pizza club - october 2016 - lisa
TRANSCRIPT
Why 3D cell culture? What are organoids?
• Multiple organ-specific cell types
• Spatial organisation
• Physiological functions
Utility in research:
• Disease mechanisms
• Regenerative medicine
• Drug testing
Yin et al., 2016
Development of mDA neurons
Image adapted from Lumsden et al., 1995, Gale et al., 2008, Aguila et al., 2012
midbrain identity: OTX2, FOXA2, LMX1A
mDA neuron progenitor: FOXA2, LMX1A, NURR1
mDA neurons: TH, DAT, FOXA2, LMX1A
Characterisation of hMLOs
Ki67, EdU: proliferation
MAP2, MASH1: neuronal
OTX2: midbrain
NURR1: mDA progenitor
• transitioned from progenitors to mature neurons• developing neuroectoderm toward floor plate
Identification of mDA neurons in hMLOs
FOXA2, OTX2, LMX1A: midbrain
TH, DAT: dopamine
• time-dependent induction of floor plate precursors • differentiation into mDA neurons within developing hMLOs
Transcriptional characterisation of hMLOs
in vivo
in vitro
• gene expression profiles of hMLOs reflects prenatal midbrain identity
• contain cells that are not present in 2D
Oligodendrocytes Glial cells
Identification of neuromelanin in hMLOs
• NM granules secreted from mDA neurons within hMLOs• role of NM unclear
Functional maturation of DA neurons in hMLOs
• mDA neurons exhibited mature neuronal properties• synapse formation with other neurons• DA production within hMLO
Biocytin/THM
• apico-basal polarity of midbrain specific cells
• identification of neuromelanin in mDA neurons
• electrophysiological activity and suppression of mDA firing
• hMLO transcriptome profile closer to prenatal midbrain compared to 2D mDA neuronal cultures
• detection of dopamine using HPLC
Summary
Thanks for your attention!