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In vivo monitoring of remnant undifferentiated neural cells following human induced pluripotent stem cell‐derived neural stem/progenitor cells transplantation

Abstract Transplantation of human‐induced pluripotent stem cell‐derived neural stem/progenitor cells (hiPSC‐NS/PCs) is a promising treatment for a variety of neuropathological conditions. Although previous reports have indicated the effectiveness of hiPSC‐NS/PCs transplantation into the injured spinal cord of rodents and nonhuman primates, long‐term observation of hiPSC‐NS/PCs post‐transplantation suggested some “unsafe” differentiation‐resistant properties, resulting in disordered overgrowth. These findings suggest that, even if “safe” NS/PCs are transplanted into the human central nervous system (CNS), the dynamics of cellular differentiation of stem cells should be noninvasively tracked to ensure safety. Positron emission tomography (PET) provides molecular‐functional information and helps to detect specific disease conditions. The current study was conducted to visualize Nestin (an NS/PC marker)‐positive undifferentiated neural cells in the CNS of immune‐deficient (nonobese diabetic‐severe combined immune‐deficient) mice after hiPSC‐NS/PCs transplantation with PET, using 18 kDa translocator protein (TSPO) ligands as labels. TSPO was recently found to be expressed in rodent NS/PCs, and its expression decreased with the progression of neuronal differentiation. We hypothesized that TSPO would also be present in hiPSC‐NS/PCs and expressed strongly in residual immature neural cells after transplantation. The results showed high levels of TSPO expression in immature hiPSC‐NS/PCs‐derived cells, and decreased TSPO expression as neural differentiation progressed in vitro. Furthermore, PET with [18F] FEDAC (a TSPO radioligand) was able to visualize the remnant undifferentiated hiPSC‐NS/PCs‐derived cells consisting of TSPO and Nestin+ cells in vivo. These findings suggest that PET with [18F] FEDAC could play a key role in the safe clinical application of CNS repair in regenerative medicine.

Transplanted "safe" NS/PCs have been found to fully differentiate into three lineages (neuron, astrocyte, and oligodendrocyte), contributing to the recovery of locomotor function in traumatic brain injury (TBI) and spinal cord injury (SCI) in experimental animal models. [5][6][7] Long-term observation of transplanted cells, however, has revealed the existence of "unsafe" NS/PCs characterized by differentiationresistant properties that could cause abnormal cell growth in the injured spinal cords of rodents due to residual immature neuronal cells after hiPSC-NS/PCs transplantation. 8,9 Therefore, it is necessary to select "safe" and "unsafe" clones in the cell manufacturing process. 7,10,11 Furthermore, since the extent of cellular differentiation and maturation depends on the host microenvironment, 12 even "safe" clones, which can be purified for homogeneity and produced as high-quality populations, may not be able to completely differentiate into neural cell types at the transplanted site. Therefore, clinical application of hiPSC-NS/PCs to central nervous system (CNS) disorders would benefit from a relatively noninvasive detection technique for monitoring the progress of cellulardifferentiation even after "safe" clone transplantation.
Among various types of in vivo imaging techniques, positron emission tomography (PET) is a useful modality because it provides molecular-functional information with high sensitivity. [13][14][15] In addition, longitudinal PET imaging could enable monitoring of dynamic changes of a target molecule in vivo.
The 18 kDa translocator protein (TSPO), also known as a peripheraltype benzodiazepine receptor, 16 is mainly expressed in an outer mitochondrial membrane, and its expression is upregulated by specific types of neoplastic cells such as gliomas, 13,17 activated microglia, [18][19][20] and reactive astrocytes 21 associated with neuroinflammation. Additionally, it has been suggested that TSPO could be a hallmark of cellular-differentiation in NS/PCs analogous to Nestin, which is widely known as an NS/PC marker. 22,23 Therefore, we focused on visualizing TSPO expression in remnant undifferentiated hiPSC-NS/PC-derived neural cells using the PET modality.
In the present study, in order to determine the efficiency of PET with TSPO ligand, we used two different types of iPSC-derived NS/PCs; "unsafe" 253G1-NS/PCs known to have differentiationresistant proliferative properties 8,9 and "safe" 414C2-NS/PCs, which were reported as a nontumorigenic hiPSC-NS/PCs. 11 First, we examined differences in TSPO expression levels in each hiPSC-NS/PCs before and after neuronal induction using immunohistochemistry, realtime reverse-transcription polymerase chain reaction (RT-PCR) and Western blot assays in vitro. Next, we transplanted each hiPSC-NS/ PCs into the brain and spinal cord of nonobese diabetic-severe com-  19 In an in vitro study, TSPO was initially expressed in both hiPSC-NS/PCs types, but decreased over time as neural differentiation progressed in "safe" 414C2-NS/PCs. Notably, "unsafe" 253G1-NS/PCs exhibited a differentiation-resistant profile and continued to express high levels of TSPO expression in vitro. Consistent with these results, PET with [ 18 F] FEDAC was able to detect the poorly differentiated neural tissues of 253G1-NS/PCs-grafted mice brains in vivo due to their high TSPO density. These results suggest that PET imaging for TSPO provides an appropriate method for monitoring the dynamics of neural differentiation and maturation following hiPSC-  described. 2,5,[24][25][26] In the analyses of neuronal differentiation, hiPSC-NS/PCs were plated onto poly-L-ornithine/fibronectin-coated 48-well chamber slides (Costar 3548; Corning, New York) at a density of 1 × 10 5 cells/mL and cultured in medium without growth factors at 37 C in 5% CO 2 and 95% air for 14 days. Differentiated cells were fixed paraformaldehyde (PFA) in 0.1 phosphate-buffered saline (PBS) and stained with the following primary antibodies for immunocytochemistry: anti-human-specific TSPO (NP157, rabbit IgG, 1:300; National Institute for Quantum and Radiological Science and Technology, Chiba, Japan), antihuman-specific Nestin protein (MAB5236, mouse IgG1, 1:500; Merck Millipore, Billerica, Massachusetts), anti-β III-tubulin (T8660, mouse IgG2b, 1:500; Sigma-Aldrich, St. Louis, Missouri), anti-NeuN (MAB377, mouse IgG1, 1:500; Merck Millipore).
All in vitro images were obtained using confocal laser scanning microscopy (LCM 700; Carl Zeiss, Jenna, Germany).

| Real-time reverse-transcription polymerase chain reaction
RNA isolation and RT-PCR were performed as described previously. 5 Detailed methods are presented in Supporting Information (SI) Materials and Methods.

| Western blotting assay
Protein isolation and Western blotting assay were performed as described previously. 27 Detailed methods are presented in SI Materials and Methods.

| Transplantation
Transplantation was performed as described previously. 8

| Bioluminescence imaging
Bioluminescence imaging (BLI) was performed as described previously, with slight modifications. 24 Detailed methods are presented in SI Materials and Methods.

| PET and computed tomography scanning
PET and computed tomography (CT) was performed as described previously. 19 Detailed methods are presented in SI Materials and Methods.

| Production of [ 18 F] FEDAC
Radiosynthesis of [ 18 F] FEDAC was conducted in accord with a previous report. 19 Detailed methods are presented in SI Materials and Methods.

| Magnetic resonance imaging
Magnetic resonance imaging (MRI) was performed as described previously. 29 Detailed methods are presented in SI Materials and Methods.

| Ex vivo autoradiography
Ex vivo autoradiography was performed as described previously. 30 Detailed methods are presented in SI Materials and Methods.

| Histological analyses
After autoradiography, the brains and spinal cords were used for histological analyses. Detailed methods are presented in SI Materials and Methods.

| Undifferentiated/differentiation-resistant hiPSC-NS/PCs-derived neuronal cells highly expressed TSPO mRNA and protein
RT-PCR was performed to assess the levels of TSPO mRNA for each hiPSC-NS/PCs type after neuronal induction. The data were presented as expression levels relative to the U-251MG (human brain glioblastoma cell line; GBM). Since previous studies reported that GBM strongly expressed TSPO, 13 we used U-251MG as a positive control in RT-PCR and PET experiments ( Figure 2A). The expression of TSPO mRNA in the U-251MG group was significantly higher than that in the 253G1-d14 (P < .01) and 414C2-d14 groups (P < .001). Importantly, the results revealed significantly elevated levels of TSPO mRNA in the 253G1-d14 group compared to the 414C2-d14 group (P < .05).
Next, Western blot analysis was performed to examine the protein levels of TSPO in each hiPSC-NS/PCs before and after neuronal differentiation ( Figure 2B,C). Consistent with the results of RT-PCR, TSPO protein levels in the 253G1-d14 group were significantly higher than those in the 414C2-d14 group (P < .001), while maintaining high levels of Nestin expression (P < .01). In contrast, TSPO and Nestin protein levels in the 414C2-d14 group strongly decreased over time with an increase in β III tubulin expression. . The data were normalized to the reference GAPDH levels. Values are means ± SD (n = 3, each). B, Western blot results of the expression of TSPO, β III tubulin, and Nestin protein levels of 253G1-and 414C2-NS/PCs before and after neuronal differentiation using Western blot. C, Quantitative analysis of TSPO, Nestin, and β III tubulin and protein levels using Western blot. The data were normalized to the reference β-actin levels. The relative intensities on the band of 414C2-NS/PCs, 253G1-d14, and 414C2-d14 were compared to the 253G1-NS/PCs. Values are mean ± SD (n = 4, each). *P < .05, **P < .01, ***P < .001. NS/PCs, neural stem/ progenitor cells cells using BLI. These cells were lentivirally transduced with ffLuc, a fusion protein between a fluorescent Venus protein and a firefly luciferase, 31 which allowed us to monitor the growth of the grafted cells by their fluorescent Venus signals and bioluminescent luciferase signals. The photon counts of the 253G1 group increased more rapidly than that of the 414C2 group over time ( Figure S1).
Dynamic small-animal PET scanning using [ 18 F] FEDAC was performed at four to eight weeks after transplantation. The timing of PET evaluation was dependent on the health of the mice during the experimental period. Only the U-251MG group was scanned with PET two weeks after transplantation because the life span of these animals was between 2 and 4 weeks. Importantly, representative PET images, acquired by summing up the [ 18 F] FEDAC signal generated between 10 and 30 minutes and scaled with distribution volume ratio (DVR), a quantitative index for specific biding, could detect radioactive accumulation in the transplanted site (the right striatum) of the 253G1 and U-251MG groups as opposed to the contralateral side ( Figure 3A; Table S1). The 414C2 and PBS groups showed no detectable signal in both sides. We calculated the uptake of radioactivity between 10 and 30 minutes after the injection, represented as the area under the curve (AUC 10-30 min ) in the ipsilateral and contralateral sides of each mouse brain ( Figure 3B). In the 253G1 and U-251MG groups (n = 5, Values are means ± SD (n = 5, 4, 5, and 4, respectively). **P < .01, ***P < .001, N.S., not significant. DVR, distribution volume ratio; hiPSC-NS/PCs, humaninduced pluripotent stem cell-derived neural stem/progenitor cells; PBS, phosphate-buffered saline; SUV, standard uptake volume each), the AUC 10-30 min of the ipsilateral side (the transplanted side; 4.4 ± 0.4 and 8.5 ± 0.4, respectively) was significantly higher than that of contralateral side (the intact side; 3.8 ± 0.1 and 4.3 ± 0.6, respectively) (253G1 group, P < .01 and U-251MG group, P < .001).
Next, to confirm whether the radioactive signals in [ 18 F] FEDAC-PET images in each hiPSC-NS/PCs-transplanted mouse corresponded with the lesion area after transplantation, we performed contrastenhanced MRI using gadolinium-based contrast agent. In the 253G1-NS/PCs-grafted mouse brain, the contrast-enhanced areas were detected on the gadolinium injected T1-weighted MR images ( Figure 4A). Corresponding PET images revealed intense DVR signals of [ 18 F] FEDAC uptake in the same lesion ( Figure 4C). In contrast, 414C2-NS/PCs-grafted mice brains did not exhibit any detectable signal enhancement in both T1 and T2-weighted images on MRI and PET ( Figure 4B,C).

| The radioactive accumulation in PET images was supported using ex vivo autoradiography
To examine the detailed anatomical investigation of [ 18    TSPO facilitates diverse cellular functions, including mitochondrial respiration, cholesterol transport, cell proliferation, and apoptosis. 34 TSPO also plays a crucial role in neural development. 35 In addition, TSPO is expressed in NS/PCs but not in mature neurons. 22 The present study confirmed TSPO expression in hiPSC-NS/PCs which is similar to mouse neuroectodermal stem cells. Particularly, poorly differentiated hiPSC-NS/PCs-derived neural cells (ie, 253G1-d14) exhibited significantly higher levels of TSPO compared to welldifferentiated hiPSC-NS/PCs-derived neurons (ie, 414C2-d14). Immunocytochemical analyses revealed that β III tubulin + neurons derived from hiPSC-NS/PCs exhibited decreased TSPO levels, while immature Nestin + cells still expressed high levels of TSPO through neuronal induction ( Figure 1). As a reference to the analysis described in this study, it is important to accurately note the expression of TSPO in hiPSC-NS/PCs-derived neurons. In previous studies, the expression of TSPO is lost in mature neurons but immature neurons express a very low level of TSPO. 16,22 In the present study, immature β III tubulin + hiPSC-NS/PCs-derived neurons seemed to express little amounts of TSPO ( Figure 1C,E). In order to elucidate the TSPO expression status Several PET studies using [ 18 F] FEDAC in rodent models have shown in vivo specific binding with TSPO and high radioactive signals in TSPO-rich organs. 19,39 In contrast, the uptake of [ 18 F] FEDAC in healthy CNS is reported to be much lower. 40 Therefore, PET with [ 18 F] FEDAC has the potential to detect early CNS disorders related to TSPO. In this study, PET with [ 18 F] FEDAC clearly detected radioactive accumulation in the graft area of the mouse brain in only the 253G1 and U-251MG (positive control) groups. Four out of five mice in the 414C2 group were used for analysis (one mouse died before detection), and none of the mice exhibited any significant radioactive uptake ( Figure 3A). However, small-animal PET, especially rodent models, has limitations in concisely evaluating the radial uptake due to poor resolution. 41 Therefore, we put more emphasis on the comparison between the ipsilateral side and contralateral side, and ex vivo autoradiography to validate the accumulation of the tracer. Accordingly, the PET data revealed that the AUC values on the ipsilateral side of the brain (ie, the transplanted site) in the 253G1 and U-251MG groups were significantly higher than those on the contralateral side of the brain (ie, the intact side) ( Figure 3B). Furthermore, ex vivo autoradiography confirmed that the anatomical distribution of radioactive accumulation on the transplanted site ( Figure 5A) and the ICR were significantly higher in the 253G1 group compared to the PBS-injected group ( Figure 5B). On the other hand, there was no significant difference between the 253G1 group (2.7 ± 1.2, n = 5) and the 414C2 group (1.4 ± 0.2, n = 4) in the ICR, despite the relatively higher tendency for radioactive accumulation in the 235G1 group. In general, autoradiography offers an order of magnitude higher spatial resolution than PET. 42 Therefore, ex vivo autoradiography was also able to detect radial accumulation of residual immature tissues in the 414C2 group two months post-transplantation ( Figure S5). This observation is not unexpected since non-proliferative undifferentiated cells may persist for several months (2, 3) post-transplantation even when nontumorigenic hiPSC-NS/PCs are used. 5,9 In other words, device development associated with PET, such as higher resolution and sensitivity  Figure 6A-D). A few Iba1 + microglia and GFAP + astrocytes were observed within the graft area, but only partially contributed to the overall TSPO expression ( Figure 6G,H). It is likely that these results reflect a similar pattern to the TSPO expression of glioma-associated microglia/macrophages (GAMs). 13,44,45 A previous report demonstrated that GAMs contributed to the uptake in glioma PET imaging with the TSPO radioligand, but their influence on glioma TSPO expression was weak. 46 Together, these results suggested that the detected signal by PET with [ 18 F] FEDAC would be predominantly derived from Nestin + undifferentiated neural cells after hiPSC-NS/ PCs transplantation, and not microglia nor astrocytes associated with neuroinflammation. Additionally, elevated levels of TSPO in inflammatory cells in response to lesions are reported to be directly related to the extent of the damage. 47,48 In the present study, we utilized these intact models after enough time had passed post-transplantation to exclude inflammatory factors that may have contributed to the total TSPO signal. 33,49,50 In order to validate the clinical application of [ 18 33,56 Moreover, it has been reported that the activated Iba1 + microglia, which present a major cellular source of TSPO, peaks 42 days post-SCI 50 while TSPO expression peaks within a week or 72 hours after infarction, especially in the cerebral infarction models and the brain injury models, respectively. 56,57 In order to elucidate time-dependent changes of TSPO expression after SCI in rodent models, we analyzed the gene expression of TSPO in mice at the nine days marker and 42 days marker after SCI using microarray. We observed that TSPO was significantly elevated 9 days after injury (dpi) ( Figure S7) and there was no significant difference between 9 and 42 dpi. Accordingly, we showed that the TSPO signal associated with inflammation reached its peak within 6 weeks post-TBI or SCI in rodent models. In the clinical setting, transplantation is routinely per-

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available on request from the corresponding authors.