Table 3.

ENDpoiNTs in vitro methods selected to interrogate essential DNT key events.

Assay	Endpoint	Readout	Ref
Mouse C17.2 NPC line	Proliferation	LDH release	[47]
	Differentation	bIII-tubulin staining
	Neurite growth	Quantification of neurite length in cells stained with bIII-tubulin and DAPI
Mouse primary NPC (SVZ)	Proliferation	Sphere size, BrdU, Ki67/PH3 stainings	[48]
	Differentiation	DCX staining
Mouse primary neuron-oligodendrocyte co-cultures	Differentiation	MBP staining	[49]
	Myelination	MBP staining
Rat NPCs (different brain regions)	Proliferation	Sphere Size, BrdU staining
	Differentiation	GFAP (HCIA), O4 staining (HCIA)
	Migration	HCIA of the bIII-tubulin or O4 stained cells which migrate from the sphere core stained with DAPI
	Neurite growth	DAPI staining, MAP2, bIII-tubulin staining (HCIA)
	Myelination	qRT-PCR for expression of markers of oligodendrocyte differentiation and maturation
	Network formation and activity	Synapsin/PSD95 staining
Rat primary cortical cultures	Network formation and activity	MEA	[50]
Human primary NPC	Proliferation	Sphere Size, BrdU staining	[42]
	Differentiation	GFAP staining (HCIA), O4 staining (HCIA), bIII-tubulin staining (HCIA)	[42]
	Migration	HCIA of the bIII-tubulin or O4 stained cells which migrate from the sphere core stained with DAPI	[42]
	Neurite growth	DAPI staining, MAP2 staining, bIII-tubulin staining	[51]
	Myelination	rtRT-PCR for expression of markers of oligodendrocyte differentiation (and maturation?)	[42]
	Network formation and activity	MEA
Human iPSC-derived NPC (different brain regions)	Proliferation	Sphere Size, BrdU
	Differentiation	GFAP staining
	Migration	HCIA of the bIII-tubulin or O4 stained cells which migrate from the sphere core stained with DAPI	[42]
	Neurite growth	DAPI staining, bIII-tubulin staining
	Network formation and activity	Synapsin/PSD95 staining, MEA
Human iPSC-derived NCC cMINC (UKN2)	Migration	HCIA of viable cells (stained with Hoechst and calcein) which migrate to a previously unpopulated plate area	[42]
Human iPSC-derived NCC PeriTox (UKN5)	Neurite growth	HCIA of cells stained with Hoechst and calcein	[42]
Human immortalized primary NPC NeuriTox (UKN4)	Neurite growth	HCIA of cells stained with Hoechst and calcein	[42]
Human iPSC-derived neuronal co-cultures	Network formation and activity	MEA
Human iPSC-derived cortical brain organoids	Network formation and activity	MEA
	Molecular changes	Single cell transcriptomics, epigenomics	[52]

DAPI: 4′,6-diamidino-2-phenylindole, BrdU: bromodeoxyuridine, DCX: doublecortin, GFAP: glial fibrillary acidic protein, hiPSCs: human induced pluripotent stem cells, LDH: lactate dehydrogenase, MEA: microelectrode arrays, MAP2: microtubule-associated protein 2, MBP: myelin basic protein, NPCs: neural progenitor cells, PSD95: postsynaptic density protein 95, and SVZ: subventricular zone.