Table 1.

Current protocols of neuronal differentiation using NGN2 overexpression

Neuronal subtype	Source/species	Transcription factors used and method of delivery	NGN2 induction duration	Time to functional neurons	Developmental patterning	Growth factors and co-culture	Reference
Bipolar neurons	Human ESCs and iPSCs	Ngn2 and Ngn1 via lentiviral gene delivery	1–4 days	4 days for single action potentials, 14 days for action potentials trains	NA	Co-cultured with rat astrocytes	(Busskamp et al., 2014)
Glutamatergic excitatory neurons	Human ESC and iPSCs	Ngn2 via lentiviral gene delivery	Entire duration	2 weeks	NA	BDNF	(Zhang et al., 2013)
	Mouse ESCs	Transient Ngn2 expression and clonal transgenic line	Entire duration	7 days	NA	NA	(Thoma et al., 2012)
	NPCs derived from human iPSCs	Ngn2 or NGN2 via lentiviral gene delivery	2 days–3 weeks	2 weeks	SB431542 and LDN193189	NA	(Ho et al., 2016)
	Human iPSCs	NGN2 transgenic line (i3 neurons)	72 h	2 weeks	NA	BDNF, NT-3 co-cultured with primary mouse astrocytes (also recommend using astrocyte conditioned media)	(Fernandopulle et al., 2018)
	Human ESCs and iPSCs	Ngn2 via lentiviral gene delivery	Entire duration	2–3 weeks	SB431542, XAV939, and LDN193189	Co-cultured with mouse primary cortical glial cells	(Nehme et al., 2018)
	Human ESCs or ESC-derived anterior and posterior NPCs	NGN2 alone or in addition to one of the following: EMX1, EMX2, OTX1, OTX2, EOMES (TBR2), LHX2, or FOXG1	4 days or for single-cell RNA sequencing 14 days	28 days	SB431542, LDN193189 and CHIR99021	Co-cultured with mouse glia for long term cultures	(Ang et al., 2020)
Dopaminergic neuron-like	Human fibroblasts	NGN2, ASCL1, SOX2, NURR1, PITX3 via lentiviral gene delivery	Transient	20 days	SHH, FGF8	NA	(Liu et al., 2012)
Midbrain dopaminergic neurons	Human iPSCs	NGN2, ATOH1 via synthetic mRNA delivery	Multiple mRNA transfections: ATOH1 for 3 days, NGN2 for 1 day	20–49 days	SHH, FGF8b, and DAPT	BDNF, GDNF, TGFb-3, cAMP, ascorbic acid, and DAPT	(Xue et al., 2019)
Dopaminergic neuron	Mouse ESC-derived NPCs	Ngn2, Nurr1 via retroviral gene delivery	3–10 days	9–16 + days	bFGF, EGF	NA	(Park et al., 2008)
Serotonergic neurons	Human dermal fibroblasts	NGN2, ASCL1, NKX2.2, FEV, GATA2, LMX1B via lentiviral gene delivery	3–4 weeks	6 weeks	Dibutyryl cyclic-AMP, noggin, LDN193189, A83-1, CHIR99021, SB431542, forskolin	GDNF, BDNF, dibutyryl cyclic-AMP co-culture with primary rodent astrocytes for electrophysiological analyses	(Vadodaria et al., 2016)
Motor neurons	Human ESC and iPSCs	NGN2 NGN1, NGN3, ND1, ND2 via synthetic mRNA delivery	2–4 transient transfections	7–10 days	Forskolin, SB431542, dorsomorphin, retinoic acid	BDNF, GDNF, NT-3	(Goparaju et al., 2017)
	Human ESC and iPSC-derived NPCs	NGN2, ISL1, LHX3 via adenoviral gene delivery	Transduce day 0 and 4	21 days	Retinoic acid, forskolin, SHH	Retinoic acid, SHH	(Hester et al., 2011)
Spinal motor neurons	Mouse ESC-derived EBs	Ngn2, Isl1, Lhx3 via inducible transgenic line	2 days	7–11 day	NA	Co-culture with mouse astrocytes 7–10 days	(Mazzoni et al., 2013)
	Mouse and human fibroblasts	Ngn2, Ascl1, Brn2, Mytl1, Lhx3, Hb9, Isl1 via retroviral transduction	Undefined	21 + days	NA	GDNF, BDNF, CNTF	(Son et al., 2011)
	Human iPSCs	Ngn2, Isl1, Lhx3 inducible line via a piggyBac transposon vector	5 days	13 days	DAPT, SU5402	BDNF, GDNF, L-ascorbic acid	(Garone et al., 2019)
Cranial motor neurons	Mouse ESCs-derived EBs	Ngn2, Isl1, Phox2a via inducible transgenic line	2 days	11 days	NA	Co-culture with mouse astrocytes 7–10 days	(Mazzoni et al., 2013)
	Human iPSCs	Ngn2, Isl1, Phox2a inducible line via a piggyBac transposon vector	5 days	13 days	DAPT, SU5402	BDNF, GDNF, L-ascorbic acid	(Garone et al., 2019)
Lower motor neurons	Human iPSCs	NGN2, ISL1, LHX3 inducible transgenic line	72 h	14 + days	NA	Optional co-culture of astrocytes	(Fernandopulle et al., 2018)
Cholinergic motor neurons	Human fetal lung fibroblasts or human postnatal and adult skin fibroblasts	NGN2 (+Sox11 for skin fibroblasts) using retroviral gene delivery	2–35 days	50 days	Dorsomorphin and forskolin	Co-cultured with C2C12 murine myoblast line	(Liu et al., 2013)
Mixed population of sensory neurons	Human and mouse fibroblasts	Ngn2 and BRN3A or Ngn1 and BRN3A via lentiviral transduction	8 days	14–22 days	NA	BDNF, GDNF, NGF	(Blanchard et al., 2015)
Mixed population of sensory neurons	Human ESC neural crest progenitors	NGN2 via lentiviral transduction	4 days	32 days	SB431524, CHIR99021, BMP2, FGF2	BDNF, GDNF, NT-3, bNGF, Y-27632	(Hulme et al., 2020)
Cold-sensing mechanoreceptors	Human iPSCs or iPSC-derived neural crest cells	NGN2 and BRN3A via an inducible line	14 days	20 days	Y-27632, bFGF, EGF, SB431524	BDNF, GDNF, NT-3, bNGF, Y-27632	(Nickolls et al., 2020)
Mechanoreceptors	Human iPSC-derived neural crest cells	NGN2 and BRN3A via an inducible line	1 day	20 days	Y-27632, bFGF, EGF, SB431524	BDNF, GDNF, NT-3, bNGF Retinoic acid	(Nickolls et al., 2020)
	Human ESC and iPSC neural crest progenitors	NGN2 via lentiviral transduction	1 day	21– 30 days	FGF, EGF	BDNF, GDNF, NT-3, bNGF Retinoic acid	(Schrenk-Siemens et al., 2015)