research Institute (TRI), Brisbane, Australia.
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12
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A human SIRPA knock-in xenograft mouse model to study human hematopoietic and cancer stem cells. Blood 2020; 135:1661-1672. [PMID: 32206775 DOI: 10.1182/blood.2019002194] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2019] [Accepted: 02/20/2020] [Indexed: 12/15/2022] Open
Abstract
In human-to-mouse xenogeneic transplantation, polymorphisms of signal-regulatory protein α (SIRPA) that decide their binding affinity for human CD47 are critical for engraftment efficiency of human cells. In this study, we generated a new C57BL/6.Rag2nullIl2rgnull (BRG) mouse line with Sirpahuman/human (BRGShuman) mice, in which mouse Sirpa was replaced by human SIRPA encompassing all 8 exons. Macrophages from C57BL/6 mice harboring Sirpahuman/human had a significantly stronger affinity for human CD47 than those harboring SirpaNOD/NOD and did not show detectable phagocytosis against human hematopoietic stem cells. In turn, Sirpahuman/human macrophages had a moderate affinity for mouse CD47, and BRGShuman mice did not exhibit the blood cytopenia that was seen in Sirpa-/- mice. In human to mouse xenograft experiments, BRGShuman mice showed significantly greater engraftment and maintenance of human hematopoiesis with a high level of myeloid reconstitution, as well as improved reconstitution in peripheral tissues, compared with BRG mice harboring SirpaNOD/NOD (BRGSNOD). BRGShuman mice also showed significantly enhanced engraftment and growth of acute myeloid leukemia and subcutaneously transplanted human colon cancer cells compared with BRGSNOD mice. BRGShuman mice should be a useful basic line for establishing a more authentic xenotransplantation model to study normal and malignant human stem cells.
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13
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Preciado S, Muntión S, Corchete LA, Ramos TL, de la Torre AG, Osugui L, Rico A, Espinosa-Lara N, Gastaca I, Díez-Campelo M, Del Cañizo C, Sánchez-Guijo F. The Incorporation of Extracellular Vesicles from Mesenchymal Stromal Cells Into CD34 + Cells Increases Their Clonogenic Capacity and Bone Marrow Lodging Ability. Stem Cells 2019; 37:1357-1368. [PMID: 31184411 PMCID: PMC6852558 DOI: 10.1002/stem.3032] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2018] [Revised: 04/11/2019] [Accepted: 04/20/2019] [Indexed: 12/22/2022]
Abstract
Mesenchymal stromal cells (MSC) may exert their functions by the release of extracellular vesicles (EV). Our aim was to analyze changes induced in CD34+ cells after the incorporation of MSC‐EV. MSC‐EV were characterized by flow cytometry (FC), Western blot, electron microscopy, and nanoparticle tracking analysis. EV incorporation into CD34+ cells was confirmed by FC and confocal microscopy, and then reverse transcription polymerase chain reaction and arrays were performed in modified CD34+ cells. Apoptosis and cell cycle were also evaluated by FC, phosphorylation of signal activator of transcription 5 (STAT5) by WES Simple, and clonal growth by clonogenic assays. Human engraftment was analyzed 4 weeks after CD34+ cell transplantation in nonobese diabetic/severe combined immunodeficient mice. Our results showed that MSC‐EV incorporation induced a downregulation of proapoptotic genes, an overexpression of genes involved in colony formation, and an activation of the Janus kinase (JAK)‐STAT pathway in CD34+ cells. A significant decrease in apoptosis and an increased CD44 expression were confirmed by FC, and increased levels of phospho‐STAT5 were confirmed by WES Simple in CD34+ cells with MSC‐EV. In addition, these cells displayed a higher colony‐forming unit granulocyte/macrophage clonogenic potential. Finally, the in vivo bone marrow lodging ability of human CD34+ cells with MSC‐EV was significantly increased in the injected femurs. In summary, the incorporation of MSC‐EV induces genomic and functional changes in CD34+ cells, increasing their clonogenic capacity and their bone marrow lodging ability. stem cells2019;37:1357–1368
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Affiliation(s)
- Silvia Preciado
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Salamanca, Spain.,Department of Medicine, Universidad de Salamanca, Salamanca, Spain.,RETIC TerCel, ISCIII, Salamanca, Spain
| | - Sandra Muntión
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Salamanca, Spain.,RETIC TerCel, ISCIII, Salamanca, Spain
| | - Luis A Corchete
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain
| | - Teresa L Ramos
- RETIC TerCel, ISCIII, Salamanca, Spain.,Laboratorio de Terapia Celular, Instituto de Biomedicina de Sevilla (IBIS), UGC-Hematología, Hospital Universitario Virgen del Rocío/CSIC/CIBERONC, Sevilla, Spain
| | - Ana G de la Torre
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain.,Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain
| | - Lika Osugui
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain
| | - Ana Rico
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Salamanca, Spain
| | - Natalia Espinosa-Lara
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Salamanca, Spain
| | - Irene Gastaca
- Servicio de Ginecología, Hospital Universitario de Salamanca, Salamanca, Spain
| | - María Díez-Campelo
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain.,Department of Medicine, Universidad de Salamanca, Salamanca, Spain.,RETIC TerCel, ISCIII, Salamanca, Spain
| | - Consuelo Del Cañizo
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Salamanca, Spain.,Department of Medicine, Universidad de Salamanca, Salamanca, Spain.,RETIC TerCel, ISCIII, Salamanca, Spain.,Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain
| | - Fermín Sánchez-Guijo
- Servicio de Hematología, IBSAL-Hospital Universitario de Salamanca, Salamanca, Spain.,Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Salamanca, Spain.,Department of Medicine, Universidad de Salamanca, Salamanca, Spain.,RETIC TerCel, ISCIII, Salamanca, Spain.,Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain
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14
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Charlesworth CT, Camarena J, Cromer MK, Vaidyanathan S, Bak RO, Carte JM, Potter J, Dever DP, Porteus MH. Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting. MOLECULAR THERAPY. NUCLEIC ACIDS 2018; 12:89-104. [PMID: 30195800 PMCID: PMC6023838 DOI: 10.1016/j.omtn.2018.04.017] [Citation(s) in RCA: 79] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 03/11/2018] [Revised: 04/27/2018] [Accepted: 04/28/2018] [Indexed: 12/11/2022]
Abstract
Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.
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Affiliation(s)
| | - Joab Camarena
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - M Kyle Cromer
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | | | - Rasmus O Bak
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA
| | - Jason M Carte
- Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA
| | - Jason Potter
- Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008, USA
| | - Daniel P Dever
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
| | - Matthew H Porteus
- Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
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15
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Futrega K, Mosaad E, Chambers K, Lott WB, Clements J, Doran MR. Bone marrow-derived stem/stromal cells (BMSC) 3D microtissues cultured in BMP-2 supplemented osteogenic induction medium are prone to adipogenesis. Cell Tissue Res 2018; 374:541-553. [PMID: 30136155 PMCID: PMC6267724 DOI: 10.1007/s00441-018-2894-y] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2017] [Accepted: 07/18/2018] [Indexed: 12/11/2022]
Abstract
Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.
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Affiliation(s)
- K Futrega
- Stem Cell Therapies Laboratory, Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI), Translational Research Institute (TRI), Brisbane, Australia.,Science and Engineering Faculty (SEF), Translational Research Institute (TRI), Brisbane, Australia
| | - E Mosaad
- Stem Cell Therapies Laboratory, Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI), Translational Research Institute (TRI), Brisbane, Australia.,Australian Prostate Cancer Research Centre - Queensland (APCRC-Q), Institute of Health and Biomedical Innovation (IHBI) & School of Biomedical Sciences, Queensland University of Technology (QUT), Translational Research Institute (TRI), Brisbane, Australia.,Biochemistry Division, Chemistry Department, Faculty of Science, Damietta University, Damietta, Egypt
| | - K Chambers
- Quadram Institute Bioscience, Norwich Research Park, Norwich, UK
| | - W B Lott
- Stem Cell Therapies Laboratory, Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI), Translational Research Institute (TRI), Brisbane, Australia.,Science and Engineering Faculty (SEF), Translational Research Institute (TRI), Brisbane, Australia
| | - J Clements
- Science and Engineering Faculty (SEF), Translational Research Institute (TRI), Brisbane, Australia
| | - M R Doran
- Stem Cell Therapies Laboratory, Queensland University of Technology (QUT), Institute of Health and Biomedical Innovation (IHBI), Translational Research Institute (TRI), Brisbane, Australia. .,Australian Prostate Cancer Research Centre - Queensland (APCRC-Q), Institute of Health and Biomedical Innovation (IHBI) & School of Biomedical Sciences, Queensland University of Technology (QUT), Translational Research Institute (TRI), Brisbane, Australia. .,Mater Research Institute - University of Queensland (UQ), Translational Research Institute (TRI), Brisbane, Australia. .,Australian National Centre for the Public Awareness of Science, Australian National University (ANU), Canberra, Australia.
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16
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Early assessment of dosimetric and biological differences of total marrow irradiation versus total body irradiation in rodents. Radiother Oncol 2017; 124:468-474. [PMID: 28778346 DOI: 10.1016/j.radonc.2017.07.018] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2017] [Revised: 07/15/2017] [Accepted: 07/15/2017] [Indexed: 11/22/2022]
Abstract
PURPOSE To develop a murine total marrow irradiation (TMI) model in comparison with the total body irradiation (TBI) model. MATERIALS AND METHODS Myeloablative TMI and TBI were administered in mice using a custom jig, and the dosimetric differences between TBI and TMI were evaluated. The early effects of TBI/TMI on bone marrow (BM) and organs were evaluated using histology, FDG-PET, and cytokine production. TMI and TBI with and without cyclophosphamide (Cy) were evaluated for donor cell engraftment and tissue damage early after allogeneic hematopoietic cell transplantation (HCT). Stromal derived factor-1 (SDF-1) expression was evaluated. RESULTS TMI resulted in similar dose exposure to bone and 50% reduction in dose to bystander organs. BM histology was similar between the groups. In the non-HCT model, TMI mice had significantly less acute intestinal and lung injury compared to TBI. In the HCT model, recipients of TMI had significantly less acute intestinal injury and spleen GVHD, but increased early donor cell engraftment and BM:organ SDF-1 ratio compared to TBI recipients. CONCLUSIONS The expected BM damage was similar in both models, but the damage to other normal tissues was reduced by TMI. However, BM engraftment was improved in the TMI group compared to TBI, which may be due to enhanced production of SDF-1 in BM relative to other organs after TMI.
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17
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Futrega K, Atkinson K, Lott WB, Doran MR. Spheroid Coculture of Hematopoietic Stem/Progenitor Cells and Monolayer Expanded Mesenchymal Stem/Stromal Cells in Polydimethylsiloxane Microwells Modestly Improves In Vitro Hematopoietic Stem/Progenitor Cell Expansion. Tissue Eng Part C Methods 2017; 23:200-218. [PMID: 28406754 PMCID: PMC5397247 DOI: 10.1089/ten.tec.2016.0329] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
While two-dimensional (2D) monolayers of mesenchymal stem/stromal cells (MSCs) have been shown to enhance hematopoietic stem/progenitor cell (HSPC) expansion in vitro, expanded cells do not engraft long term in human recipients. This outcome is attributed to the failure of 2D culture to recapitulate the bone marrow (BM) niche signal milieu. Herein, we evaluated the capacity of a novel three-dimensional (3D) coculture system to support HSPC expansion in vitro. A high-throughput polydimethylsiloxane (PDMS) microwell platform was used to manufacture thousands of uniform 3D multicellular coculture spheroids. Relative gene expression in 3D spheroid versus 2D adherent BM-derived MSC cultures was characterized and compared with literature reports. We evaluated coculture spheroids, each containing 25-400 MSCs and 10 umbilical cord blood (CB)-derived CD34+ progenitor cells. At low exogenous cytokine concentrations, 2D and 3D MSC coculture modestly improved overall hematopoietic cell and CD34+ cell expansion outcomes. By contrast, a substantial increase in CD34+CD38- cell yield was observed in PDMS microwell cultures, regardless of the presence or absence of MSCs. This outcome indicated that CD34+CD38- cell culture yield could be increased using the microwell platform alone, even without MSC coculture support. We found that the increase in CD34+CD38- cell yield observed in PDMS microwell cultures did not translate to enhanced engraftment in NOD/SCID gamma (NSG) mice or a modification in the relative human hematopoietic lineages established in engrafted mice. In summary, there was no statistical difference in CD34+ cell yield from 2D or 3D cocultures, and MSC coculture support provided only modest benefit in either geometry. While the high-throughput 3D microwell platform may provide a useful model system for studying cells in coculture, further optimization will be required to generate HSPC yields suitable for use in clinical applications.
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Affiliation(s)
- Kathryn Futrega
- 1 Stem Cell Therapies Laboratory, Translational Research Institute, Queensland University of Technology , Brisbane, Australia
| | - Kerry Atkinson
- 1 Stem Cell Therapies Laboratory, Translational Research Institute, Queensland University of Technology , Brisbane, Australia
| | - William B Lott
- 1 Stem Cell Therapies Laboratory, Translational Research Institute, Queensland University of Technology , Brisbane, Australia
| | - Michael R Doran
- 1 Stem Cell Therapies Laboratory, Translational Research Institute, Queensland University of Technology , Brisbane, Australia .,2 Mater Research Institute - University of Queensland, Translational Research Institute , Brisbane, Australia
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18
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Nowlan B, Futrega K, Brunck ME, Walkinshaw G, Flippin LE, Doran MR, Levesque JP. HIF-1α-stabilizing agent FG-4497 rescues human CD34 + cell mobilization in response to G-CSF in immunodeficient mice. Exp Hematol 2017; 52:50-55.e6. [PMID: 28527810 DOI: 10.1016/j.exphem.2017.05.004] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2017] [Revised: 05/09/2017] [Accepted: 05/10/2017] [Indexed: 12/13/2022]
Abstract
Granulocyte colony-stimulating factor (G-CSF) is used routinely in the clinical setting to mobilize hematopoietic stem progenitor cells (HSPCs) into the patient's blood for collection and subsequent transplantation. However, a significant proportion of patients who have previously received chemotherapy or radiotherapy and require autologous HSPC transplantation cannot mobilize the minimal threshold of mobilized HSPCs to achieve rapid and successful hematopoietic reconstitution. Although several alternatives to the G-CSF regime have been tested, few are used in the clinical setting. We have shown previously in mice that administration of prolyl 4-hydroxylase domain enzyme (PHD) inhibitors, which stabilize hypoxia-inducible factor (HIF)-1α, synergize with G-CSF in vivo to enhance mouse HSPC mobilization into blood, leading to enhanced engraftment via an HSPC-intrinsic mechanism. To evaluate whether PHD inhibitors could be used to enhance mobilization of human HSPCs, we humanized nonobese, diabetic severe combined immune-deficient Il2rg-/- mice by transplanting them with human umbilical cord blood CD34+ HSPCs and then treating them with G-CSF with and without co-administration of the PHD inhibitor FG-4497. We observed that combination treatment with G-CSF and FG-4497 resulted in significant mobilization of human lineage-negative (Lin-) CD34+ HSPCs and more primitive human Lin-CD34+CD38- HSPCs into blood and spleen, whereas mice treated with G-CSF alone did not mobilize human HSPCs significantly. These results suggest that the PHD inhibitor FG-4497 also increases human HSPC mobilization in a xenograft mouse model, suggesting the possibility of testing PHD inhibitors to boost HSPC mobilization in response to G-CSF in humans.
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Affiliation(s)
- Bianca Nowlan
- Stem Cell Therapies Laboratory, Translational Research Institute, Queensland University of Technology, Woolloongabba, Queensland, Australia; Mater Research Institute, Translational Research Institute, University of Queensland, Woolloongabba, Queensland, Australia
| | - Kathryn Futrega
- Stem Cell Therapies Laboratory, Translational Research Institute, Queensland University of Technology, Woolloongabba, Queensland, Australia
| | - Marion E Brunck
- Mater Research Institute, Translational Research Institute, University of Queensland, Woolloongabba, Queensland, Australia
| | | | | | - Michael R Doran
- Stem Cell Therapies Laboratory, Translational Research Institute, Queensland University of Technology, Woolloongabba, Queensland, Australia; Mater Research Institute, Translational Research Institute, University of Queensland, Woolloongabba, Queensland, Australia; Australian National Centre for the Public Awareness of Science - Australian National University, Australia.
| | - Jean-Pierre Levesque
- Mater Research Institute, Translational Research Institute, University of Queensland, Woolloongabba, Queensland, Australia.
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19
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Zhang XY, Zhang PY. Stem cell transplantation during cancer. Oncol Lett 2017; 12:4297-4300. [PMID: 28105145 PMCID: PMC5228504 DOI: 10.3892/ol.2016.5260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Accepted: 09/27/2016] [Indexed: 11/05/2022] Open
Abstract
Hematological malignancies account for approximately 9.5% of new cancers diagnosed annually. Lymphoma is the most frequent of all known categories of hematological malignancies. Worldwide, extensive research has focused on this type of cancer. However, new treatments are investigated in various clinical as well as pre-clinical studies. Hematopoietic stem cell transplantation (HSCT) is a recent and upcoming treatment strategy for patients with hematopoietic malignancies and inborn errors of metabolism or immune deficiencies. Recent studies have revealed that successful clinical outcome of this treatment strategy depends on multiple factors including the protocol applied, disease under treatment, health of the patient, source of the grafts, severity of complications such as graft versus host disease during grafting and associated infections. The scope of this review is to achieve greater understanding of various clinical effects of the disease and related mechanisms. The electronic database Pubmed was searched for pre-clinical as well as clinical controlled trials reporting efficacy of the HSCT against hematological malignancies.
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Affiliation(s)
- Xiao-Ying Zhang
- Nanjing University of Chinese Medicine, Information Institute, Nanjing, Jiangsu 221009, P.R. China
| | - Pei-Ying Zhang
- Department of Cardiology, Xuzhou Central Hospital, The Affiliated Xuzhou Hospital of Medical College of Southeast University, Xuzhou, Jiangsu 221009, P.R. China
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20
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In vitro and in vivo assessment of direct effects of simulated solar and galactic cosmic radiation on human hematopoietic stem/progenitor cells. Leukemia 2016; 31:1398-1407. [PMID: 27881872 DOI: 10.1038/leu.2016.344] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2016] [Revised: 10/10/2016] [Accepted: 10/21/2016] [Indexed: 12/13/2022]
Abstract
Future deep space missions to Mars and near-Earth asteroids will expose astronauts to chronic solar energetic particles (SEP) and galactic cosmic ray (GCR) radiation, and likely one or more solar particle events (SPEs). Given the inherent radiosensitivity of hematopoietic cells and short latency period of leukemias, space radiation-induced hematopoietic damage poses a particular threat to astronauts on extended missions. We show that exposing human hematopoietic stem/progenitor cells (HSC) to extended mission-relevant doses of accelerated high-energy protons and iron ions leads to the following: (1) introduces mutations that are frequently located within genes involved in hematopoiesis and are distinct from those induced by γ-radiation; (2) markedly reduces in vitro colony formation; (3) markedly alters engraftment and lineage commitment in vivo; and (4) leads to the development, in vivo, of what appears to be T-ALL. Sequential exposure to protons and iron ions (as typically occurs in deep space) proved far more deleterious to HSC genome integrity and function than either particle species alone. Our results represent a critical step for more accurately estimating risks to the human hematopoietic system from space radiation, identifying and better defining molecular mechanisms by which space radiation impairs hematopoiesis and induces leukemogenesis, as well as for developing appropriately targeted countermeasures.
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Verbiest T, Finnon R, Brown N, Finnon P, Bouffler S, Badie C. NOD Scid Gamma Mice Are Permissive to Allogeneic HSC Transplantation without Prior Conditioning. Int J Mol Sci 2016; 17:E1850. [PMID: 27827995 PMCID: PMC5133850 DOI: 10.3390/ijms17111850] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2016] [Revised: 10/14/2016] [Accepted: 11/02/2016] [Indexed: 11/16/2022] Open
Abstract
Scid hematopoietic stem cells (HSCs) have an intrinsic defect in their maintenance within the bone marrow (BM) niche which facilitates HSC transplantation without the absolute requirement of prior conditioning. Nevertheless, NOD scid mice have a significantly altered life span due to early development of thymic lymphomas, which compromises the ability to study the long-term fate of exogenous HSCs and their progeny. Here, we present data on the transplantation of HSCs into NOD scid gamma (NSG) mice to achieve long-term engraftment without prior conditioning. We transplanted allogeneic HSCs constitutively expressing the mCherry fluorescent marker into age-matched NSG mice and assessed donor chimerism 6 months post-transplantation. All transplanted NSG mice showed long-term myeloid and lymphoid cell chimerism. Also, in vivo irradiated HSCs showed long-term engraftment, although overall white blood cell (WBC) donor chimerism was lower compared with non-irradiated HSCs. Using this novel NSG transplantation model, we will be able to study the effects of low dose in vivo X-ray exposure on the long-term fate of HSCs, without the requirement of prior radio-ablation of the recipient, and thus leaving the recipient's BM microenvironment uncompromised. In conclusion, we demonstrated for the first time that allogeneic HSCs from a different inbred strain can compete for niches in the BM compartment of NSG mice.
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Affiliation(s)
- Tom Verbiest
- Cancer Mechanisms and Biomarkers Group, Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Didcot OX11 ORQ, UK.
- CRUK & MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK.
| | - Rosemary Finnon
- Cancer Mechanisms and Biomarkers Group, Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Didcot OX11 ORQ, UK.
| | - Natalie Brown
- Cancer Mechanisms and Biomarkers Group, Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Didcot OX11 ORQ, UK.
| | - Paul Finnon
- Cancer Mechanisms and Biomarkers Group, Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Didcot OX11 ORQ, UK.
| | - Simon Bouffler
- Cancer Mechanisms and Biomarkers Group, Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Didcot OX11 ORQ, UK.
| | - Christophe Badie
- Cancer Mechanisms and Biomarkers Group, Radiation Effects Department, Centre for Radiation, Chemical and Environmental Hazards, Public Health England, Didcot OX11 ORQ, UK.
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