Differentiation of human ES and Parkinson's disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid

Abstract

The cardinal motor symptoms of Parkinson's disease (PD) are caused by the vulnerability to dysfunction and degeneration of ventral midbrain (VM) dopaminergic (DA) neurons. A major limitation for experimental studies of current ES/iPS cell differentiation protocols is the lack of VM DA neurons with a stable phenotype as defined by an expression marker code of FOXA2/TH/β-tubulin. Here we demonstrate a combination of three modifications that were required to produce VM DA neurons. Firstly, early and specific exposure to 10(-)(8)M (low dose) retinoic acid improved the regional identity of neural progenitor cells derived from human ES cells, PD or healthy subject-specific iPS cells. Secondly, a high activity form of human sonic hedgehog established a sizeable FOXA2(+) neural progenitor cell population in vitro. Thirdly, early exposure to FGF8a, rather than Fgf8b, and WNT1 was required for robust differentiation of the FOXA2(+) floor plate-like human neural progenitor cells into FOXA2(+) DA neurons. FOXA2(+) DA neurons were also generated when this protocol was adapted to feeder-free conditions. In summary, this new human ES and iPS cell differentiation protocol using FGF8a, WNT1, low dose retinoic acid and a high activity form of SHH can generate human VM DA neurons that are required for relevant new bioassays, drug discovery and cell based therapies for PD.

Fig 1

Early exposure to mShh-N and 10⁻⁸M retinoic acid promotes midbrain regionalization of human ES cell-derived neural progenitor cells. (A) Human ES cells were differentiated by 4 concentrations of RA with mouse Shh for 12 days before exposure to BDNF, ascorbic acid, mouse Shh and Fgf8b for 8 days. (B) Quantitative PCR determined that 10⁻⁸M retinoic acid with mouse Shh induced a midbrain-like transcriptional profile by significantly increasing the levels of EN1 without changing the levels of OTX2, HOXB1 and PAX2 (* p < 0.05 ANOVA).

Fig 2

High activity form of recombinant SHH promotes the dose-dependent differentiation of FOXA2 expressing neural progenitor cells from 10⁻⁸M RA treated human ES cells. (A) Human ES cells were exposed for 12 days to a SHH-C24II and a further 5 days to neural expansion medium. (B–E) Immunocytochemistry at DIV19 revealed few FOXA2⁺ (red)/β-tubulin^Low (green) neural progenitor cells that had differentiated from human ES cells after exposure to mouse Fgf8b with (B) or without mouse Shh (E). In contrast, 500 ng/ml SHH-C24II generated many FOXA2⁺/β-tubulin^Low neural progenitor cells in the presence of either FGF8a (C) or Fgf8b (D). (F) Cell counts revealed a significantly greater percentage of human FOXA2⁺ neural progenitor cells after exposure to 500 ng/ml SHH-C24II and either FGF8a or Fgf8b (* p < 0.05 ANOVA). Scale bar = 50 µm.

Fig 3

Phenotypic characterization of FOXA2 expressing neural progenitor cells from RA-treated human ES/iPS cells. (A–L) Immunocytochemistry at DIV19 revealed that human ES cell-derived neural progenitor cells formed rosette-like structures and coexpressed OTX2 (red) but not FOXA2 (green) in response to Fgf8b with (A, E, I) or without mouse Shh (D, H, L). In contrast, isolated neural rosette-like structures were observed in cultures exposed to SHH-C24II and FGF8a (B, F, J). These rosette-like structures coexpressed FOXA2 and OTX2 (B). In cultures exposed to SHH-C24II and Fgf8b, very few rosette-like structures were observed and cells coexpressed FOXA2 and OTX2 (C, G, K). (M) Human ES cells exposed to 500 ng/ml SHH and FGF8a or Fgf8b differentiated into FOXA2⁺ cells (green) that expressed human-specific nestin (red). (N) In similar culture conditions, FOXA2⁺ cells (green) coexpressed 3CB2 (red). (O) In parallel cultures, all human iPS cell lines differentiated into FOXA2⁺ (red)/β-tubulin^Low (green) neural progenitor cells. Scale bar A–L, O = 50 µm, M = 20 µm, N = 10 µm.

Fig 4

Expansion of human ES cell-derived FOXA2⁺ neural progenitor cells by SHH-C24II and human FGF8a for 28 days leads to the generation of FOXA2 expressing DA neurons. (A) Human ES cell-derived neural progenitor cells were grown in medium supplemented with Shh and FGF8 splice variants before neuronal differentiation. (B–E) Immunocytochemistry at DIV49 revealed many β-tubulin⁺ neurons (green) and TH⁺ neurons (blue) but very few FOXA2⁺ cells (red) differentiated from human ES cells after exposure to Fgf8b with (B) or without mouse Shh (E). In contrast, 500 ng/ml SHH-C24II with FGF8a (C) or Fgf8b (D) generated many FOXA2⁺ cells. Importantly, FOXA2⁺/β-tubulin^High/TH⁺ neurons were only observed after exposure to FGF8a (C, arrowheads). (F) Cell counts revealed a significantly greater percentage of human FOXA2⁺ cells (* p < 0.05 ANOVA) and FOXA2⁺ neurons (# p < 0.05 ANOVA) after exposure to 200 or 500 ng/ml SHH-C24II and FGF8a. A significant increase in the percentage of FOXA2⁺ DA neurons was observed after exposure to 500 ng/ml SHH-C24II and FGF8a (δ p < 0.05 ANOVA). Scale bar = 50 µm.

Fig 5

Phenotypic characterization of FOXA2⁺ dopaminergic neurons generated by SHH-C24II and FGF8a from RA-treated human ES/iPS cells. (A) Human PD-iPS cell lines (PDC^3F-1) were competent to generate FOXA2⁺ (red) dopaminergic neurons (TH, blue; β-tubulin, green; arrowheads). (B,C) In cultures differentiated with 500 ng/ml SHH-C24II and FGF8a, cells coexpressed TH (blue), FOXA2 (red) and calbindin (green, B) or weakly GIRK2 (green, C), indicative of an A10 or A9 DA neuron phenotype, respectively. Scale bar A = 50 µm, B,C = 10 µm.

Fig 6

Differentiation of human FOXA2 expressing DA neurons from ES cells requires recombinant WNT1 but not Noggin. (A) Human ES cells were differentiated with or without WNT1 and/or Noggin during the first 14 days of the protocol. (B–E) At DIV49, FOXA2⁺/TH⁺/β-tubulin⁺ cells were observed after exposure to SHH-C24II, FGF8A and RA with (B) or without Noggin (C). FOXA2⁺/TH⁺/β-tubulin⁺ cells were not observed at DIV49 without early exposure to WNT1 (D), or WNT1 and Noggin (E). (F) Cell counts revealed a significantly greater percentage of human FOXA2⁺ neurons (# p < 0.05 ANOVA) and FOXA2⁺ DA neurons (δ p < 0.05 ANOVA) after exposure to WNT1 with or without Noggin. Scale bar = 50 µm.

Fig 7

Feeder cell-free differentiation of stem cell-derived human FOXA2 expressing DA neurons. (A) Human pluripotent stem cells were differentiated without coculture on mouse MS5 feeder cells. (B) At DIV16, large clusters of cells formed that exhibited prominent radial outgrowth. (C) At higher magnification (see inset in B), small clusters of cells were observed adjacent to radial cell processes. (D–E) At DIV49, immunocytochemistry and cell counts revealed many FOXA2 expressing cells (red) that coexpressed TH (blue) and β-tubulin (green). Scale bar = 50 µm.