Midbrain-like organoids from Human Pluripotent Stem Cells contain functional dopaminergic and neuromelanin-producing neurons

1. Journal Club - Lisa Smits
25th October 2016

2. 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

3. 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

4. Method - Derivation of hMLOs

5. 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

6. 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

7. 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

8. Identification of neuromelanin in hMLOs
• NM granules secreted from mDA neurons within hMLOs
• role of NM unclear

9. Functional maturation of DA neurons in hMLOs
• mDA neurons exhibited mature neuronal properties
• synapse formation with other neurons
• DA production within hMLO
Biocytin/THM

10. • 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!