First published online February 2, 2006
Stem Cells Vol. 24 No. 6 June 2006, pp. 1458-1466
doi:10.1634/stemcells.2005-0413; www.StemCells.com
© 2006 AlphaMed Press

Embryonic Stem Cell-Derived Neuronally Committed Precursor Cells with Reduced Teratoma Formation After Transplantation into the Lesioned Adult Mouse Brain
Marcel Dihnéa,b, Christian Bernreuthera, Christian Hagelc, Kai O. Weschea, Melitta Schachner

The therapeutic potential of embryonic stem (ES) cells in neurodegenerative disorders has been widely recognized, and methods are being developed to optimize culture conditions for enriching the cells of interest and to improve graft stability and safety after transplantation.

Whereas teratoma formation rarely occurs in xenogeneic transplantation paradigms of ES cell-derived neural progeny, more than 70% of mice that received murine ES cell-derived neural precursor cells develop teratomas, thus posing a major safety problem for allogeneic and syngeneic transplantation paradigms.

Here we introduce a new differentiation protocol based on the generation of substrate-adherent ES cell-derived neural aggregates (SENAs) that consist predominantly of neuronally committed precursor cells.

Purified SENAs that were differentiated into immature but postmitotic neurons did not form tumors up to four months after syngeneic transplantation into the acutely degenerated striatum and showed robust survival.

...Cell therapy is a promising approach to treat neurodegenerative diseases [1–3]. Whereas primary tissue transplants are limited in number and thus not applicable to the numerous individuals suffering from neural disorders, in vitro expandable populations of neural precursors represent an attractive alternative source for neural transplantation [4]. Embryonic stem (ES) cells can proliferate extensively in an undifferentiated state, thus providing an almost unlimited source of neural precursors after predifferentiation [5–7]. However, ES cell-derived neural precursor cell populations have been observed to be tumorigenic, particularly after allogeneic or syngeneic transplantations [8]. Thus, there is a need to predifferentiate ES cells such that their tumorigenic potential is minimized.

We have developed an optimized protocol for predifferentiation of ES cells and isolation of ES cell-derived neural aggregates, resulting in a high yield of neuronally committed cells in vitro and after transplantation in vivo without tumor formation in a syngeneic transplantation paradigm in which predifferentiated ES cell-derived neural aggregates from C57BL/6J mice were injected into the quinolinic acid-lesioned striatum of adult C57BL/6J recipient mice.

DISCUSSION:

In this study, we developed a protocol for generating and enriching SENAs. In contrast to ES cell-derived neural precursors differentiated with the conventional 5-stage protocol [12, 13], cells within SENAs differentiated into an almost pure (> 90%) population of neurons in vitro.

For transplantation experiments, we harvested SENAs at a stage of differentiation at which SENAs consist predominantly of postmitotic ß-tubulin+ neurons (> 75%) that do not yet express mature neuronal markers. When undissociated SENAs of this stage were syngeneically grafted into the quinolinic acid-lesioned striatum, no teratomas were found for up to 4 months after transplantation—the latest time point tested—whereas neural precursors generated with the conventional embryoid body-based, 5-stage differentiation protocol led to the formation of teratomas in 70% or 17% of syngeneic transplantation experiments, depending on their state of maturation.

When SENAs start to form in stage 4 at 6+, they consist mainly of proliferating nestin+, Pax-6+, and NCAM+precursors (from 6+ until 12+), which subsequently (from 12+ until 18+) differentiate into immature, postmitotic, ß-tubulin+/NF–/GABA– neurons (about 76% at 18+). After continued differentiation at 18+/6–, the fraction of ß-tubulin+ neurons within SENAs, generated from the EGFP+C57BL/6J or R1 ES cell lines, had increased further, to more than 90% with nearly half the cells expressing mature neuronal markers, such as NF-200, GAD-6, SV-2, or ChAT, indicating the predominantly neuronal commitment of SENAs (only ~8% are glial cells). In comparison, when applying the 5-stage differentiation protocol, the EGFP+ C57BL/6J or R1 ES cell lines, in agreement with previous studies [12, 13], yielded only about 13% or 55% neurons, respectively, whereas 33%–43% glial cells were detected pointing to an approximately equally sized potential of stage 4 cell populations to generate neurons or glial cells. To additionally compare the differentiation potential of SENAs with another type of neural precursor cells in their aggregated state, we generated neurospheres from the central nervous system of 14-day-old embryos and found, also in agreement with previous studies [17], a predominantly astrocytic commitment of neurospheres (> 70%). Thus, a clear increase in neuronal yield is achieved when applying the SENA differentiation protocol, indicating that the SENA-based differentiation strategy that can be applied to different ES cell lines is able to generate highly enriched, neuronally committed cell populations. In addition, it could be shown that ES cell-derived SENAs are fundamentally different from CNS-derived neurospheres, as SE-NAs cannot be generated in a free-floating fashion and as SENA cells are predominantly neuronally committed.

Purified SENAs at 18+/3– were chosen for transplantation into the acutely lesioned striatum of adult mice. Thus, SENA grafts consisted of numerous committed, immature neurons, but only a few nestin+ proliferating precursors. In comparison, stage 4 grafts, and, to a lesser degree, stage 5 grafts, consisted predominantly of proliferating nestin+ , Pax-6+, and NCAM+ precursors (Table 1Go). Although neurons are generally regarded to be more vulnerable toward toxic influences at the lesion site in comparison to immature nestin+ cells [18], most likely due to expression of NMDA receptors, which make them sensitive to excitotoxic insults [19], survival of cells in SENA grafts was robust, as indicated by the low percentage of caspase+ cells, which was comparable to that of stage 5 grafts. Indeed, determination of the number of NF+ (~57,000) and ß-tubulin+ (~60,000) cells in SENA grafts revealed neuronal survival of approximately 80% of all grafted neurons. In comparison, neuronal survival of 3%–5% or 22% has been reported in previous experiments [20, 21]. Robust neuronal survival in SENA grafts might be related at least in part to the grafting of nondissociated cellular aggregates. As ~50,000 transplanted SENA-derived cells had adopted a GABAergic phenotype, as assessed by GAD-65/67 immunohistochemistry (~83% of all SENA-derived neurons), SENAs provide a particular neuronal subtype after transplantation that can be used for neurodegenerative diseases with a loss of this cell type.

An additional benefit of SENAs was the absence of teratoma formation in the syngeneic transplantation paradigm used in this study. Erdö and colleagues [8] reported that murine ES cells and ES cell-derived stage 4 neural precursors formed tumors in 86% of recipient mice but not in rats, questioning the applicability of allogeneic or syngeneic transplantation strategies in humans. The reasons for tumor formation after syngeneic transplantation but not after xenogeneic transplantation are not known, but it is likely that immune rejection of the tumor occurs after xenogeneic transplantation. Assessment of possible teratoma formation after xenogeneic transplantation into immunodeficient recipients, for instance into nude rats [22], should clarify the role of a potentially teratoma-preventing immune response. In agreement with the results of Erdö and colleagues, we found that stage 4 grafts formed teratomas in 70% of recipient mice but not in recipient rats (unpublished observations), suggesting that the therapeutically most relevant allogeneic or syngeneic transplantation paradigms [23] can reveal the tumorigenic potential of ES cell-derived neural progeny that would not be recognized in xenogenic transplantations.

In our study, teratoma formation after syngeneic transplantation was decreased to 17% when predifferentiated stage 5 cells were transplanted, suggesting a relation between graft maturity and tumor formation. This notion was further supported by the observation that syngeneic SENA grafts did not generate teratomas during a post-transplantation period of up to 4 months. A plausible reason for the reduced teratoma formation of SENAs might be that some immature teratoma-forming ES cells may have escaped pre-differentiation toward stages 4 or 5 but not toward the SENA stage. However, assessment of possible Oct-4+ cells within stages 4 or 5 and SENA cell populations revealed that immature ES cells can be found only rarely in all of these cell populations, with less than 5 Oct-4+ cells/100,000 stage 4, stage 5, or SENA cells. Thus, it is unlikely that the very few immature ES cells in stages 4 and 5 and in SENA populations would give rise to teratomas only after grafting stage 4 or 5 cells.

Alternatively, one may argue that proliferating, immature, nestin+ /Pax-6+ /NCAM+ neural precursors in stage 4 and 5 grafts, induced by cues from the host tissue, regress more easily into teratoma-forming cells than ß-tubulin+ neurons within SENA grafts. Our assessment of the proliferation and maturity of stage 4 and 5 cells and cells in SENAs before transplantation revealed a high correlation between proliferation and maturity in vitro and tumor formation in vivo, supporting the possibility that at least some ES cell-derived nestin+ neural precursor cells can be re-programmed in vivo toward an immature, teratoma-forming state. Thus, this study suggests that even purification of ES cell-derived nestin+ /Pax-6+ /NCAM+ neural precursors does not prevent teratoma formation and that ES cells should rather be differentiated toward a more mature state. However, as also SENAs contain some proliferating nestin+ cells, it has to be speculated if they also have a minimal tumorigenic risk.

Our observations are thus noteworthy with regard to long-term safety and efficacy of ES cell therapy in humans. Although most transplantation studies have used immature, nestin+ neural precursors or slightly predifferentiated neural precursors we have grafted ES cell-derived aggregates consisting predominantly of ß-tubulin+ neurons. Our study demonstrates that the state of maturity of ES cell-derived transplants critically determines tumorigenicity and provides a platform for further experiments aimed at using ES cell-derived transplants to treat neurological disorders.