Efficient Derivation of Functional Floor Plate Tissue from Human Embryonic Stem Cells

Developmental Biology Program, Sloan-Kettering Institute, 1275 York Ave., New York, NY 10065, USA.
Cell stem cell (Impact Factor: 22.27). 04/2010; 6(4):336-47. DOI: 10.1016/j.stem.2010.03.001
Source: PubMed


The floor plate (FP) is a critical signaling center during neural development located along the ventral midline of the embryo. Little is known about human FP development because of the lack of tissue accessibility. Here we report the efficient derivation of human embryonic stem cell (hESC)-derived FP tissue capable of secreting Netrin-1 and SHH and patterning primary and hESC derived tissues. FP induction in hESCs is dependent on early SHH exposure and occurs at the expense of anterior neurectoderm (AN). Global gene expression and functional studies identify SHH-mediated inhibition of Dkk-1 as key factor in FP versus AN specification. hESC-derived FP tissue is shown to be of anterior SIX6+ character but is responsive to caudalizing factors suppressing SIX6 expression and inducing a shift in usage of region-specific SHH enhancers. These data define the early signals that drive human FP versus AN specification and determine regional identity in hESC-derived FP.

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    • "As the hypothalamus is an anterior and ventral neural structure (Fig. 1A,B), we differentiated hPSCs in the presence of small molecule inhibitors of the BMP and TGFβ/NODAL/activin signaling pathways to promote neural differentiation (Fig. 1D) (Chambers et al., 2009) and a small molecular inhibitor of the WNT pathway (Huang et al., 2009), because WNTs induce the formation of posterior brain regions (Yamaguchi, 2001). To ventralize these putative forebrain progenitors, we exposed them to small molecule agonists of the SHH pathway, including purmorphamine (Pur) (Sinha and Chen, 2006) and SAG (Chen et al., 2002), which can induce ventral neural characteristics (Wilson and Rubenstein, 2000), particularly when exposed to differentiating hPSCs at early time points (Fasano et al., 2010; Maroof et al., 2013). "
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    • "Next, we asked if the technology can be applied to hES cells, and to this end devised an experiment to compare the differentiation of mouse and human ES cells towards a common endpoint: we performed a pair of more focused screens to compare known protocols and discover new methods to make dopaminergic neurons. These are lost in Parkinson's disease and their differentiation from ES cells has been studied intensively [3], [42]–[46]. We devised two 10,000-plex screening matrices comprising published neurogenic media (some known to promote generic neural differentiation, others more directed dopaminergic specification) and new formulations based on subtle variations thereof. "
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    • "As shown by treatments with Wnt/β-catenin inhibitors, this endogenous Wnt/β-catenin activation restrained induction of rostral forebrain fates in favor of caudal forebrain and/or midbrain fates [52, 88]. Other studies, in which neuralized hESCs predominantly acquired rostral forebrain fates, reported upregulation of endogenous Wnt antagonists during neural induction [77, 89]. Similar results have been reported in mESCs [44]. "
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