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Enrichment of Skeletal Stem Cells from Human Bone Marrow bioRxiv preprint doi: https://doi.org/10.1101/2019.12.19.882563; this version posted January 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Enrichment of Skeletal Stem Cells from Human 2 Bone Marrow Using Spherical Nucleic Acids 3 Miguel Xavier,a Maria-Eleni Kyriazi,b Stuart A. Lanham,a Konstantina Alexaki,b Afaf H. El- 4 Sagheer,d,e Tom Brown,d Antonios G. Kanarasb,c,* and Richard O.C. Oreffo.a,c,* 5 a Bone and Joint Research Group, Centre for Human Development, Stem Cells and 6 Regeneration, Human Development and Health, Institute of Developmental Sciences, Faculty of 7 Medicine, University of Southampton, Southampton, SO16 6YD, UK. 8 b School of Physics and Astronomy, Faculty of Engineering and Physical Sciences, University of 9 Southampton, Southampton SO17 1BJ, UK. 10 c Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, UK. 11 d Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield 12 Road, Oxford OX1 3TA, UK. 13 e Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining 14 Engineering, Suez University, Suez 43721, Egypt. 15 16 KEYWORDS. Gold Nanoparticles, DNA, Human Bone Marrow, mRNA, spherical nucleic acid, 17 skeletal stem cell 18 19 1 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.19.882563; this version posted January 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 20 ABSTRACT. Human bone marrow (BM) derived stromal cells contain a population of skeletal 21 stem cells (SSCs), with the capacity to differentiate along the osteogenic, adipogenic and 22 chondrogenic lineages enabling their application to clinical therapies. However, current methods, 23 to isolate and enrich SSCs from human tissues remain, at best, challenging in the absence of a 24 specific SSC marker. Unfortunately, none of the current proposed markers, alone, can isolate a 25 homogenous cell population with the ability to form bone, cartilage, and adipose tissue in 26 humans. Here, we have designed DNA-gold nanoparticles able to identify and sort SSCs 27 displaying specific mRNA signatures. The current approach demonstrates the significant 28 enrichment attained in the isolation of SSCs, with potential therein to enhance our understanding 29 of bone cell biology and translational applications. 30 INTRODUCTION 31 In the last decade, progress in colloid chemistry has enabled the synthesis of advanced, 32 functional nanoparticles (NPs) that can survive complex biological media and perform 33 programmed tasks including cell targeting, sensing, and drug release.1, 2 Mirkin and co-workers 34 were the first to demonstrate how the surface of gold NPs could be functionalised with a shell of 35 synthetic oligonucleotides attached to the gold surface via a thiol bond.3 This new design of 36 oligonucleotide – coated NPs, also termed spherical nucleic acids (SNAs) have subsequently 37 emerged as live cell probes for the detection of targets including RNA, molecules and ions.4-12 38 The extensive use of SNAs within the biomedical field is primarily due to their high specificity, 39 binding of complementary oligonucleotide strands and improved resistance towards DNA 40 enzymatic degradation as well as the enhanced uptake of SNAs by multiple types of cells.13, 14 41 Seferos et al. were the first to show how SNAs could be hybridised to shorter fluorophore 42 bearing oligonucleotides for the targeted detection of mRNA. The authors demonstrated the 2 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.19.882563; this version posted January 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 43 detection of survivin mRNA, where a significant increase in fluorescence intensity was recorded 44 compared to treatment with non – targeting NPs.15 Following this work, several studies have 45 demonstrated the use of SNAs for the detection of mRNA targets with high accuracy and 46 specificity. Lahm et al. demonstrated the use of SNAs for the detection of nanog and gdf3 47 mRNA in embryonic stem cells as well as induced pluripotent stem (iPS) cells of murine, 48 porcine and human origin. Target positive cells were sorted after which nanog – specific probes 49 identified reprogrammed murine iPS cells.16 The detection of nanog mRNA was also taken 50 advantage of by Owen et al. for the detection and isolation of cancer stem cells.17 In contrast, 51 Wang et al. developed SNAs to enable self – activating and imaging of mRNA sequential 52 expression (tubb3 and fox 3 mRNA) during the neural stem cell differentiation process.18 53 Recently, we have demonstrated that SNAs can be used to detect vimentin mRNA in skin 54 wounds as vimentin mRNA is expressed during the epithelial to mesenchymal transition that 55 occurs during wound healing.19 In addition, we have designed SNAs to detect hymyc1 mRNA, 56 responsible for the regulation of the balance between stem cell self – renewal and differentiation, 57 in real time in live Hydra Vulgaris.20 We have also shown the development of SNAs and dimer 58 SNAs, which can detect up to two different mRNA targets whilst coordinating the release of two 59 different types of drugs within the same local microenvironment.21, 22 60 Recently, the SNA design has evolved to include mRNA detection directed by an external 61 stimulus. Lin et al. demonstrated the use of such a strategy for the specific detection of 62 manganese superoxide dismutase (MnSOD) mRNA. Following application of oligonucleotides 63 modified with a photoactivated linker, mRNA detection was present only in select cells at a 64 desired time point via the use of two-photon illumination. By illuminating specific cells of 3 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.19.882563; this version posted January 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 65 interest, mRNA detection was visualized only in these specific cells, while neighboring cells 66 remained fluorescently dormant.6 67 The design of SNAs has been further exploited for the detection of other targets including 68 miRNAs and, recently, for the delivery of oligonucleotides or peptides to activate the immune 69 system.5, 23-28 Zhai et al. reported the detection of microRNA – 1246, a breast cancer biomarker, 70 in human plasma exosomes, whereas, Zhao et al. used the same design for the direct detection of 71 microRNA – 375.29 In contrast, Ferrer et al. developed a dual targeting SNA that could 72 coordinate the delivery of a nucleic acid specific for toll like receptor 9 (TLR9) inhibition and a 73 small molecule (TAK – 242) that inhibited TLR4; with the goal of attenuating inflammation by 74 downregulating pro-inflammatory markers downstream of each receptor.24 Undoubtedly, SNAs 75 have emerged as a versatile tool, with significant potential within the biomedical field. 76 An area of research that has come to the fore in recent years is the application of stem cells in 77 regenerative medicine. The ready accessibility of human bone marrow stromal cells (BMSCs) 78 from bone marrow and their ability to differentiate into bone-forming osteoblasts when 79 implanted in vivo, has garnered significant scientific interest and driven the application of SSCs 80 in the clinic.30 Bone marrow (BM) is the soft connective tissue within bone cavities that 81 functions to produce blood cells and as a store for fat. Traditionally BM has been seen as an 82 organ composed of two main systems: the hematopoietic tissue and the supportive stroma.31 83 Hematopoietic stem cells (HSCs) sustain the generation of all blood cell types and are thus 84 invaluable for the treatment of hematopoietic disorders.32 In contrast, the BM stroma contains 85 mesenchymal stem/stromal cells (MSCs). These are defined as unspecialized cells that lack 86 tissue – specific characteristics and maintain an undefined phenotype.33 The term MSC was 87 originally coined in reference to a hypothetical common progenitor of a wide range of 4 bioRxiv preprint doi: https://doi.org/10.1101/2019.12.19.882563; this version posted January 14, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 88 “mesenchymal” (non-hematopoietic, non-epithelial, mesodermal) tissues. The term represents a 89 heterogeneous cell population when cultured in vitro and are likely to include cells with a wide 90 spectrum of regenerative potential from specific terminal cell type progenitors to multipotent 91 stem cells, named SSCs.34, 35 The SSC is used here to refer specifically to the self-renewing stem 92 cell of the bone marrow stroma responsible for the regenerative capacity inherent to bone with 93 osteogenic, adipogenic and chondrogenic differentiation potential in vivo.34, 36 However, a 94 number of challenges limit SSC application including the limited numbers of SSCs in BM 95 aspirates (1 in 10-100,000), and the absence of specific markers limiting facile homogenous 96 isolation of the SSC from human tissue. Thus, there remains limited understanding of SSC fate, 97 immunophenotype and simple selection criteria; all of which have proven to be limiting factors 98 in the widespread clinical application of these cells.34 99 Current techniques employed for SSC sorting are based on fluorescence and magnetic activated 100 cell sorting (respectively named FACS and MACS) combined with plastic culture adherence.
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