Midbrain dopaminergic (mDA) neurons undergo progressive degeneration in patients with Parkinson’s disease (PD). Cell replacement therapy is a promising strategy for PD treatment. However, the efficacy of mDA neuron replacement therapy remains limited due to challenges involving the isolation of relatively homogenous dopaminergic neuron populations. To enhance the effectiveness of mDA neuron replacement therapy for PD patients, it is crucial to understand human mDA neuron development. Desulfation mediated by sulfatase (SULF) 1 and 2, which are highly expressed in radial glia of the midbrain, plays an essential role in neuronal development. These sulfatases remove sulfate groups from heparan sulfate (HS), a key glycosaminoglycan (GAG) involved in modulating signaling pathways. However, the specific role of SULF1 and SULF2 in mDA neuron development remains unclear. To investigate the function of SULF1 and SULF2 in mDA neuron development, we will generate SULF1/2-knockout human-induced pluripotent stem cells (hiPSCs). The differentiated mDA neurons will be collected for RNA sequencing and GAG profiling to examine HS biosynthesis gene expression and sulfation patterns. This study will provide valuable insights into the GAG-related molecular mechanisms regulating neurogenesis and may contribute to the improvement of cell-based therapies for PD.