Longitudinal tracking of human fetal cells labeled with super paramagnetic iron oxide nanoparticles in the brain of mice with motor neuron disease

Abstract

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.

Figure 1. In vitro measurements of SPIOn.

(A) Size distribution and (B) zeta-potential measured in distilled water, saline solution and Amniomax II by DLLS. (C–D) Representative images of SPIOn spotted on the mica and visualized by AFM. (C) SPIOn are mainly monodispersed although a small number of chain-like clusters (red arrow) were found. (D) AFM picture shows the spheroid shape of SPIOn. (E) EFTEM images of SPIOn. Single particles were detectable, despite aggregation caused by magnetic forces, confirming data obtained by AFM. Red spots indicate the ESI analysis for iron. Scale bars: C = 1 µm; D–E = 40 µm.

Figure 2. Detection of SPIOn internalization in hAFCs.

(A) Representative picture of Prussian blue staining. Internalization process of SPIOn in hAFCs involved the whole cell population regardless the different morphologies. (B) Higher magnification figure of Prussian blue staining. (C) Representative figure of Hoechst 33258 (blue) and anti-carboxydextran staining (green). (D) Higher magnification picture of carboxydextran and Hoechst 33258 internalization. (E) Aggregates of SPIOn not enclosed by a membrane in the cytoplasm of hAFCs (red arrows). (F) TEM analysis in iron-free hAFCs (F). Scale bars: A–C = 100 µm; B–D = 20 µm. E = 1 µm.

Figure 3. FACS analysis of hAFCs.

Evaluation of cell cycle revealed that there were no differences between unlabeled (A) and SPIOn/Hoechst 33258 positive hAFCs (B). In all, 50,000 events per histogram were analyzed (horizontal axis: linear fluorescence intensity; vertical axis: relative cell number). Additional data are reported in Table 2.

Figure 4. Evaluation of vital parameters in labeled hAFCs.

(A) Representative figure of apoptosis measurement by Annexin V/PI staining and FACS analysis. The lower left fractions of each panel indicate viable cells (Annexin negative-PI negative, Q3). Early (Annexin positive-PI negative, Q1) and end stage (Annexin positive- PI positive, Q2) apoptotic cells are in the upper left and upper right fractions respectively. Dead cells (Annexin negative-PI positive, Q4) are in the lower right fractions. No significant differences were found between the two experimental groups (see also Table 2). (B) Proliferation of hAFCs. SPIOn and Hoechst 33258 internalization did not influence the proliferative capability of cells. T is expressed as days in vitro. Data are mean ± SD from 2 replicates of 3 different experiments. Statistical analysis: Two-Way ANOVA and Bonferroni post test for multiple comparisons. (C) Metabolic activity assessment. MTS assay showed no differences between control and SPIOn/Hoechst 33258 labeled hAFCs, at either T0 or 10 days after labeling. Data are expressed as mean ± SD from 2 replicates of 3 different experiments. Statistical analysis: Two-Way ANOVA and Bonferroni post test for multiple comparisons.

Figure 5. Representative longitudinal MRI of wobbler mouse brains 1 day after ICV administration of PBS (A), hAFCs labeled with SPIOn (B) and iron-free hAFCs (C).

Representative longitudinal MRI of the same mouse brain 28 days after ICV administration of PBS (D), hAFCs labeled with SPIOn (E) and iron-free hAFCs (F). Arrows indicate the different compartments of brain ventricles: L.V.: lateral ventricle; CB: cerebellum; S.C.: spinal cord. Representative longitudinal MRI of wobbler mouse brains before (G), 1 (H), 24 (I) and 48 hours (J) after SPIOn administration. For each panel, the sagittal slice is representative of the region comprising lateral ventricles (L.V.), cerebellum (CB) and fourth ventricle at the spinal cord level (S.C.), as indicated by arrows.

Figure 6. MRI of labeled hAFCs. Representative pictures of MRI of healthy (A) and wobbler (B) mice one day before hAFC transplantation.

Axial MRI analysis of the same healthy (C) and wobbler mice (D) at 1, 14 and 56 days after graft. For each panel, the coronal slices are indicative of different regions of the ventricular system including the site close to cell administration (L.V.), the region corresponding to the ventral hippocampus (HP), the region between the brainstem and the cerebellum (CB) and the cervical spinal cord region (S.C.).

Figure 7. Relative quantification of MRI signal.

(A) Acquisition scheme of serial slices selected for the quantification of volumes. (B) Representative slice showing the ROI manually defined for the volume quantification. Light blue areas show the hypo-intense signal, while yellow area show the brain parenchyma. (C) Quantification of the percentage of hypo-intense volume in the brain. No significant difference was found between wobbler mice and healthy controls 1, 14 and 56 days after hAFC transplantation. Data are expressed as mean ± SD (n = 4) for each group. Statistical analysis: Two-Way ANOVA and Bonferroni post test for multiple comparisons.

Figure 8. Ex vivo analyses.

(A) MRI pictures showing axial slices corresponding to the lateral ventricle close to the site of administration. (B) Histological section observed by UV shows the presence of Hoechst 33258 positive nuclei in the ventricular area. (C) MRI axial slice close to the ventral hippocampus. (D) Fluorescent microscopy of histological section revealed a co-localization between Hoechst 33258 and MRI signal. (E) Iron accumulation in the brain section adjacent to that shown in figure D. (F) High magnification picture showing the merge between iron accumulation and Hoechst 33258 positive nuclei. (G) Merge between the carboxydextran immunoreactivity (green) and Hoechst 33258 positive nuclei (blue). (H) Merge between the HLA-I (red) and Hoechst 33258 positive nuclei (blue). Scale bars: B–D–E = 600 µm; F–G = 45 µm; H = 30 µm.