Abstract
Generation of mesencephalic dopamine (mesDA) neurons from human embryonic stem cells (hESCs) requires several stages of signaling from various extrinsic and intrinsic factors. To date, most methods incorporate exogenous treatment of Sonic hedgehog (SHH) to derive mesDA neurons. However, we and others have shown that this approach is inefficient for generating FOXA2+ cells, the precursors of mesDA neurons. As mesDA neurons are derived from the ventral floor plate (FP) regions of the embryonic neural tube, we sought to develop a system to derive FP cells from hESC. We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to FP lineage. The GLI1+ cells coexpressed FP markers, FOXA2 and Corin, and displayed exocrine SHH activity by ventrally patterning the surrounding neural progenitors. This system results in 63% FOXA2+ cells at the neural progenitor stage of hESC differentiation. The GLI1-transduced cells were also able to differentiate to neurons expressing tyrosine hydroxylase. This study demonstrates that GLI1 is a determinant of FP specification in hESC and describes a highly robust and efficient in vitro model system that mimics the ventral neural tube organizer. Stem Cells 2010;28:1805–1815
Highlights
In the last decade, since the derivation of human embryonic stem cell lines, there has been much effort in generating mesencephalic dopamine neurons from human embryonic stem cells (hESCs) for developing cell replacement therapies to treat Parkinson’s disease [1,2,3]
We show that forced expression of the transcription factor GLI1 in hESC at the earliest stage of neural induction, resulted in their commitment to floor plate (FP) lineage
We showed that human recombinant Sonic hedgehog (SHH) treatment of hESC during neural induction is capable of inhibiting a dorsal neural fate and promotes expression of the basal neural tube marker, NKX6.1
Summary
Since the derivation of human embryonic stem cell (hESC) lines, there has been much effort in generating mesencephalic dopamine (mesDA) neurons from hESC for developing cell replacement therapies to treat Parkinson’s disease [1,2,3]. The first postmitotic mesDA neurons can be identified by Nurr expression at E10.5 [4] and later, at E11.5, are immunoreactive for tyrosine hydroxylase (TH) [5]. These TH neurons arise from the ventral regions of the mesencephalon [6, 7], as identified by the expression of transcription factors known to be critical for the development of mesDA progenitor cells. FoxA2 is one such gene expressed in the mesDA progenitor cells and its expression is maintained in adult mesDA neurons [8, 9]. Heterozygous FoxA2 mutants show a progressive loss of mesDA neurons in adult mice indicating its requirement for mesDA survival [8]
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