Midbrain dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) provide an excellent source for disease modeling and drug screening for Parkinson’s disease. During brain development, midbrain floor plate (mFP) is formed during 21-28 days of gestation along the ventral midline of developing neural tube and it has been shown that midbrain DA neurons are differentiated from mFP cells. Recent reports have focused on identifying the appropriate in vitro conditions to differentiate hPSCs to properly regionalized floor plate precursors, rather than a more general neural stem cell population, in order to create authentic DA neurons. However, published protocols are quite lengthy and complicated leading to increased variability in differentiation efficiencies. Also, few reports describe whether specified progenitors can be expanded and cryopreserved. Our objective is to develop a culture media system designed to simplify and standardize this process while compressing timelines and adding increased flexibility in this complex differentiation workflow. Here we describe our results which have broken the process down into 3 distinct steps: (1) specification of hPSC to midbrain floor plate (mFP) cells, (2) expansion and cryopreservation of derived mFP cells, and (3) maturation to DA neurons. Characterization of floor plate cells and mature DA neurons was performed by immunostaining for the presence of specific markers including Lmx1, Otx2, FoxA2 and TH, additional qPCR analysis included expanded lists of genes to help define these cell populations. Electrophysiological characteristics of differentiated neurons were assessed by Multi-electrode array and spontaneous and depolarization induced dopamine release was measured with HPLC. In comparison to published protocols, our new system has several advantages including ease of use, significant expansion and preservation of progenitors in relatively short culture duration. This efficient system will benefit researchers with increased scale and flexibility in targeted studies.

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