Comparative strategies for stem cell biodistribution in a preclinical study

Abstract

Stem cell therapy represents the potential alternative effective strategy for some diseases that lack effective treatment currently. Correspondingly, it is crucial to establish high-sensitive and reliable quantification assay for tracing exogenous cell migration. In the present study, we first used both bioluminescence imaging (BLI) indirect labeling (human norepinephrine transporter-luciferase reporter system) and ⁸⁹zirconium (⁸⁹Zr)-hNSCs direct labeling combined with positron emission tomography/computer tomography (PET/CT) system for tracking human neural stem cells (hNSCs) migration into the brain via nasal administration in preclinical study. But the above two methods failed to give the biodistribution profile due to their low sensitivity. Considering its superior sensitivity and absolute quantitation capability, we developed and validated the droplet digital PCR (ddPCR) targeting species-specific gene in frozen and paraffin sections, slices, and whole blood with the sensitivity of 100-200 hNSCs. Accurate and high throughput quantification could be performed using ddPCR with the coefficient of variation (CVs) of lower quality control (LQC) below 30%. In combination with immunohistochemistry and ddPCR, we confirmed the migration of hNSCs into the brain via nasal administration, which supported the efficacy of hNSCs in MPTP-treated mice, an animal model of Parkinson's disease. In conclusion, the present study is the first to report the application of ddPCR in the pharmacokinetics profile description of tracking of hNSCs in preclinical studies.

Fig. 1

Data explanation of ddPCR. a Cutoff value of the positive droplet per analytical batch. b ddPCR data report process

Fig. 2

Typical imaging of in vitro and in vivo intrastriatal administration. a Different cell numbers (1 × 10³ − 1 × 10⁵) of hNET-Luc-hNSCs were added to a 24-well plate for fluorescence imaging. The in vitro detection threshold was ~10⁵ cells. b Typical imaging of in vivo intrastriatal administration (10⁶ hNET-Luc-hNSCs). The in vivo detection threshold was ~10⁶ cells

Fig. 3

Biodistribution of ⁸⁹Zr-hNSCs at 4 h after intrastriatal administration. a Different cell numbers (1 × 10² − 1 × 10⁵) of ⁸⁹Zr-hNSCs were added to a 96-well plate for PET/CT imaging. The detection threshold was ~10³ cells. b Typical brain imaging of the biodistribution of ⁸⁹Zr-hNSCs at 4 h after intrastriatal administration of 10³ and 10^{4 89}Zr-hNSCs (individual G1-F-01). c %ID/g representing the ⁸⁹Zr-hNSC biodistribution in the region of interest of the brain after intrastriatal administration of 10³, 10⁴, and 10^{5 89}Zr-hNSCs. *P < 0.05, **P < 0.01, ***P < 0.001 vs 10³ intrastriatal administration group. ^#P < 0.05 vs. 10⁴ intrastriatal administration group. VTM, midbrain ventral tegmental area

Fig. 4

Biodistribution of ⁸⁹Zr-hNSCs postnasal administration. a Typical images of ⁸⁹Zr-hNSCs in the nasal cavity and olfactory bulb at 11 min, 35 min, 1 h, 4 h, 24 h, and 72 h after nasal administration of 2 × 10^{6 89}Zr-hNSCs per rat (individual G4-F-02). b Cortex (parietal lobe), cortex (frontal lobe), hippocampus, striatum, and midbrain ventral tegmental area (VTM) scanning using PET/CT and whole brain MPI imaging at 35 min (left) and 4 h (right) postnasal administration of 2 × 10^{6 89}Zr-hNSCs per rat (individual G4-F-01)

Fig. 5

Typical immunofluorescence images of hNSCs postnasal administration. The rats were nasally administered 2×10⁶ hNSCs, and the brain (striatum, cortex, and hippocampus regions) immunofluorescence was determined at 24 h, 3 days, 4 days, and 7 days (STEM121, 1:500). Only 24h immunofluorescence images were shown

Fig. 6

Biodistribution profile of hNSCs using ddPCR. The hNSC concentration (copies per 20 μL PCR)-time curves of mice after intranasal administration of 10⁶ cells. Data are shown as the mean ± SD from two independently analyzed reactions. The copies were derived from the ~20 μL (300 ng gDNA) of frozen section tissues (striatum, hippocampus, cerebellum) and 20 μL (80 ng gDNA) of FFPE section tissues (heart, liver, spleen, lung, kidney). Grp1: MPTP model + hNSC intranasal administration; Grp2: Normal model + hNSC intranasal administration; Grp3: MPTP model + NaCl intranasal administration