Journal Club - Lisa Smits25th October 2016

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

Method - Derivation of hMLOs

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!