Biological Characteristics of Fluorescent Superparamagnetic Iron Oxide Labeled Human Dental Pulp Stem Cells

Abstract

Tracking transplanted stem cells is necessary to clarify cellular properties and improve transplantation success. In this study, we investigate the effects of fluorescent superparamagnetic iron oxide particles (SPIO) (Molday ION Rhodamine-B™, MIRB) on biological properties of human dental pulp stem cells (hDPSCs) and monitor hDPSCs in vitro and in vivo using magnetic resonance imaging (MRI). Morphological analysis showed that intracellular MIRB particles were distributed in the cytoplasm surrounding the nuclei of hDPSCs. 12.5-100 μg/mL MIRB all resulted in 100% labeling efficiency. MTT showed that 12.5-50 μg/mL MIRB could promote cell proliferation and MIRB over 100 μg/mL exhibited toxic effect on hDPSCs. In vitro MRI showed that 1 × 10⁶ cells labeled with various concentrations of MIRB (12.5-100 μg/mL) could be visualized. In vivo MRI showed that transplanted cells could be clearly visualized up to 60 days after transplantation. These results suggest that 12.5-50 μg/mL MIRB is a safe range for labeling hDPSCs. MIRB labeled hDPSCs cell can be visualized by MRI in vitro and in vivo. These data demonstrate that MIRB is a promising candidate for hDPSCs tracking in hDPSCs based dental pulp regeneration therapy.

Figure 1

Isolation and characterization of human dental pulp stem cells (DPSCs). (a) The morphological observation of primary culture expanded dental pulp stem cells (DPSCs). (b) Odontogenic/osteogenic differentiation of DPSCs. (c) Adipogenic differentiation of DPSCs. (d and e) Representative flow cytometry analysis of cell surface markers in unlabeled and labeled hDPSCs. Cell surface markers (d) on unlabeled hDPSCs in P3 and (e) on MIRB-labeled hDPSCs in P3. Data show that both labeled and unlabeled hDPSCs are negative for CD34 and CD45 while they are positive for CD29, CD90, and CD44.

Figure 2

Morphological observation of hDPSCs labeled with various concentrations of MIRB. Light microscopy images: (a) Prussian blue staining of unlabeled control hDPSCs; (f, k, p, u) Prussian blue staining of 12.5 μg/mL–100 μg/mL MIRB-labeled hDPSCs. The scale bar indicates 1 mm. Fluorescence microscopy images: (b, g, l, q, v) the nuclei of hDPSCs are stained with DAPI (blue); (c, h, m, r, w) the cytoskeleton is stained with FITC-phalloidin (green); (d, i, n, s, x) the MIRB particles show red; (e, j, o, t, y) merged images of nuclei, cytoskeleton, and MIRB. The scale bar indicates 100 μm.

Figure 3

(a) and (b) TEM images of MIRB internalized in hDPSCs; (b) rRepresents several vesicles loaded with MIRB selected from the boxed area of (a). The magnification of image (b) is 40000x. The bar in image (a) is 2 µm; in (b) it represents 500 nm.

Figure 4

Biological effects of MIRB on hDPSCs. (a) Intracellular iron content analysis. (b) Proliferation capacity of hDPSCs measured with MTT assay. MIRB (12.5 μg/mL–50 μg/mL) promotes hDPSCs proliferation. ^∗P < 0.05. (c) Promotion effect of MIRB (12.5 μg/mL–100 μg/mL) on cell cycle progression. ^∗P < 0.05. (d) Effect of MIRB labeling on cell apoptosis. 100 μg/mL MIRB is toxic to hDPSCs. ^∗P < 0.05.

Figure 5

Odonto-/osteogenic differentiation analysis on MIRB-labeled and unlabeled hDPSCs. (a) Images of the ALP staining in labeled and unlabeled groups after 7 and 14 days of osteogenic induction. (b) Quantitative results of ALP staining. (c) Images of the mineral deposits of the labeled and unlabeled hDPSCs after 14 days of osteogenic induction. (d) Quantitative results of Alizarin Red staining. The scale bar indicates 100 μm. (e) Expression of ALP, BSP, DSPP, and OPN by hDPSCs after 7 and 14 days of osteogenic induction. ^∗P < 0.05.

Figure 6

MRI of MIRB-labeled hDPSCs in vitro. (a) MRI of 1 × 10⁶ cells labeled with various concentrations of MIRB. (b) MRI of 1 × 10⁵ cells labeled with various concentrations of MIRB.

Figure 7

MRI and histological examination of MIRB-labeled hDPSCs cell sheets/root fragments complex in vivo. (a) In vivo MR images of nude mice immediately after transplantation. (b) In vivo MR images of nude mice 30 days after transplantation. (c) In vivo MR images of nude mice 60 days after transplantation. Left boxed area indicates the unlabeled cell sheets (high signal image) and right boxed area indicates the labeled cell sheets. (d) Quantification analysis of the signal intensity of MIRB from the boxed area of (a), (b), and (c). ^∗P < 0.05. (e) Prussian blue staining of the MIRB-labeled group immediately after transplantation. (f) Prussian blue staining of the MIRB-labeled group 30 days after transplantation. (g) Prussian blue staining of the MIRB-labeled group 60 days after transplantation. (h) Prussian blue staining of the control group 60 days after transplantation. The scale bar of (e–h) indicates 500 μm.