Generation of Cholinergic and Dopaminergic Interneurons from Human Pluripotent Stem Cells as a Relevant Tool for In Vitro Modeling of Neurological Disorders Pathology and Therapy

Figure 1

Comparison of different protocols for deriving of midbrain dopaminergic neurons from hESCs and hiPSCs. (a) Methods based on mechanical neural rosettes selection. Neural differentiation was induced by coculture of hPSCs on stromal cells MS5 or S2 [8] and dual inhibition of SMAD signaling pathway (NOGGIN + TGFB inhibitor: SB431542) in the presence of knockout serum replacement (KSR) and N2 medium [9]. Rosettes structures were harvested mechanically and gently replated in the presence of growth factors. In the final step, newly generated neural progenitor cells were differentiated into DA neurons in the absence of SHH and FGF8. (b) Methods based on the floor plate (FP) induction. Dual SMAD inhibition (BMP inhibitor: LDN193189 + TGFB inhibitor: SB431542) and activation of WNT signaling by SHH and GSK3B inhibitor (GSK3Bi), CHIR99021, were used for midbrain FP cell generation from hPSCs [10]. Purmorphamine treatment was applied for FP cell patterning. hPSCs induced with LDN193189 and A83-01 (inhibitor of TGFB type I receptor ALK5) were cultured in media supplemented with purmorphamine and FGF8 to induce floor plate cells. FP cells under stimulation with growth factors generated DA neurons; recombinant E8 fragments of human laminin 511 (LM511-E8) supported the neural differentiation and cell survival [11]. Final concentration of growth factors, supplements, and inhibitors may be different in the specified protocols.

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