Long-term monitoring of transplanted human neural stem cells in developmental and pathological contexts with MRI

Abstract

Noninvasive monitoring of stem cells, using high-resolution molecular imaging, will be instrumental to improve clinical neural transplantation strategies. We show that labeling of human central nervous system stem cells grown as neurospheres with magnetic nanoparticles does not adversely affect survival, migration, and differentiation or alter neuronal electrophysiological characteristics. Using MRI, we show that human central nervous system stem cells transplanted either to the neonatal, the adult, or the injured rodent brain respond to cues characteristic for the ambient microenvironment resulting in distinct migration patterns. Nanoparticle-labeled human central nervous system stem cells survive long-term and differentiate in a site-specific manner identical to that seen for transplants of unlabeled cells. We also demonstrate the impact of graft location on cell migration and describe magnetic resonance characteristics of graft cell death and subsequent clearance. Knowledge of migration patterns and implementation of noninvasive stem cell tracking might help to improve the design of future clinical neural stem cell transplantation.

Fig. 1.

In vitro analysis of SPIO-labeled hCNS-SCns. SPIO labeling was efficient and did not affect proliferation, differentiation and electrophysiology of hCNS-SCns in vitro. (A) hCNS-SCns stained with an anti-dextran antibody (DX-1 in green) and a human-specific cytoplasmic marker (SC121, red; DAPI, blue) 6 days after in vitro proliferation. (Inset) hCNS-SCns stained for DX-1 for quantitative analysis of iron content. (B) Proliferation of SPIO-labeled (filled bars) and unlabeled hCNS-SCns (open bars) analyzed 48 h after labeling. (C) The SPIO content is reduced by 50% with each division over 9 days. (D) Analysis of labeled and unlabeled hCNS-SCns grown under differentiation conditions for 10 days revealed no difference in cell fate. Results are means ± SEM. (E–F) Electrophysiological cell properties were not changed after SPIO labeling of hCNS-SCns. (E) Action potential in a neural progenitor cells-derived neuron recorded in response to a current injection of 80 pA. (F) Voltage-activated sodium and potassium currents in a neural progenitor cells-derived neuron, steps of 35–65 mV.

Fig. 2.

Migration and integration of SPIO-labeled hCNS-SCns. MRI detects widespread migration of SPIO-labeled hCNS-SCns after intraventricular injection in the P0/P1 NOD-SCID mouse brain. (A and D) (A) Three weeks after transplantation, sagittal MRI shows hypointensities representing SPIO-labeled hCNS-SCns in the lateral ventricle (asterisk), along the RMS, (arrowheads) toward the OB (arrow) and in the 4th ventricle (black arrow). (D) corresponding section stained with the human-specific cytoplasmic marker SC121. (Inset) Shows RMS in adjacent section. (B and E) (B) sagittal MRI 18 weeks after transplantation showing that hCNS-SCns have integrated in the ventricular wall (arrowhead), in the core of the OB (white arrow) and in the fourth ventricle (black arrow). (Insert) Demonstrates migration along the corpus callosum (arrowheads). (E) Corresponding histological section. (C and F) (C) sagittal MRI of a control animal transplanted with unlabeled hCNS-SCns 18 weeks after transplantation shows no cell signal. (F) Corresponding histological section. (G–H) Higher magnification sagittal images (areas boxed in Fig. 2E) show hCNS-SCns in the CA1, CA3, and dentate gyrus of the hippocampus (G) and in the OB (H) of the NOD-SCID mouse. (I) There was a robust cell survival at 18 weeks after transplantation without statistically significant difference between SPIO-labeled (n = 4 animals) and unlabeled cells (n = 3 animals). Results are mean ± SEM. (J and K) Quantification of SC101-positive human cells coexpressing β-tubulin or GFAP overall (J) and coexpressing β-tubulin in anatomical subregions in NOD-SCID mice 18 weeks after intraventricular transplantation of hCNS-SCns (K). There was no statistically significant difference between SPIO-labeled and -unlabeled cells in terms of cell fate. OB, olfacatory bulb; RMS, rostral migratory stream; SVZ, subventricular zone. Results are mean ± SEM.

Fig. 3.

Migration in the stroked brain, 3D graft morphology, and graft location. MRI detects intraparenchymal-targeted migration of hCNS-SCns toward the ischemic brain. (A–F) Rats were transplanted 7 days after distal middle cerebral artery occlusion, with 3 × 10⁵ cells in three deposits. (A and C) two consecutive coronal sections showing the bolus of hCNS-SCns (arrowhead) medial to the hyperintense stroke area in T2-weighted images 1 week (A) and 5 weeks (C) after transplantation. There is some callosal (arrow) but no intraparenchymal migration at 1 week (A). At 5 weeks after transplantation, robust migration of hCNS-SCns toward the lesion is visible as a hypointense area on the edge of the bolus extending laterally (arrows) (C). Three-dimensional reconstruction and surface rendering of the rat brain based on high resolution T2-MRI as illustrated in (E). Posterior view of the rat brain 1 (B) and 5 (D) weeks after transplantation showing the graft (pink) and the stroked area (green). Note the broad migration of hCNS-SCns along the medial border of the stroke in the anterior posterior and craniocaudal direction, resulting in a significant increase in graft volume. (E) Three-dimensional reconstruction of the rat brain illustrating the segmentation process of the stroked area (green) and the graft (pink) based on coronal T2-MRI. (F) Histological section corresponding to the MRI (C) at 5 weeks stained with the human-specific nuclear marker SC101, showing the lateral bolus edge on the left side (asterisk) and robust migration (arrows) toward the infarct.

Fig. 4.

Graft undergoing necrosis. Immunosuppressed rats were transplanted with viable hCNS-SCns into the right striatum and dead hCNS-SCns (three freeze–thaw passages) into the left striatum. Early loss of MR signal and graft volume indicates graft/cell death. (A–C) MRI showing a left sided intrastriatal graft of killed cells at days 2 (A), 7 (B), and 35 (C) after transplantation. Loss of graft signal and reduction in graft size is noted on the left side, whereas the right side remains unchanged. (D–F) Confocal immunofluorescence image (D), showing number and morphology of Iba-1-positive cells (green) in close relation to hCNS-SCns (red) in a viable graft. In the dead graft (E), morphologically activated Iba-1-positive cells (green) are more abundant and have phagocytosed hCNS-SCns (red) at 35 days after transplantation. (F) Iba-1 expression in healthy brain tissue (insert at higher magnification). Nuclei counterstained with DAPI (blue). (G) Volumes of dead grafts (filled bar) and viable grafts (open bar) based on MR images at 2, 7, and 35 days after transplantation clearly demonstrates the early volume loss (∗, P < 0.05). (H) quantification of Iba-1-positive cell density surrounding the dying grafts (DG), viable grafts (VG), and in normal parenchyma (NP) at 35 days after transplantation. Results are means ± SEM.