Rescue of radiation-induced cognitive impairment through cranial transplantation of human embryonic stem cells

In Vitro and in Vivo Immunostaining of hESCs.

Before transplantation, hESCs were maintained on matrigel in an undifferentiated state. Pluripotency of hESCs was verified by immunostaining for Oct-4 (transcription factor Octamer-4) and SSEA-4 (glycolipid surface - Stage Specific Embryonic Antigen-4), and under our culture conditions, cells showed abundant expression of these markers (Fig. 1 A–D). For the studies presented, hESCs were used at passages 42–49.

To trace hESCs after grafting, hESCs were labeled in vitro with BrdU before transplantation. In vitro controls verified the colabeling of hESCs with BrdU and a human specific nuclear antigen (Fig. 1E). These labels were also used to verify the presence of the BrdU positive human cells in tissue sections of animals engrafted with hESCs (Fig. 1F). The correspondence between cells colabeled with BrdU and human specific markers indicated that grafted cells did not undergo significant proliferation postgrafting, and validates these markers for tracking of grafted hESCs in the host tissue. A schematic representation of our study is illustrated in Fig. 1G.

Transplanted hESCs Rescue Cognitive Deficits.

Four months postgrafting, rats were habituated and trained on a novel place recognition task (NPR). The NPR task uses the innate tendency of rats to explore novelty. First, rats are exposed to two identical objects in specific spatial locations within a test arena. Following a retention interval, one of the objects is moved to a new spatial location and rats are placed in the test arena again. Rats that remember the previous spatial arrangement of the objects will spend more time exploring the object that has been moved to the novel spatial location. Successful performance of the task has been shown to rely on intact hippocampal function (46, 47).

As expected, irradiated rats (IRR) showed impaired novel place recognition at the short, 5-min retention interval. Irradiated animals spent significantly less time exploring the novel place compared to control animals (Fig. 2A). In contrast, after the 5-min retention interval, irradiated, hESC-transplanted rats (IRR+hESC) did not differ from control animals and spent more time than expected by chance (indicated by the dashed line in Fig. 2A) exploring the novel spatial location (Fig. 2A). Similar trends were observed in terms of total time spent exploring the novel spatial location (Fig. 2B), and in frequency of visits to the novel spatial location and latency to explore the novel spatial location. These results suggested that hESC transplantation attenuated post-irradiation cognitive impairments and preserved short-term memory for spatial information in a hippocampal-dependent task.

At the 24-h retention interval, IRR rats again showed impaired NPR compared to control animals (Fig. 2C). By contrast, IRR+hESC rats did not differ significantly from control animals. However, IRR+hESC animals did not spend more time than expected by chance exploring the novel spatial location. Again, we observed similar trends when we analyzed total exploration of the novel spatial location (Fig. 2D), as well as frequency of visits and latency to first explore the novel location. The results suggest that hESC transplantation may also improve long-term retention of spatial information, although the treatment appeared to be more effective at the shorter, 5-min retention interval. During the 24-h test phase only, control animals receiving hESCs spent significantly less time exploring the novel place compared to controls receiving vehicle injections (Fig. 2C). This result suggests that hESC implantation in control animals disrupts long-term but not short-term recognition memory, although further behavioral testing is necessary to confirm this finding. These data suggest that in the intact hippocampus, hESC transplantation may have a distinct dose-response (i.e., cell number) or may actually disrupt hippocampal function as compared to an irradiated microenvironment that might be more conducive to integration of grafted hESCs.

To better understand why IRR animals showed impaired short- and long-term NPR abilities, we analyzed total time spent exploring both objects during the familiarization phase given before the retention intervals. We found a significant difference between the groups; IRR animals tended to spend less time exploring during the familiarization phase compared to both the control and IRR+hESC animals (Fig. 2E). This result suggested that the impairments seen in the IRR animals at 5min and 24 h post-familiarization might be related to lower exploration during initial exposure to the objects.

Location and Migration of hESCs in the Irradiated Rat Hippocampus.

At 4 months posttransplant and after cognitive testing, animals in each group were euthanized for immunocytochemical analyses. An additional cohort of animals not subjected to cognitive tests was analyzed at 1 month post-transplant, to assess early patterns of incorporation and differentiation of the grafted hESCs. An examination of BrdU immunostained sections from animals that received transplants revealed that the majority of grafted cells were located within the hippocampal formation and adjacent to the CA1 subfield and corpus callosum (Fig. 3). Representative sections showed the intrahippocampal location of grafted hESCs at 1 (Fig. 3 A–C) and 4 months post-transplant (Fig. 3 D–F). Similar localization and migration patterns of hESCs were found at both post-transplant times in all animals receiving grafts. Moreover, the data show that grafted hESCs migrated extensively into the dentate gyrus (DG), hilus (DH), and hippocampal CA1 subfield, (Fig. 3). In both irradiated and unirradiated animals, grafted hESC were also found to exhibit significant homing to the SGZ, populating this neurogenic niche throughout the septo-temporal axis of the host hippocampus.

Neural Differentiation of Transplanted hESCs.

To analyze the differentiation of engrafted hESCs, dual immunofluorescence and Z-section analyses using confocal microscopy were performed on representative sections at 1 and 4 month post-transplant. Analysis of BrdU colabeling with NeuN or GFAP demonstrated that a portion of cells showed neural and astrocytic differentiation at both posttransplant times in all animals that received hESC grafting. Grafted cells positive for both BrdU and NeuN displayed mature neuronal morphology (Fig. 4A, B, E, and F). Grafted cells expressing these neuronal markers were predominately found at the SGZ, as opposed to other hippocampal subfields. Assessment of astrocytic differentiation via BrdU and GFAP dual immunofluorescence revealed the presence of GFAP positive astrocytes at each posttransplant time analyzed (Fig. 4 C, D, G, and H). Grafted cells expressing astrocytic phenotypes were uniformly distributed throughout the hippocampus, with no apparent preference for a particular hippocampal region.

Animals and Irradiation.

Young (2-month-old) athymic nude rats acquired from National Cancer Institute (strain 02N01 Cr:NIH-rnu) were used in this study. All animal procedures were performed in strict accordance with the National Institutes of Health and Institutional IACUC guidelines. Anesthetized rats were subjected to head only γ-irradiation (10 Gy) using a ¹³⁷Cs irradiator (J.L. Shepard and Associates Mark I) at the dose rate of 2.07 Gy/min. Growth and characterization of hESC and the processing of all tissue sections followed standard procedures (see SI Methods for further details).

Intrahippocampal Transplantation of hESCs.

Rats received bilateral intrahippocampal injections of BrdU labeled hESCs 48 h after cranial or sham irradiation. 1.0 × 10⁵ live cells were injected in 1 μL of the cell suspension using a 5-μL Hamilton microsyringe (33-gauge) at an injection rate of 0.25 μL/min. Each hippocampi received four distinct graft injections using the following stereotaxic coordinates: (i) AP: 3.0 mm from bregma, ML: 1.8 mm from midline, and ventral DV: 3.2 mm from the surface of the brain; (ii) AP: 3.6 mm, ML: 2.5 mm, DV: 3.2 mm; (iii) AP: 4.2 mm, ML: 3.2 mm, DV: 3.2 mm and (iv) AP: 4.8 mm, ML: 4.0 mm, DV: 3.2 mm. Thus, the hippocampus of each hemisphere received a total of 4.0 × 10⁵ live hESCs. Control and irradiated rats receiving vehicle (sterile conditioned medium) at the same stereotaxic coordinates served as sham surgery groups.

Behavioral Analysis.

At 4 months post-grafting, a blinded observer examined rats in all groups [control (Con+hESC) and irradiated (IRR+hESC) rats receiving hESC grafts, control (Con) and irradiated (IRR) rats receiving sham surgery and vehicle injections], for hippocampal-dependent spatial memory using a novel place recognition (NPR) task according to a standard protocol (46) (SI Methods).

Measures and Analyses.

Exploration ratio, or the percentage of total exploration time spent exploring the novel spatial location (t_novel/t_novel+t_familiar) was used as the main dependent measure. We chose to analyze the behavior of the animals during minute 1 of the 3-min, 5-min, and 24-h test phases because previous research has shown that preference for the novel place diminishes after the first minute, as the spatial locations become equally familiar to the animals (46). For completeness, repeated measures ANOVA that included all data from each of the three test sessions were performed. These analyses confirmed our behavioral findings found during the first minute. We also analyzed the total time, frequency of visits, and latency to first explore the novel place during the test phases. Additionally, time spent exploring both familiar places was recorded during the familiarization phase.

Exploration ratio data were analyzed using univariate ANOVAs for the 5-min and 24-h test phases (PASW Statistics version 17.0, SPSS Inc.). When a statistically significant (i.e., P ≤ 0.05) overall group effect was found, Fisher Protected Least Significant Difference (FPLSD) post hoc tests were run to compare the four groups.