Tracking the engraftment and regenerative capabilities of transplanted lung stem cells using fluorescent nanodiamonds

Lung stem/progenitor cells are potentially useful for regenerative therapy, for example in repairing damaged or lost lung tissue in patients. Several optical imaging methods and probes have been used to track how stem cells incorporate and regenerate themselves in vivo over time. However, these approaches are limited by photobleaching, toxicity and interference from background tissue autofluorescence. Here we show that fluorescent nanodiamonds, in combination with fluorescence-activated cell sorting, fluorescence lifetime imaging microscopy and immunostaining, can identify transplanted CD45–CD54+CD157+ lung stem/progenitor cells in vivo, and track their engraftment and regenerative capabilities with single-cell resolution. Fluorescent nanodiamond labelling did not eliminate the cells’ properties of self-renewal and differentiation into type I and type II pneumocytes. Time-gated fluorescence imaging of tissue sections of naphthalene-injured mice indicates that the fluorescent nanodiamond-labelled lung stem/progenitor cells preferentially reside at terminal bronchioles of the lungs for 7 days after intravenous transplantation. Supplementary information The online version of this article (doi:10.1038/nnano.2013.147) contains supplementary material, which is available to authorized users.

[1]  Huan-Cheng Chang,et al.  In vivo imaging and toxicity assessments of fluorescent nanodiamonds in Caenorhabditis elegans. , 2010, Nano letters.

[2]  Patrick Georges,et al.  Detection of single photoluminescent diamond nanoparticles in cells and study of the internalization pathway. , 2008, Small.

[3]  A. Gill,et al.  Mesenchymal stem cells: isolation, characterisation and in vivo fluorescent dye tracking. , 2008, Heart, lung & circulation.

[4]  B. Grimes,et al.  Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver. , 2001, Blood.

[5]  J. McQualter,et al.  Evidence of an epithelial stem/progenitor cell hierarchy in the adult mouse lung , 2010, Proceedings of the National Academy of Sciences.

[6]  M. Rosen,et al.  Finding Fluorescent Needles in the Cardiac Haystack: Tracking Human Mesenchymal Stem Cells Labeled with Quantum Dots for Quantitative In Vivo Three‐Dimensional Fluorescence Analysis , 2007, Stem cells.

[7]  U. Khoo,et al.  A novel subset of putative stem/progenitor CD34+Oct-4+ cells is the major target for SARS coronavirus in human lung , 2007, The Journal of experimental medicine.

[8]  W. Liu,et al.  Degradation or excretion of quantum dots in mouse embryonic stem cells , 2010, BMC biotechnology.

[9]  Huan-Cheng Chang,et al.  Bright fluorescent nanodiamonds: no photobleaching and low cytotoxicity. , 2005, Journal of the American Chemical Society.

[10]  Hsiao-Yun Wu,et al.  Characterization and application of single fluorescent nanodiamonds as cellular biomarkers , 2007, Proceedings of the National Academy of Sciences.

[11]  Sanjiv S. Gambhir,et al.  Trafficking Mesenchymal Stem Cell Engraftment and Differentiation in Tumor‐Bearing Mice by Bioluminescence Imaging , 2009, Stem cells.

[12]  A. B. Lyons,et al.  Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. , 2000, Journal of immunological methods.

[13]  F. Watt,et al.  Stem cells are dispensable for lung homeostasis but restore airways after injury , 2009, Proceedings of the National Academy of Sciences.

[14]  N. Billinton,et al.  Seeing the wood through the trees: a review of techniques for distinguishing green fluorescent protein from endogenous autofluorescence. , 2001, Analytical biochemistry.

[15]  Alessandro Esposito,et al.  Fluorescence Lifetime Imaging Microscopy , 2004, Current protocols in cell biology.

[16]  Stefanie Dimmeler,et al.  Homing and engraftment of progenitor cells: a prerequisite for cell therapy. , 2008, Journal of molecular and cellular cardiology.

[17]  Huan-Cheng Chang,et al.  The long-term stability and biocompatibility of fluorescent nanodiamond as an in vivo contrast agent. , 2012, Biomaterials.

[18]  D. Chistiakov,et al.  Endogenous and exogenous stem cells: a role in lung repair and use in airway tissue engineering and transplantation , 2010, Journal of Biomedical Science.

[19]  D. Maclaurin,et al.  Quantum measurement and orientation tracking of fluorescent nanodiamonds inside living cells. , 2011, Nature nanotechnology.

[20]  Aniruddh Solanki,et al.  Nanotechnology for regenerative medicine: nanomaterials for stem cell imaging. , 2008, Nanomedicine.

[21]  Erik Pierstorff,et al.  Active nanodiamond hydrogels for chemotherapeutic delivery. , 2007, Nano letters.

[22]  B. Stripp,et al.  Terminal bronchioles harbor a unique airway stem cell population that localizes to the bronchoalveolar duct junction. , 2002, The American journal of pathology.

[23]  Winfried Wiegraebe,et al.  Detection of functional haematopoietic stem cell niche using real-time imaging , 2009, Nature.

[24]  T. Webster Safety of nanoparticles , 2009 .

[25]  L. Bjermer,et al.  Fibrocytes and the tissue niche in lung repair , 2011, Respiratory research.

[26]  Carla F. Kim,et al.  Paving the road for lung stem cell biology: bronchioalveolar stem cells and other putative distal lung stem cells. , 2007, American journal of physiology. Lung cellular and molecular physiology.

[27]  Huan-Cheng Chang,et al.  The biocompatibility of fluorescent nanodiamonds and their mechanism of cellular uptake , 2009, Nanotechnology.

[28]  Allison N. Lau,et al.  Stem cells and regenerative medicine in lung biology and diseases. , 2012, Molecular therapy : the journal of the American Society of Gene Therapy.

[29]  E. Palik Handbook of Optical Constants of Solids , 1997 .

[30]  Huan-Cheng Chang,et al.  Mass production and dynamic imaging of fluorescent nanodiamonds. , 2008, Nature nanotechnology.

[31]  Ching-Fang Chang,et al.  The exocytosis of fluorescent nanodiamond and its use as a long-term cell tracker. , 2011, Small.

[32]  Robert Langer,et al.  New opportunities: the use of nanotechnologies to manipulate and track stem cells. , 2008, Cell stem cell.

[33]  J. McQualter,et al.  Concise Review: Deconstructing the Lung to Reveal Its Regenerative Potential , 2012, Stem cells.

[34]  Takaya Suzuki,et al.  Evidence for human lung stem cells. , 2011, The New England journal of medicine.

[35]  B. Stripp,et al.  Plasticity of airway cell proliferation and gene expression after acute naphthalene injury. , 1995, The American journal of physiology.

[36]  B. Brockway,et al.  Airway Epithelial Progenitors Are Region Specific and Show Differential Responses to Bleomycin‐Induced Lung Injury , 2012, Stem Cells.

[37]  Tsai-Jung Wu,et al.  Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARS-CoV) infection in vitro. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[38]  John J. Schlager,et al.  Cytotoxicity and Genotoxicity of Carbon Nanomaterials , 2009 .

[39]  Kuang-Kai Liu,et al.  Endocytic carboxylated nanodiamond for the labeling and tracking of cell division and differentiation in cancer and stem cells. , 2009, Biomaterials.

[40]  Yury Gogotsi,et al.  The properties and applications of nanodiamonds. , 2011, Nature nanotechnology.

[41]  David W. Rowe,et al.  Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche , 2009, Nature.

[42]  M. Chalfie,et al.  Green fluorescent protein as a marker for gene expression. , 1994, Science.

[43]  B. Hogan,et al.  Preparing for the first breath: genetic and cellular mechanisms in lung development. , 2010, Developmental cell.

[44]  S. Gambhir,et al.  Noninvasive cell-tracking methods , 2011, Nature Reviews Clinical Oncology.

[45]  T. Jacks,et al.  Identification of Bronchioalveolar Stem Cells in Normal Lung and Lung Cancer , 2005, Cell.