ES cell-derived renewable and functional midbrain dopaminergic progenitors

Purification of mDA NPs using coexpression of Otx2GFP and Corin. (A–P) Immunocytochemistry on FACS-purified cells using anti-Corin antibody and anti-Otx2 antibody. (Q and R) FACS purification of mDA NPs efficiently removed residual pluripotent (SSEA1⁺) cells. (S–V) Purified Otx2⁺Corin⁺ cells express other mDA NP markers such as FoxA2 and Lmx1b. (W–BB) Purified Otx2⁺Corin⁺ cells also express Nestin and Glast, whereas there are only few cells that express mature neural markers β-tubulin and GFAP. (Scale bar: 50 μm.)

Purified Otx2⁺Corin⁺ cells generate mDA neurons. (A–D) TH immunocytochemistry. (E–J) Purified Otx2⁺Corin⁺ cells generated mature mDA neurons that coexpress Pitx3, Lmx1b, Nurr1, DAT, and DDC. (K and L) Purified cells generated DA neurons with both A9-like (Aldh1a1⁺) and A10-like (Calbindin⁺) phenotype. (M–R) FACS purification significantly enrich DA neurons, and significantly decrease GABAergic neurons. (S and T) Serotonergic neurons were efficiently removed after double FACS sorting. (Scale bar: 50 μm.) (U) Cell-counting analysis before and after FACS (mean ± SEM; n = 4). Unsorted or Otx2⁺Corin⁺ cells at ND stage were analyzed for DA differentiation, GABAergic differentiation, or serotonergic differentiation. (V) DA release analysis (mean ± SEM; n = 4). (W) Specific DA uptake by DAT (mean ± SEM; n = 4). (X) The injection of depolarizing current (10 pA) to purified cells at the ND stage led to the firing of action potentials by the recorded cell under current-clamp conditions. (Y) The first spike from X on a faster time scale. The action potential is followed by the large AHP. (Z) Representative traces showing sEPSCs under control conditions. (AA) Traces show sEPSCs recorded from a single cell, which were aligned by their onset.

bFGF and FGF8 support proliferation of purified mDA NPs. (A) Fold increase of purified mDA NPs in the presence of various signaling molecules (mean ± SEM; n = 4). (B–K) Cells treated with bFGF or FGF8 show robust proliferation as shown by Ki67 staining. (L–U) Immunocytochemistry analysis showing that in the absence of an mDA NP proliferating signal, bFGF, or FGF8, purified cells spontaneously differentiated into DA neurons, as shown by TH staining. (Scale bar: 50 μm.)

Purified mDA NPs can be expanded in vitro without losing their developmental potential. (A) Growth curve of purified mDA NPs in the presence of bFGF (mean ± SEM; n = 3). (B–I) The majority of mDA NPs express FoxA2, Otx2, Nestin, and Glast after 4 wk of expansion. (J–O) Purified mDA NPs can generate DA neurons after 4 wk of expansion in bFGF coexpressing TH and β-tubulin. (P–T) The proportion of neural subtypes does not change significantly over expansion (mean ± SEM; n = 4). (U–HH) Further characterization of ND cells derived from 4 wk-expanded mDA NPs coexpressing TH with Pitx3, DAT, DDC, Nurr1, and Lmx1b. Expanded cells also generate both A9 and A10 phenotype. (Scale bar: 50 μm.)