Universitas Indonesia Conferences, Asian Federation for Pharmaceutical Sciences (AFPS) 2019

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Understanding the role of The Wnt signalling pathway in generating midbrain dopa-minergic neurons of the substantia nigra pars compacta /A9 subtype neurons
Shanti Marlina Sibuea

Last modified: 2019-06-15


Background: Cell replacement therapy is one of the most promising strategies for the treatment of Parkinson Disease (PD) and for this purpose, there has been a great interest in deriving dopaminergic (DA) neurons from human pluripotent stem cells (hPSCs). Protocols for directed differentiation of human ESCs into midbrain dopamine neurons have been developed based around the embryonic midbrain differentiation program.  However, several challenges remain, such as the need to develop strategies that can enrich for selective subtypes of midbrain dopamine neurons (mDA) and the need to modulate the maturation and survival of the cells. Wnt signalling has been reported to play important role in dopaminergic neuron specification, including for directing the differentiation to the A9 subtype located in substantia nigra pars compacta (SNc), which degenerates in Parkinson’s disease (PD). The purified and enriched cultures of A9 neurons will be an ideal sources for cells based model of PD and transplantation therapy. Materials and Methods: Dopaminergic neuron were grown from pluripotent stem cell reporter line LmX1a-AMP-IRES-eGFP to track the expression of Lmx1a. Developing cultures were incubated with the Wnt mimetic, CHIR99021 (CHIR) or Wnt signalling modulator dickkopf-3 (DKK3) at days 3-11 and/or CHIR between days 40-65 of culture maturation. Dopaminergic phenotypes were examined using immunocytochemistry and quantitative PCR (qPCR) Results: Some neural phenotype specific markers were altered by incubation of CHIR, Wnt signalling mimetic both in early phase and maturation stage of culture. Conclusion: Up to date, this study suggest that concentration-dependent activation of Wnt signalling in hESCs alters some key markers of mDA neuron both in early and maturation stage of differentiation.