CRISPR/Cas9-Engineered Triple-Fusion Reporter Gene Imaging System for Monitoring Transplanted Neural Progenitor Cells in Ischemic Stroke.

Background Neural progenitor cell therapy holds great potential for repairing brain damage induced by ischemic stroke, and molecular imaging plays a crucial role in evaluating the therapeutic efficacy of neural progenitor cell transplantation. However, the presence of the blood-brain barrier significantly limits the effectiveness of such imaging methods. Purpose To enable long-term monitoring of transplanted human neural progenitor cells (hNPCs) in a rat model of ischemic stroke by combining a clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9)-engineered triple-fusion (TF) reporter gene system with a noninvasive adenosine agonistic micelle (AM)-based probe delivery strategy. Materials and Methods Between January 2021 and May 2025, 60 male rats that were 2 months old were included. Thirty-seven rats with stroke were administered either TF human NPCs (hNPCs) or vehicle (culture media) and underwent MRI, bioluminescence imaging, PET/CT, and neurologic assessments at weeks 1, 2, 4, and 8 after transplantation. Comparisons between groups were determined by tests, one-way analysis of variance, linear regression, and linear mixed-effects model. Results TF-hNPCs proliferated within the ischemic rat brain (week 8 vs week 1, bioluminescence imaging and PET: < .001 and = .02, respectively) and exhibited progressive migration and maturation by 8 weeks after transplantation (proportion of microtubule-associated protein 2-positive TF-hNPCs at week 8 vs week 4: 94.08% ± 3.02 vs 85.47% ± 6.54, respectively [ = .04]; proportion of doublecortin-positive TF-hNPCs at week 4 vs week 2: 83.90% ± 2.84 vs 59.74% ± 0.55, respectively [ = .02]). Moreover, TF-hNPC transplantation increased glucose (fluorine 18 fluorodeoxyglucose) uptake in the ischemic brain (TF-hNPCs vs vehicle at week 4, 0.58 ± 0.04 vs 0.37 ± 0.05, respectively [ = .008]; TF-hNPCs vs vehicle at week 8, 0.52 ± 0.06 vs 0.29 ± 0.02, respectively [ = .01]) and attenuated neurologic deficits compared with the vehicle group (neurologic score, TF-hNPCs vs vehicle at week 8: 9.6 ± 0.25 vs 7.6 ± 0.3, respectively; = .003). Conclusion A CRISPR/Cas9-engineered TF reporter gene imaging system combined with a noninvasive AM-based approach enabled in vivo monitoring of transplanted human NPCs in a rat model of ischemic stroke. © RSNA, 2025 See also the editorial by Chapelin in this issue.