Up to this point, we have discussed the necessity of starting with an animal model that mimics the clinical pathology for the neurological disease of interest and supports engraftment as well as long-term survival, explored lessons learned from the hematopoietic transplantation field and mechanisms of allogenic and xenogeneic rejection, and reviewed early classifications of the CNS, as well as embryonic and neural stem cells, as ‘immunoprivileged’. We now present a comprehensive review of the literature regarding achieving engraftment and cell survival with xenografts in the CNS. We focused our literature review on studies xenotransplanting human cells into rodent models of neurological diseases/trauma or into the normal brain. For purposes of this review, we define ‘engraftment’ as the percentage of animals that demonstrate surviving cells (total number of animals at sacrifice with human cells still present divided by the total number of initially transplanted animals × 100). In parallel, we define ‘cell survival’ as the total number of cells present at sacrifice. The total number of cells must have been assessed either via unbiased stereology (typically via optical fractionator) or bioluminescence for the percentage cell survival report to be included in our tables. Unbiased stereological techniques permit rigorous quantitative analysis of tissue, including accurate volume-corrected estimates of cell number; because changes in tissue and structure volume due to disease/injury pathogenesis can be a significant experimental confound, stereological analysis is the gold standard for determining cell number, lesion volume and other variables vulnerable to these artifacts (see [114
] for a review of the use of stereology in neuroscience). A limitation to the cell survival data generated by stereological analysis in the CNS is how the region of interest is defined. If the analysis is confined to an anatomically defined region (e.g., the striatum) [116
], but the transplanted cell population has migrated beyond this artificial boundary, the determination of estimated cell number will only be accurate within the striatum, and the total number of surviving cells will be underestimated. Because of this limitation, papers that have this constraint in stereological data collection have been included in the overall tables, indicated by a symbol, and in the calculation of percentage engraftment, but have not been included in the calculation of percentage cell survival. Bioluminescence also permits quantitative analysis of cell survival within tissue, however, there are two critical limits for cell detection. First, sensitivity; while stereology permits an estimate of total cell number based on the detection of every cell visualized by immunoperoxidate, immunofluorescence, or promotor driven fluorescence in a transplanted tissue, bioluminescence has a clear threshold for detection that is affected by many factors, including transplantation site/depth. In the neural stem cell transplantation field, at least one study has demonstrated that the number of luciferase-expressing cells necessary to generate a detectable bioluminescence signal is in the order of 1000 [117
]. Coupled with this detection threshold limit, the propensity of transplanted cell populations for migration will significantly affect the accuracy of total cell survival quantification by this method. Finally, the long-term stability of luciferase expression has not been established, and decrements in signal may, in some cases, result from promoter downregulation [117
]. Owing to the limitations of bioluminescence in providing accurate total cell survival quantification, papers using this method of quantification have been included in the overall tables, indicated by a symbol, and in the calculation of percentage engraftment, but have not been included in the calculation of percentage cell survival.
Literature searches for this analysis of xenotransplantation in the CNS were performed in January 2011 with the keywords ‘human stem cell’ in combination with other keywords in series: ‘transplantation’, ‘brain’, ‘CNS’, ‘spinal cord’, ‘spinal cord injury’, ‘stroke’, ‘middle cerebral artery occlusion’, ‘ischemia’, ‘brain injury’, ‘brain trauma’, ‘multiple sclerosis’, ‘Parkinson disease’ and ‘Huntington disease’. Additional references were added when found cited in the initial round of papers retrieved via MedLine. No papers were excluded from our analysis, a priori. Using these criteria, we found 133 unique, relevant papers. It should be noted that the primary focus of any given paper need not have been the key variables discussed in this article (i.e., engraftment and cell survival). Rather, many papers compared a cell line with and without an experimental treatment or other component in an injured environment, or the effects of a cell line on functional outcome, and did claim to assess either engraftment or quantify total surviving cells. We grouped these papers into three primary categories based on common features of the model: models of normal neonatal or adult brain; models of acute/traumatic injury (SCI, traumatic brain injury or stroke); and models of chronic/atraumatic injury (Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, amyotropic lateral sclerosis, allodynia or demyelination). Within each primary category, references were subdivided into those using immunocompetent animals with immunosuppression, those using immunocompetent animals without immunosuppression and those using immunodeficient animals. A summary of the primary categories and the number of papers using immunocompetent versus immunodeficient animals is shown in .
We recorded 25 variables for each paper: citation, URL, model, primary category of model, paradigm or method, cell type, host species/sex, immunocompetent or immunodeficient host, location of injury, transplant time (time post-injury), final dose of cells, volume of injection, route/location of transplant, immunosuppressant, dose and duration, detection method for human cells, quantification method (stereology or number of sections), treatment groups, survival time (post-transplant), total number of transplanted animals in a group, numbers of animals with cells at sacrifice, percentage engraftment, and behavioral outcome. Factors that were not applicable for a given model or not reported were noted as well. In order to accurately assess the influence of immunocompetent (with or without immunosuppression) or immunodeficient models on successful engraftment and cell survival, we included only those studies using cell suspensions and excluded studies using solid grafts where total cell number (dose) or surviving cell number could not be determined. Individual tables are provided for each of the three primary categories (models of normal neonatal or adult brain, models of acute/traumatic injury and models of chronic/atraumatic injury, as defined previously); these primary categories are further subdivided by whether cells in the identified studies were transplanted into immunocompetent animals without immunosuppression, immunocompetent animals with immunosuppression, and immunodeficient animals (–).
Normal CNS: immunocompetent animals with immunosuppression.
shows that of the 133 unique papers, six papers examined xenotransplantation in normal brain (5%), 95 papers examined xenotransplantation in acute/traumatic models (71%), and 32 papers examined xenotransplantation in chronic/atraumatic models (24%). A total of 79 papers (59%) reported neither engraftment nor survival, while only 11 papers (8%) reported both engraftment and survival. For example, in the primary category ‘acute/traumatic’, 37 out of 95 papers (40%) used no immunosuppression in immunocompetent animals. Seven of these 37 papers reported the percentage of animals engrafted, only one reported the percentage of cell survival based on stereology, no papers reported both engraftment and percentage of cell survival, and 30 reported neither variable. In this category (acute/traumatic), the one paper to report cell survival (based on stereology) in a stroke model without the use of immunosuppression reported that only 35% of the initial cell dose survived 8 weeks post-transplant across four treatment groups [118
] (see for additional details).
Acute/traumatic: immunocompetent animals with no immunosuppression.
Several key conclusions can be drawn from & . First, the majority of CNS xenografts have used immunocompetent animals coupled with immunosuppression (n = 69 or 52%). The next most common paradigm is to use immunocompetent animals and no immunosuppression (n = 47 or 35%). Only 19 papers (or 14%) used immunodeficient animals in CNS xenograft studies using human cells. Second, the percentage of animals engrafted (when reported) is usually highest in immunodeficient animals. Third, the paucity of studies in the normal CNS and chronic/atraumatic cohorts that have employed immunodeficient models and quantitatively assessed engraftment/cell survival makes acute/traumatic models the only category in which these variables can be compared between immunocompetent and immunodeficient animals, and makes this the most robust category from which to draw relative conclusions for xenotransplantation success. In the acute/traumatic/cohort, the highest percentage of cell survival is in immunodeficient animal models (263%, n = 4). Fourth, although the studies available for comparison are limited, the data suggest that an uninjured niche (normal brain) may be no better than an injured niche in terms of engraftment or cell survival. Finally, caution should be exercised in interpreting papers that report surviving cell numbers. Many papers extrapolate total cell number from a limited number of histological sections. Moreover, even when systematic random sampling and stereological assessment of total cell number is performed, the number of animals assessed can be insufficient to yield interpretable numbers. For example, Suzuki et al.
report the stereological assessment of total cell number in an amyotropic lateral sclerosis model where 114% of the initial dose of cells was detected 6 weeks post-transplant; but quantification was performed in only one animal [119
Proportion of papers from that use immunocompetent or immunodeficient models.
Looking at those papers that reported either human cell engraftment or survival numbers, several interesting issues are evident. First, in immunocompetent animals given immunosuppression, cell survival decreases over time. Yashuhara et al.
transplanted green fluorescent protein (GFP)-expressing human HB1.F3 fetal-derived neural stem cells in a 6-OHDA toxicity model of Parkinson’s disease and quantified GFP+
/human nuclear antibody (hNuc)+
cells in every fifth section through the entire striatum [57
]. Animals received 200,000 cells in 3 μl phosphate-buffered saline into the ipsilateral striatum immediately following the 6-OHDA lesion. Sets of animals (n=8 per time point) were sacrificed 3, 7, 14 or 28 days post-transplant. All animals received daily intraperitoneal injections of cyclosporin A (10 mg/kg). Cell survival within the striatum was 11.2, 4.7, 1.7 and 1.1% across the four time points, respectively. Although quantification was not a true stereological assessment (neither random counting frames nor stereological dissectors were used), these cell survival numbers are directly comparable within the study and demonstrate that a host rejection response was likely active and involved in rapidly killing the human cells over time. Notably, it is possible that GFP expression may have also been downregulated over time in conjunction with a rejection response, resulting in the observed reduction in total cell numbers over time; however, antibodies to a human nuclear antigen were also used to detect the transplanted cells [57
Second, while direct comparisons within a single study between groups of immunocompetent animals receiving either immunosuppression or no immunosuppression are rare, not using immunosuppression in immunocompetent animals significantly reduces successful engraftment. Wennersten et al.
transplanted 210,000 human fetal-derived neural stem cells into a contusion model of SCI immediately postinjury [120
]. Animals received cyclosporin A for 3 or 6 weeks post-transplant (4 mg/kg Monday and Wednesday, and 8 mg/kg on Friday), while a third group received no cyclosporin A post-transplant. The three groups were sacrificed 6 weeks post-transplant and the presence or absence of humans cells was confirmed using hNuc immunohistochemistry. All animals receiving cyclosporin A, regardless of length of administration, were successfully engrafted, but only one of six animals (16.7%) contained human cells in the no cyclosporin A group. A fourth group of animals (n = 8) received cyclosporin A for 3 weeks but was allowed to survive 6 months instead of 6 weeks; five out of eight (62.5%) exhibited successful engraftment at 6 months. Quantification of cell numbers was not performed [120
]. While this study suggests that transient immunosuppression may suffice to achieve long-term engraftment, it also demonstrates that using no immunosuppression in immunocompetent animals significantly reduces the rate of successful engraftment. Unfortunately, without quantification of total cell numbers in such a study, it is impossible to ascertain the effect of short versus long-term immunosuppression on cell survival.
Third, in the few studies that conducted direct comparisons, human cell engraftment was shown to be higher in immunodeficient animals than immunocompetent ones. Deng et al.
transplanted human olfactory ensheathing cells into a hemisection model of SCI using either athymic nude (immunodeficient) or Sprague–Dawley (immunocompetent) rats [121
]. Their data demonstrate that survival of human olfactory ensheathing cells transplanted into immunocompetent animals was minimal at 24 h post-transplant and no surviving cells were identified by 7 days post-transplant; robust macrophage infiltration was found at the injection site by 7 days and engraftment was 0%. Conversely, human olfactory ensheathing cells transplanted into athymic nude rats survived and migrated away from the site of injection at 24 h and 7 days post-transplant. Engraftment was observed in 40% of the athymic nude rats, while no engraftment was observed in Sprague–Dawley rats [121
]. Although this study transplanted olfactory ensheathing cells rather than a strictly defined stem cell population, the proliferative properties of olfactory ensheathing cells in vitro
are well known, suggesting that activation of the host immune system initiates a rejection response.
The goal of this comprehensive literature review was to assess the status of the field in achieving adequate engraftment and survival in xenotransplantation models to predict clinical translation. As noted earlier, it should be acknowledged that the principal end point of a given study may not have been the assessment of cell survival per se, and such studies can still contribute meaningful data to the literature. This review of 133 xenotransplantation papers shows that the field of regenerative medicine has focused heavily on the administration of cyclosporin A alone in immunocompetent animals as a strategy for proof-of-concept experiments, resulting in both poor engraftment and low to very low cell survival (when reported). Furthermore, this summary shows that xenografted stem cells retain proliferative capacity in immunodeficient versus immunosuppressed models within the acute/traumatic category. While other differences in the acute/traumatic CNS niche could contribute to differences in the retention of proliferative capacity, it is likely that these differences provide insight into the capacity to initiate immunorejection mechanisms in these conditions. Again, given that acute/traumatic models represent the only category in which multiple studies have quantified engraftment and survival in immunodeficient animals, these data represent the most robust category from which to draw relative conclusions for xenotransplantation success. Combined with our historical survey of the broader xenotransplantation field, this analysis clearly suggests that it will be necessary to administer a multimodal course of pharmacological immunosuppression to achieve meaningful engraftment of a transplanted human cell population when using immunocompetent animals. Alternatively, immunodeficient animals yield much higher engraftment and cell survival numbers than using immunocompetent animals (with or without immunosuppression).