Defining conditions required for the derivation of midbrain dopaminergic neurons from embryonic stem cells using stem cell reporter lines
thesisposted on 15.05.2017, 01:56 by Hartley, Brigham Jay
The efficient and robust derivation of midbrain dopaminergic (mbDA) cells from embryonic stem cells (ESCs) may pave the way for cell replacement therapy (CRT) for Parkinson’s Disease (PD) patients whose disease is refractory to current pharmacotherapy. It may also facilitate investigation of the molecular mechanisms underlying the pathogenesis of PD through in vitro modeling. However, a major hindrance to the application of ESC-derived cells for in vitro and in vivo applications is the observation that current differentiation protocols yield heterogeneous cultures. Reporter cell lines offer the ability to isolate cells from differentiating cultures. The LIM homeobox transcription factor 1 α (LMX1A) is a prominent regulator of early mbDA and forebrain GABAergic specification. In this thesis I utilize both mouse and human ESCs expressing EGFP under the control of the endogenous LMX1A promoter to investigate aspects of fate specification following derivation under a chemically defined monolayer (CDML) or PA6 stromal cell differentiation protocols. ESC differentiation under PA6 conditions results in a population of mbDA progenitors that can be isolated based on EGFP expression, which subsequently gives rise to high yields of mbDA neurons. ESCs differentiating under CDML neural induction cues only, default to an anterior neuroectoderm phenotype. However, timed exposure to morphogens that mimic in vivo mbDA developmental conditions (SHH & WNT signalling) derives functional DA neurons with a protein profile indicative of a midbrain phenotype. The cultivation and subsequent subtype specification of these defined neural progenitors holds promise for the derivation of mbDA cells for PD CRT, in vitro disease modelling and pharmacotherapeutic screening platforms.