Efficient derivation of neural precursors and dopamine neurons from human embryonic stem cells and their application to a Parkinsonian rat model

Abstract

[한글]

[영문]Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain-type neural precursor (NP) cells that subsequently generate high yields of dopamine (DA) neurons. These findings indicate that hES-derived NP cells can be a prospective cell source to be used in cell replacement strategies for Parkinson’s disease (PD). To further clarify the prospect, we have scrutinized the properties of hES-NP cells in the points of (1) capability of these cells in stably providing human DA neurons in a large scale (2) cell survival and tumorigenic potentials which are the most critical issues for an effective and safe cell therapy.First, we investigated the potential of hES-NP cells for the large-scale generation of human DA neurons for functional analyses and therapeutic applications. To address this, hES-NP cells were expanded in vitro for 1.5 months with 6 passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES-NP cell number was increased by >2×104-folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity towards neuronal cells, exhibiting in vitro presynaptic DA neuronal functionality. Furthermore, the hES-NP cells can be cryopreserved without losing their proliferative and developmental potential. Taken together, these findings indicate that hES-NP cell expansion is exploitable for a large-scale generation of experimental and transplantable DA neurons of human-origin.In second part of this thesis, we describe cell survival and tumorigenic aspects of these hES-NP cells that are critical issues in cell therapeutic approaches for PD. Neuroepithelial rosettes, a potentially tumorigenic structure, disappeared during hES-NP cell expansion in vitro. Although a minor population of cells positive for Oct3/4, a marker specific for undifferentiated hES cells, persisted in culture during hES-NP cell expansion, they could be completely eliminated by subculturing hES-NPs under differentiation-inducing conditions. Consistently, no tumors/teratomas formed in rats grafted with multi-passaged hES-NPs. However, extensively expanded hES-NP cells easily underwent cell death during differentiation in vitro and after transplantation in vivo. Transgenic expression of Bcl-XL and sonic hedgehog (SHH) completely overcame the cell survival problems without increasing tumor formation. These findings indicate that hES-NP cell expansion in conjunction with Bcl-XL+SHH transgene expression may provide a renewable and safe source of DA neurons for transplantation in PD.In conclusion, we provide evidences for hES-NP cells as a continuous, stable, and on-demand source for human DA neurons. Multiple passaging of these NP cells helped for a large scale generation of human DA neuron free from tumor formation potentials. However, survival of the multi-passaged hES-NP cells was drastically decreased in vitro and in vivo after transplantation. Although transgene expression of Bcl-XL+SHH overcome the cell survival problem, the cell survival issue remain to be further studied.