Here we report that intrahippocampal engraftment of hNSCs prevented the development of radiation-induced cognitive impairment, showing that engraftment of multipotent stem cells can protect the brain from a serious side effect of cranial irradiation. Following irradiation and engraftment, hNSCs survived, migrated extensively throughout the hippocampus, and expressed neuronal and astrocytic markers (–). Engrafted cell survival quantified by unbiased stereology revealed that 23% and 12% of the transplanted cells survived at 1 and 4 months, respectively (). Quantification of phenotypic fate of the engrafted hNSCs at these times revealed the addition of both immature and mature neurons and astroglial cell types in the irradiated hippocampus (). Thus, as few as approximately 100,000 surviving cells (15% neuronal, 45% astroglial) in the irradiated brain were sufficient to prevent the cognitive deficits observed in irradiated rats receiving no engraftment. Although the mechanism(s) of stem cell–based cognitive rescue has not been elucidated, it is likely that some combination of functional replacement and/or trophic support is playing a role. The identification of Arc-positive engrafted cells suggests that the transplanted hNSCs can functionally integrate into the hippocampal circuitry (), which points to a possible mechanism for stem cell–based effects on cognition after cranial irradiation.
The assessment of cognitive performance was accomplished using an NPR task. Successful performance of this task depends on intact hippocampal function (26
). The present findings corroborate prior behavioral findings and suggest that radiation-induced alterations in cognition are caused, in part, by injury to the hippocampus (28
). Irradiated animals that received vehicle injections exhibited significantly impaired NPR, showing that short- (5 minutes) and long-term (24 hours) memory for a specific spatial arrangement of the objects was impaired by exposure to 10 Gy head-only irradiation (). In contrast, irradiated animals transplanted with hNSCs (IRR + hNSC) did not display impaired spatial memory and their performance was comparable with that of unirradiated controls, showing the ability of engrafted cells to prevent radiation-induced cognitive dysfunction.
The behaviorally induced immediate early gene Arc
, and its protein product, is induced in the hippocampus by spatial exploration (30
). Arc is rapidly activated by robust patterned synaptic activity related to learning and memory behavior (33
), and altering Arc expression impairs spatial learning and long-term potentiation (34
). Therefore, Arc expression has been used extensively to map neuronal networks that underlie information processing and plasticity (30
). Our data showing the presence of BrdUrd+
engrafted cells suggest that some of the transplanted cells integrated into existing neuronal circuits (), although the full extent of such integration and its functional significance remains to be determined. A recent report found a gradual, time-dependent increase in Arc expression in newly born DG cells that eventually differentiated into mature neurons and incorporated into the hippocampal circuitry (35
). Stereologic estimates derived from the 1-month postgrafting time showed an average survival of 123,000 engrafted cells per hippocampus. Extrapolating this number yields approximately 13,500 cells expressing Arc in each hippocampus or approximately 27,000 per brain. Whether these engrafted cells prevent cognitive decline or actually improve cognition is uncertain at this time, but the recent finding of super-connected “hub” cells in the brain (36
) suggests the intriguing possibility that engrafted stem cells may be preferentially recruited as such cells to foster and preserve the functional connectivity of the CNS (37
Functional integration of engrafted hNSCs within the hippocampus may not be necessary or sufficient for cognitive rescue. Restoration of damaged synaptic circuits may also require the presence of glial cells to support preexisting neurons. Grafted glial cells may serve to repair or improve the function of existing cells either directly, or indirectly, by mediating the remodeling of the irradiated microenvironment (38
). We found 32% and 46% astroglial differentiation of engrafted hNSCs at 1- and 4-month postengraftment group, respectively (). This resulted in the addition of 78,000 and 45,000 astrocytes () in the irradiated hippocampus. Significant past work using various injury and disease models suggests that engrafted cells secrete a range of beneficial growth factors, such as glial cell line–derived neurotrophic factor (40
), and brain-derived neurotrophic factor (42
). The positive influence that engrafted cells have on cognition is noteworthy given the duration of the postirradiation intervals and the multifaceted nature of learning and memory. Future studies will determine whether engrafted cells exert longer-lasting changes in synaptic plasticity, if that effect is dependent on integration of cells into the hippocampal circuitry and/or if it is facilitated through trophic support.
The hNSCs used here were multipotent based on marker expression in vitro
), and the BrdUrd labeling indices of the cells was approximately 90%. Noteworthy too was the absence of any overt adverse cognitive sequelae (e.g., ataxia) in the cohort of animals receiving hNSC grafting, suggesting that intracranial teratogenesis and/or hyperproliferation was not problematic over the duration of this study. This was supported by the small fraction of transplanted cells that remained multipotent (nestin+
) and/or retained their capacity to proliferate (Ki67+
; ). Analysis of brain sections revealed that engrafted cells migrated extensively throughout the hippocampus (). Engrafted cells near the SGZ or at other hippocampal sites such as the CA1 showed characteristic signs of differentiation, as mature neurons and glia developed morphology that is distinct to those lineages ( and ).
Our current studies have now clearly shown the benefits of engrafted stem cells for reversing cognitive impairments following cranial irradiation. Moving these promising results into the clinic for the treatment of radiation-induced cognitive dysfunction along with other degenerative conditions will require careful consideration of many potential limitations including immunorejection and tumorigenesis (24
). Use of patient-derived induced pluripotent stem cells may one day alleviate certain practical concerns (43
), but considerable effort is still required to further define mechanisms and optimal treatment parameters. These efforts will help determine the timeline and feasibility of using similar stem cell–based therapies as safe and effective options in humans. The capability to minimize the adverse cognitive sequelae associated with cranial radiotherapy is encouraging and points to the promise of using stem cell–based strategies for minimizing normal tissue damage.