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Prolonged Maturation Culture Favors a Reduction in the Tumorigenicity and the Dopaminergic Function of hESC-Derived Neural Cells in a Primate Model of Parkinson’s Disease

 

Other than purity and optimal functionality, one of the main concerns with the transplantation of embryonic stem cell (ESC)- or induced pluripotent stem cell (iPSC)-derived cells is their potential tumourigenicity in vivo. To address this, researchers from the group of Jun Takahashi from Kyoto University, Japan studied the effect of transplantation of neural progenitor cells (NPCs) derived from human ESCs (hESCs) into mouse brain and then into the brain of primate models of Parkinson’s disease. Interestingly, their findings conclude that prolonged in vitro culture of NPCs significantly reduces the risk of tumourigenesis, and that dopaminergic (DA) neurons derived from transplanted cells can lead to behavioural improvements (Doi and Morizane et al).

The initial part of the study entailed the investigation of a modified protocol (Hayashi et al and Kawasaki et al) for the generation of DA neurons from hESCs. hESCs were grown on Matrigel in the presence of PA6 stromal cell conditioned medium and formed spheroidal colonies with a large number of nestin (NES) positive NPCs by day 7, and were then expanded as floating spheres in the presence of FGF2 (Hayashi et al).   By day 28, the culture contained no undifferentiated hESCs as measured by the lack of OCT4+ NANOG+ cells.   From day 14-28, cells were treated with FGF8 and Shh, followed by replating on poly-L-ornithine/laminin coated dishes and grown in Neurobasal medium with BDNF, GBDF, ascorbic acid and dibutyryl cyclic AMP, for either one or two weeks. The authors note that through using this modified technique, neural induction and subsequent DA differentiation was more efficient than in previously published reports; a neural cell marker (SOX1) was maximal at day 14 while a post-mitotic neuronal marker (MAP2AB) gradually increased and markers for DA neural progenitors (LMXA1 and EN1) decreased after 28 days as markers of immature DA neurons (NURR1) and DA neurons (TH) increased. Differentiation of these cells to a midbrain DA neuron cell type was confirmed through the gradual increase in the ratio of TH+ to TUJ1+ cells up to day 42, while TH+ cells grown to day 56 extended neurites, expressed midbrain DA neuron markers (AADC, NURR1, PITX3 and GIRK2) and could secrete dopamine in response to high potassium stimulation.

In vivo growth of spheroids grown for differing times in vitro was then studied by comparing the growth of day 14, 28, 35 and 42 spheroids grafted into the right putamen of SCID mouse brain. Analysis at 8 weeks showed no teratoma formation for any grafts, but in brains grafted with day 14 and 28 spheres, neural epithelial cell-containing rosette formation was observed. NES- cells apparent within day 14 spheroid grafts were proliferative, although the majority of cells were NES+ with some proliferative capacity. In comparison, day 42 spheroid grafts were smaller than other grafts, with less proliferative cells, suggesting that day 14 grafts had higher tumourigenic potential.

This in vivo analysis was then extended to MPTP-treated cynomolgus monkeys, a Parkinson’s Disease model system (Burns et al), with day 14 and day 28 spheres (with no Shh or FGF8 added) injected through multiple tracts bilaterally. MRI studies at 6 months found that the lesions which formed were larger in the day 14 sphere injections, with such cells having a faster doubling time than the day 28 spheres. PET analysis of 2-[18F]fluoro-2-deoxy-glucose uptake in the brain found that glucose metabolism was also increased in the day 14 grafts while day 28 spheroids were similar to control monkeys. 3'- [18F]fluoro-3'-deoxythymidine ([18F]FLT), a fluorinated thymidine analog, is a PET tracer for evaluating cell proliferation activity, and analysis of this showed a high focal uptake only in the monkey that received day 14 sphere grafts. Histological analysis of money brains at 9 months found that those grafted with day 14 spheroids exhibited no signs of malignancy with only pigmentation and epithelial-like structures, but no hairs, cartilage, or muscle fibers were discovered. Regions of high [18F]FLT uptake were focused in small patches of cells with a high nuclear to cytoplasmic ratio, indicative of an undifferentiated hESC-like identity, but the vast majority of cells were neural cell-like. Day 28 grafts also exhibited no signs of malignancy, but importantly lacked the accumulation of undifferentiated cells observed in the day 14 grafts. Evaluation of spheroids cultured for longer periods of time (day 35 and 42) found no signs of tumours and in many cases grafts ceased growing at 6 months and in other cases, the cell doubling time became gradually longer indicating slower expansion. At 12 months, no focal uptake of [18F]FLT was observed, but cell-dense clusters were found in the grafts, which mainly contained NES+ NPCs, and not NeuN+ post-mitotic neurons, with no evidence of OCT4+ or NANOG+ cells and very low levels of proliferation. Histological analysis of the in vivo DA differentiation of the grafts found clusters of TH+ neurons extending neurites into the graft and surrounding brain tissue thus suggesting re-enervation to the putamen by the grafted cells. Many TH+ cells were also positive for midbrain neuron markers (VMAT2, AADC and PITX3) and most neurons were GABAergic. DA synthesis, as measured by PET using 6-[18F]fluoro-L-3,4-dihydroxyphenylalanine ([18F]DOPA) revealed increased uptake of [18F]DOPA in the grafts correlative with the neurological scores in each monkey, suggesting that dopamine production by the grafted cells contributed to their behavioural improvements. Neurological symptoms in the monkeys were measured using a neurological rating scale (Takagi et al) and a video-based analysis of spontaneous movements (Saiki et al) and this revealed that only the monkeys implanted with day 42 spheres showed significant behavioural recovery after three months.

Overall this study suggests that extended maturation ­in vivo allows for the loss/conversion of undifferentiated pluripotent cells during differentiation, reducing the risk of tumourigenesis associated with transplantation of NPCs in this case. Previous studies had found that extended culture of stem cells in vitro may allow for the accumulation of culture-associated genetic aberrations potentially leading to a lack of functionality. However, this study appears to show that transplanted cells still allow for some rescue of behavioural function in a Parkinson ’s disease model through the generation of functional DA-neurons.

 

References

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Doi et al.
Prolonged Maturation Culture Favors a Reduction in the Tumorigenicity and the Dopaminergic Function of Human ESC-Derived Neural Cells in a Primate Model of Parkinson's Disease. Stem Cells 2012; 30: 935–945.

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