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Heparan sulfate Sulfatases are essential for the patterning of stem cell–derived midbrain dopaminergic neurons

ABSTRACT: Midbrain dopaminergic (mDA) neuron loss underlies Parkinson’s disease (PD), making it critical to understand how mDA identity is established during development for cell replacement therapy. While intrinsic transcriptional programs and morphogen gradients driving mDA are known, the contribution of extracellular modulators remains less explored. Here, we show that the heparan sulfate (HS)–modifying enzymes SULF1 and SULF2 are essential for proper mDA neuron differentiation in vitro. Using CRISPR/Cas9-engineered human iPSCs, we demonstrate that SULF1/2 double knockout (DKO) cells fail to acquire ventral midbrain identity and instead adopt dorso-caudal fates, marked by ectopic expression of CDX2, HOX genes, neural crest, and Schwann cell markers. Mechanistically, SULF1/2 loss leads to increased 6-O-sulfation of HS chains, enhancing FGF signaling. FGF inhibition in SULF1/2 DKO cells suppresses CDX2+ axial progenitors and caudal derivatives, but does not completely restore ventral midbrain identity. Instead, it redirects cells toward dorsal lineages, including the emergence of ependymal-like cells. Our findings identify SULF1/2 as key modulators of anterior-posterior and dorsal-ventral patterning during mDA neuron development, possibly acting through a tight control of signaling molecules balance, and offer insights to refine differentiation protocols for PD cell therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE300452 | GEO | 2026/07/13

REPOSITORIES: GEO

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Project description:We aim to understand the role that Cdx2 plays in specifying the rostro-caudal identity of differentiating motor neurons. We find that expressing Cdx2 in combination with FGF signaling is sufficient to produce motor neurons with a more caudal identity. ChIP-seq analysis of Cdx2 finds that it binds extensively throughout the Hox regions in progenitor motor neurons. Analysis of polycomb-associated chromatin over Hox regions in the subsequently generated motor neurons finds that Cdx2 binding corresponds to chromatin domains encompassing de-repressed caudal Hox genes. These results suggest a direct role for Cdx2 in specifying caudal motor neuron identity. ChIP-seq studies: We characterize the binding of Cdx2 in progenitor motor neurons using a V5 tagged doxycycline inducible Cdx2 ESC line (iCdx2). Progenitor motor neurons were generated after 4 days of in vitro differentiation of mouse embryonic stem cells using retinoic acid (RA) and hedgehog (Hh) signaling exposure at day 2. On day 3, the cells are exposed to Dox with and without accompanying FGF signaling. The genome-wide binding of the induced Cdx2 transcription factor is profiled using ChIP-seq with an anti-V5 antibody. An appropriate whole-cell extract control experiment for these ChIP-seq experiments is also included. We also examine the effect of induced Cdx2 expression on polycomb-associated chromatin structure in the resulting cellular populations by profiling the H3K27me3 chromatin mark using ChIP-seq. H3K27me3 experiments were performed after 5 days of in vitro differentiation using cells exposed to either: 1) RA & Hh to derive progenitor motor neurons, followed by Dox & FGF; 2) Dox & FGF alone; or 3) RA and Hh alone. There are 6 Illumina sequencing datasets included in this submission: two biological replicates of iCdx2 ChIP-seq in the presence of FGF; one sample of iCdx2 ChIP-seq in the absence of FGF; one H3K27me3 ChIP-seq in the presence of RA, Hh, Dox, and FGF; one H3K27me3 ChIP-seq in the presence of Dox and FGF; and one H3K27me3 ChIP-seq in the presence of RA and Hh.

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Heparan sulfate Sulfatases are essential for the patterning of stem cell–derived midbrain dopaminergic neurons

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