ES cell-derived renewable and functional midbrain dopaminergic progenitors

Abstract

During early development, midbrain dopaminergic (mDA) neuronal progenitors (NPs) arise from the ventral mesencephalic area by the combined actions of secreted factors and their downstream transcription factors. These mDA NPs proliferate, migrate to their final destinations, and develop into mature mDA neurons in the substantia nigra and the ventral tegmental area. Here, we show that such authentic mDA NPs can be efficiently isolated from differentiated ES cells (ESCs) using a FACS method combining two markers, Otx2 and Corin. Purified Otx2 + Corin + cells coexpressed other mDA NP markers, including FoxA2, Lmx1b, and Glast. Using optimized culture conditions, these mDA NPs continuously proliferated up to 4 wk with almost 1,000-fold expansion without significant changes in their phenotype. Furthermore, upon differentiation, Otx2 + Corin + cells efficiently generated mDA neurons, as evidenced by coexpression of mDA neuronal markers (e.g., TH, Pitx3, Nurr1, and Lmx1b) and physiological functions (e.g., efficient DA secretion and uptake). Notably, these mDA NPs differentiated into a relatively homogenous DA population with few serotonergic neurons. When transplanted into PD model animals, aphakia mice, and 6-OHDA-lesioned rats, mDA NPs differentiated into mDA neurons in vivo and generated well-integrated DA grafts, resulting in significant improvement in motor dysfunctions without tumor formation. Furthermore, grafted Otx2 + Corin + cells exhibited significant migratory function in the host striatum, reaching >3.3 mm length in the entire striatum. We propose that functional and expandable mDA NPs can be efficiently isolated by this unique strategy and will serve as useful tools in regenerative medicine, bioassay, and drug screening.neural precursors | dopaminergic neurons | transplantation

Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors

“…S1). Nonetheless, recent studies using mouse (35) or human (36) pluripotent stem cells show that, when the initial cell pool is sufficiently large (i.e., as afforded by highly expandable stem cell populations), Corin can be used to isolate enough DA progenitors to generate functional grafts in rodent models of PD. This finding is an important advance for clinical application of pluripotent stem cells in neural grafting procedures, where some form of cell selection will almost certainly be required to eliminate potentially dangerous cell types, for example those capable of uncontrolled growth after transplantation.…”

Dopamine Receptors in Human Embryonic Stem Cell Neurodifferentiation

“…Our working hypothesis stated that facilitated neuroproliferation and en masse commitment to dopaminergic neuron type leaves neurons with less time and resources to build fully excitable membranes capable of AP generation. In the human fetus (16)(17)(18)(19)(20)(21), the postmitotic neurons mature gradually by inserting voltage-gated sodium channels into the plasmalemma, by decreasing their electrical input resistance, and by making their resting membrane potential more and more negative as neural maturation progresses [28]. The insertion of sodium channels in developing human neurons is manifested in electrophysiological recordings by an increase in peak amplitude of the fast inactivating sodium current paralleled by neuronal ability to generate APs [28].…”

“…Large numbers of transplanted neurons are necessary for their survival and integration into the host brain [15,16]. The number of surviving neurons in the host brain is considered the primary determinant of the efficacy of any cell replacement therapy, including the therapy of Parkinson's disease [17].…”

Dopamine Cell Transplantation for Parkinson's Disease: The Importance of Controlled Clinical Trials

“…The ideal markers for positive selection would be on the cell surface. Corin has been identified on the surface of dopamine cell precursors and antibodies to corin can isolate populations of dopamine precursors, which are co-expressing green fluorescent protein (GFP) under control of the otx2 promoter [71]. This kind of dual selection has not been worked out for human dopamine neurons.…”