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. 2013 Sep;8(9):682-9.
doi: 10.1038/nnano.2013.147. Epub 2013 Aug 4.

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

Tsai-Jung Wu  1 , Yan-Kai Tzeng, Wei-Wei Chang, Chi-An Cheng, Yung Kuo, Chin-Hsiang Chien, Huan-Cheng Chang, John Yu
Affiliations

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

Tsai-Jung Wu et al. Nat Nanotechnol. 2013 Sep.

Abstract

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.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Phenotypes of LSCs before and after induction for differentiation.
a, Immunofluorescence staining of the sorted CD45−CD54+CD157+ cells at the time of isolation against various types of undifferentiated and differentiated markers, including Oct-4 and Nanog (red), and cytokeratin-7, CCSP, SP-C and Aqp-5 (green). The cells expressed neither type II nor type I pneumocyte markers (SP-C and Aqp-5, respectively). Cell nuclei were stained in blue by DAPI. Scale bar, 10 µm. b, Examination for the differentiation of cultured CD45−CD54+CD157+ cells in vitro. The cells were induced to undergo differentiation by incubation in MCDB201 for 7 days, before being stained with antibodies against the type II pneumocyte marker, SP-C (green). Cells were then stained with antibodies against the type I pneumocyte marker, Aqp-5 (green), after incubation in MCDB201 for 14 days. Scale bar, 50 µm. Data are representative images from three independent experiments.
Figure 2
Figure 2. FND-labelled LSCs in vitro.
a,b, Flow cytometric analysis of FND-labelled LSCs (red) and control LSCs without FND labelling (grey), showing two distinct populations in the side scatter (SSC) and far-red fluorescence channels (a), and that the FND-labelled LSCs cultured in DMEM could be continuously monitored for 15 days post-labelling (b). D0, D2, D6 and D15 in b denote days 0, 2, 6 and 15 after FND labelling, respectively. c,d, Representative wide-field fluorescence images of differentiated type II pneumocytes derived from the control LSCs (c) and FND-labelled LSCs (d) after induction in MCDB201 medium for 7 days, followed by immunostaining with SP-C (DAPI in blue, SP-C in green and FND in red). The FND image was acquired by excitation at 515–560 nm and collection of emission at >590 nm. No FNDs were found in cell nuclei. Scale bars, 10 µm. Experiments were repeated in triplicate.
Figure 3
Figure 3. FND-labelled LSCs in uninjured mice.
a, Flow cytometric analysis of total lung cells collected from uninjured mice receiving an i.v. injection of FND-labelled LSCs for 1, 4 and 7 days (n = 3 per group). b, Representative FLIM, TGF and bright-field H&E staining images of the same lung tissue sections from mice in a. The merged H&E and TGF images show that the FND-labelled cells (denoted by black arrows) are primarily located in the subepithelium of bronchiolar airways. Scale bar, 50 µm. c, Co-localization examination of FND-labelled LSCs and macrophages in a typical lung tissue section immunostained with macrophage-specific antigen F4/80 and haematoxylin counterstain. The enlarged view in the last panel shows that the transplanted FND-labelled cells (red, denoted by red arrows) and alveolar macrophages (brown, denoted by black arrows) are located at different positions. Scale bar, 10 µm. Experiments were repeated in triplicate.
Figure 4
Figure 4. FND-labelled LSCs in lung-injured mice.
a,b, Immunohistochemical analysis of lung tissue sections (a) and flow cytometric analysis of total lung cells (b) collected from naphthalene-injured mice receiving an i.v. injection of saline (control) or FND-labelled LSCs for 1 and 7 days (n = 3 per group). The tissue sections in a were stained with CCSP for club cells (brown). c, Representative FLIM, TGF and bright-field H&E staining images of the same lung tissue sections, showing the location of FND-labelled LSCs (denoted by white and black arrows) in terminal bronchioles of the lungs. d, FLIM and H&E/TGF images of the lung tissue section of a naphthalene-injured mouse on day 7, showing engraftment of the transplanted FND-labelled LSCs (denoted by white and black arrows) to terminal bronchioles in cluster form. Scale bars, 50 µm. Experiments were repeated in triplicate.
Figure 5
Figure 5. Identification of transplanted FND-labelled LSCs in bronchiolar epithelia and their histomorphometric distribution.
a,b, Club cell markers (CCSP, a) and epithelial markers (pan-cytokeratin, b) examined by FLIM and immunostaining of lung tissue sections prepared from naphthalene-injured mice on day 7 after i.v. injection of FND-labelled LSCs. The enlarged, merged immunostaining and TGF images in the red dashed boxes show that the FND-labelled LSCs (red) co-localize with CCSP+ (brown) and pan-cytokeratin+ (brown) cells and reside at the bronchiolar epithelial region of the lung. Scale bars, 10 µm. c, Histomorphometric distribution measured by visually counting the numbers of FND-labelled LSCs nested in terminal bronchioles (black) or alveoli (white) of the lungs of uninjured and injured mice on day 7 after i.v. injection. Results are averages of more than 30 visual fields per section, with error bars representing standard deviations of the measurements (n = 6 per group).
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