Quantum dot labeling and tracking of cultured limbal epithelial cell transplants in vitro

Abstract

Purpose: Cultured human limbal epithelial cells (HLECs) have shown promise in the treatment of limbal stem cell deficiency but little is known about their survival, behavior, and long-term fate after transplantation. The aim of this research was to evaluate, in vitro, quantum dot (Qdot) technology as a tool for tracking transplanted HLECs.

Methods: In vitro cultured HLECs were labeled with Qdot nanocrystals. Toxicity was assessed using live-dead assays. The effect on HLEC function was assessed using colony-forming efficiency assays and expression of CK3, P63alpha, and ABCG2. Sheets of cultured HLECs labeled with Qdot nanocrystals were transplanted onto decellularized human corneoscleral rims in an organ culture model and observed to investigate the behavior of transplanted cells.

Results: Quantum dot labeling had no detrimental effect on HLEC viability or function in vitro. Proliferation resulted in a gradual reduction in Qdot signal but sufficient signal was present to allow tracking of cells through multiple generations. Cells labeled with Qdots could be reliably detected and observed using confocal microscopy for at least 2 weeks after transplantation in our organ culture model. In addition, it was possible to label and observe epithelial cells in intact human corneas by using the Rostock corneal module adapted for use with the Heidelberg HRA.

Conclusions: This work demonstrates that Qdots combined with existing clinical equipment could be used to track HLEC for up to 2 weeks after transplantation; however, our model does not permit the assessment of cell labeling beyond 2 weeks. Further characterization in in vivo models are required.

Figure 1

Quantum dot labeling of primary human limbal epithelial cells. Phase contrast microscopy (A&C) and fluorescence microscopy (B&D) of Qdot labeled primary human limbal epithelial cells using x 20 objective (A&B), and x40 objective (C&D). These images demonstrate that Qdots are not visible with phase contrast microscopy using white light. Images C&D demonstrate the corresponding area in A&C when viewed with a green wavelength light (450 – 550nm) and viewed using a red filter. The Qdots (red) are seen to fluoresce brightly and appear to be aggregated in clumps within individual cells.

Figure 2

Intra-cellular localisation of Qdots. Confocal microscopy (A – merged phase and fluorescence, B - fluorescence image) and electron microscopy (C & D) of Qdot labeled primary human limbal epithelial cells (HLEC) taken 24 hours post labeling showing the widespread distribution of Qdot aggregates throughout the cytoplasm. The cytoplasmic aggregates are not encapsulated by a cellular membrane but rather are free distributed. At 1 week post labeling confocal microscopy (E - merged phase and fluorescence image, F – fluorescence image) and electron microscopy (F & G) show that Qdots are primarily located in the nucleus. Some cytoplasmic aggregates are present at this time point, which on electron microscopy were found to be encapsulated by a membrane and associated with the Golgi apparatus (F). At 2 weeks post labeling Qdots were located primarily in the nucleus with few cytoplasmic aggregates (H – merged phase and fluorescence image & I - fluorescence image).

Figure 3

Results of live dead assay to evaluate the toxicity of Qdots. Unlabeled control cells (A) and cells labeled with Qdots (B) demonstrate a similar ratio of live to dead cells, scale bar 50μm. C) Quantification of the number of live (green bars) and dead cells (red bars) in both groups reveals no difference in the percentage of live or dead cells in either group. Error bars represent the standard error of the mean (n=3).

Figure 4

Effect of Qdot labeling of primary human limbal epithelial cells (HLEC) on colony forming efficiency (CFE). A) Photograph of CFE assay for HLEC labeled with Qdots demonstrating the presence of numerous colonies with classic holoclone morphology. B) Corresponding photograph for non-labeled HLEC demonstrating a similar number and size of holoclone colonies. n=3.

Figure 5

Effect of Qdots on differentiation of human limbal epithelial cells (HLEC). Confocal microscopic images of Immunofluorescence for the differentiation marker CK3 and putative stem cell “markers” p63alpha and ABCG2 at day 5 post labeling of HLEC. Scale bar = 50μm.

Figure 6

Method of culture and transplantation of a labeled sheet of human limbal epithelial cells (HLEC) in an organ culture model. A) Fluorescence microscopy image of sheet of human limbal HLEC that has been labeled with Qdot (scale bar 50μm). B) Sheet of Qdot labeled HLEC that has been separated from the culture dish using dispase. C) Decellularised human corneoscleral rims in an organ culture chamber. D) Image obtained from recombined corneoscleral rim and HLEC sheet 7 days post transplantation using a Zeiss LSM 510 confocal microscope (scale bar 10μm).

Figure 7

Results of transplanting labeled human limbal epithelial cells sheets onto decellularized corneoscleral rims. Columns A and D indicate the location from which the adjacent confocal microscopic images were collected. Columns B and C demonstrated the presence of labeled HLEC on the surface of the cornea at 7 days and 14 days post transplantation. Columns E and F demonstrate the appearance of labeled HLEC on the surface of the limbus at 7 days and 14 days post transplantation. At 7 days post transplantation an intact epithelial sheet covered the entire cornea (B) and limbus (E). A substantial proportion of these cells contained Qdots indicating that the transplanted cells had survived for the first 7 days. It was noticeable that in the limbal region, more cells still contained Qdots (E) than those on the peripheral cornea (B). Also, the limbal area contained clusters of very Qdot bright cells (E). These were not present on the cornea (B). At day 14 the number of Qdot labeled cells had decreased but there was still an intact layer of epithelium covering the cornea (C) and limbus (F). The decline in Qdot signal was greatest in the cornea (C) and less in the limbus (F). The clusters of Qdot bright cells were still present in the limbal region at 2 weeks, but they were less raised and prominent than initially (F).

Figure 8

Ex-vivo confocal microscopy of rabbit corneal epithelium labeled with Qdots Images were acquired using the Rostock corneal (Heidelberg) module modified to attach onto the Spectralis OCT. clinical imaging equipment. A&B unlabeled control rabbit cornea. Individual corneal epithelial cells can be distinguished. C&D Qdot labeled rabbit corneal epithelium. The white dots scattered throughout the field of view are the Qdot aggregates within the cytoplasm of labeled cells. Scale bar 50μm.