Cell Proliferation, Viability, Differentiation, and Apoptosis of Iron Oxide Labeled Stem Cells Transfected with Lipofectamine Assessed by MRI

Abstract

To assess in vitro and in vivo tracking of iron oxide labeled stem cells transfected by lipofectamine using magnetic resonance imaging (MRI), rat dental pulp stem cells (DPSCs) were characterized, labeled with iron oxide nanoparticles, and then transfected with lipofectamine to facilitate the internalization of these nanoparticles. Cell proliferation, viability, differentiation, and apoptosis were investigated. Prussian blue staining and MRI were used to trace transfected labeled cells. DPSCs were a morphologically spindle shape, adherent to culture plates, and positive for adipogenic and osteogenic inductions. They expressed CD73 and CD90 markers and lacked CD34 and CD45. Iron oxide labeling and transfection with lipofectamine in DPSCs had no toxic impact on viability, proliferation, and differentiation, and did not induce any apoptosis. In vitro and in vivo internalization of iron oxide nanoparticles within DPSCs were confirmed by Prussian blue staining and MRI tracking. Prussian blue staining and MRI tracking in the absence of any toxic effects on cell viability, proliferation, differentiation, and apoptosis were safe and accurate to track DPSCs labeled with iron oxide and transfected with lipofectamine. MRI can be a useful imaging modality when treatment outcome is targeted.

Keywords: MRI; dental pulp stem cells; iron oxide nanoparticles; lipofectamine; tracking.

Figure 1

Comparison of cell morphology: (A) Non-labeled and (E) Lipofectamine-transfected iron oxide labeled cells, pointed by arrow, 4×, osteogenic induction; (B) Non-labeled and (F) Lipofectamine-transfected iron oxide nanoparticles labeled cells, pointed by arrow, 4×, adipogenic differentiation; (C) Non-labeled and (G) Lipofectamine-transfected iron oxide labeled cells, pointed by arrow, 20× and RT-PCR for expression of mesenchymal and hematopoietic markers; (D) Non-labeled and (H) Lipofectamine-transfected iron oxide labeled cells).

Figure 2

MTT assay regarding cell proliferation and viability of labeled cells in different concentration of iron oxide nanoparticles (90, 180, 260 µg/mL) transfected with 1 and 2 µL/mL of lipofectamine. Data are representative of three independent experiments (p = 0.19, * p = 0.048, ** p = 0.001, *** p = 0.003, respectively), revealing the non-toxic role of nanoparticles when transfected with lipofectamine.

Figure 3

The population doubling time (PDT) in an hour and the growth curve were determined until day 6. The number of cells treated with 180 µg/mL of iron oxide transfected with 1 µL/mL of lipofectamine (Day 1: p = 0.92, day 2: p = 0.97, day 3: ** p = 0.0001, day 4: p = 0.95, day 5: p = 0.28, day 6: p = 0.155), revealing the PDT of 36 h and 33 h for non-labeled and iron oxide labeled cells (180 µg/mL) transfected with 1 µL/mL lipofectamine, respectively.

Figure 4

The T2* weighted MR imaging of in vitro samples. Group 1 contained 15% agarose gel only. In groups 2–6, 1 µL/mL of lipofectamine and 15% agarose gel were used for transfection of DPSCs and then 180, 90, 70, 50, and 30 µg/mL of iron oxide were applied for cell labeling, respectively. Group 7 was non-coated iron oxide nanoparticles and group 8 consisted of just H₂O.

Figure 5

The T2* weighted images of in vivo samples showing the hypo-intense signal of 180 µg/mL of iron oxide labeled DPSCs transfected with 1 µL/mL lipofectamine that confirms presence of iron oxide in the labeled cells in comparison to in vivo samples of non-labeled cells showing lack of this MRI appearance (Left and Right).

Figure 6

Prussian Blue staining of DPSCs labeled with 180 µg/mL of iron oxide and transfected with 1 µL/mL of lipofectamine to facilitate internalization of iron oxide nanoparticles that appear in blue color (4×).

Figure 7

The effect of 180 µg/mL of iron oxide transfected with 1 µL/mL of lipofectamine on gene expression of Bax pro-apoptotic gene (A), Bcl-2 anti-apoptotic genes (B), and Bax/Bcl-2 ratio (C) of labeled DPSCs (Bcl-2: p = 0.41_, Bax: p = 0.38, and Bax/Bcl-2 ratio: p = 0.58), revealing that 180 µg/mL of iron oxide transfected with 1 µL/mL lipofectamine in labeled DPSCs did not induce any significant apoptosis when used in cell labeling.

Figure 8

The flow cytometry analysis of annexin V+/7-AAD+ on DPSCs labeled with 180 μg/mL of iron oxide transfected with 1 µL/mL of lipofectamine. (A) Dead cells were scored as necrotic annexin V: negative/7-AAD: positive, upper left quadrants, Q1, or late apoptotic annexin V-positive/7-AAD-positive, upper right quadrants, Q2, early apoptotic annexin V-positive/7-AAD-negative, lower right quadrants, Q3, and following a gating on the normal cells that were considered viable were PE Annexin V and 7-AAD negative lower left quadrants, Q4. (B) The percentage of early and late apoptosis was indicated in a bar chart. Data presented were indicative of three independent experiments (p = 0.95).