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

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.