. 2014 Jul 14;5(4):87. doi: 10.1186/scrt476

Table 1.

Experimental protocols for motor neuron induction from human embryonic and induced pluripotent stem cells

References	Starting cells	Neural induction			MN differentiation		MN maturation		Duration
References	Starting cells	Medium	Factors	Obtained cells	Medium	Factors	Medium	Factors	Duration
Amoroso et al. [39]	ESCs and iPSCs	ESC medium^a	Y27632, bFGF, LDN193189, and SB435142	Embryoid bodies	Neural induction medium^b	RA, ascorbic acid, BDNF, C25II modified Shh, SAG, HAG, or purmorphamine	Neurobasal medium	IGF1, GDNF, CNTF, and B27	20-30 days
Boulting et al. [40]	ESCs and iPSCs	ESC medium with 10% KO replacement	bFGF and Y27632	Embryoid bodies	Neural induction medium^b supplemented with bFGF	RA, ascorbic acid, db-cAMP, and HAG	Neurobasal medium	BDNF, GDNF, and CNTF	29 days
Burkhardt et al. [41]	iPSCs	DMEM/F12 supplemented with N2, B27, and NEAA	Y27632, Dorsomorphin dihydrochloride	Embryoid bodies	DMEM/F12, GLUTAMAX, N2, B27 serum-free supplement, D-Glucose, and ascorbic acid	SAG, RA, and DAPT (only for 4 days)	DMEM/F12, GLUTAMAX	CNTF, BDNF, and GDNF	32 days
Burkhardt et al. [41]	iPSCs	DMEM/F12 supplemented with N2, B27, and NEAA	Y27632, Dorsomorphin dihydrochloride	Embryoid bodies		SAG, RA, and DAPT (only for 4 days)	N2, B27 serum free supplement, D-Glucose, and ascorbic acid	CNTF, BDNF, and GDNF	32 days
Corti et al. [25]	iPSCs	DMEM/F12, NEAA, N2, and heparin	RA	Neural rosette	DMEM/F12, NEAA, N2, and heparin	RA and Shh	DMEM/F12, NEAA, N2, and heparin	BDNF, GDNF, and IGF1	~24 days
Hester et al. [42]	ESCs and iPSCs	DMEM/F12, N2, and 10% KO serum	-	Embryoid bodies then neural rosette	DMEM/F12 with N2	RA, Shh, forskolin, B27, and adenovirus	-	-	~40 days
Hu and Zhang [43]	ESCs	DMEM/F12, KO serum replacement, NEAA, L-Glu, and BME	-	Floating embryoid bodies	Neural differentiation medium (DMEM/F12, N2, NEAA, and heparin)	Shh or purmorphamine, RA, B27, cAMP, ascorbic acid, BDNF, GDNF, and IGF1	Neural differentiation medium (DMEM/F12, N2, NEAA, and heparin)	cAMP, ascorbic acid, BDNF, GDNF, and IGF1	~40 days
Karumbayaram et al. [44]	iPSCs and ESCs	ESC medium without FGF2	-	Embryoid bodies/neural rosette	ESC medium without FGF2	RA and purmorphamine	DMEM/F12 and N2	GDNF, BDNF, CNTF, Shh, and RA	35-49 days
Reinhardt et al. [29]	iPSCs and ESCs	ESC medium^a and then expansion medium (DMEM/F12, N2, B27, pen/strep, and L-Glu)	For induction: SB435142, Dorsomorphine, CHIR, and purmorphamine	Embryoid bodies (smNPC)	Expansion medium (DMEM/F12, N2, B27, pen/strep, and L-Glu)	Purmorphamine and RA	Expansion N2, B27	BDNF, GDNF, db-cAMP, RA, and purmorphamine	>40 days
Reinhardt et al. [29]	iPSCs and ESCs		For expansion: ascorbic acid, CHIR99021, and purmorphamine	Embryoid bodies (smNPC)	Expansion medium (DMEM/F12, N2, B27, pen/strep, and L-Glu)	Purmorphamine and RA	Expansion N2, B27	BDNF, GDNF, db-cAMP, RA, and purmorphamine	>40 days
Sareen et al. [45]	iPSCs	Neural differentiation medium (DMEM, B27, vitamin A, and N2)	RA	Embryoid bodies	Neurobasal medium, B27, and N2	RA and purmorphamine	DMEM/F12 and B27	RA, purmorphamine, db-cAMP, ascorbic acid, BDNF, and GDNF	>40 days
Takazawa et al. [37]	ESCs	ESC medium^a and then DMEM/F12, N2, NEAA, L-Glu, and heparin	Y27632, bFGF, and recombinant mouse Noggin	Embryoid bodies	Wnt3a-L-cell conditioned medium	RA, ascorbic acid, db-cAMP, and recombinant mouse Shh (C25II)	Neurobasal medium with N2, B27, Glu, and NEAA	Ascorbic acid, db-cAMP, RA, Shh, BDNF, GDNF, and IGF1	>31 days
Wada et al. [46]	ESCs	ESC medium^a and FGF2	Noggin and dorsomorphin	Neural rosette	DMEM/F12, N2B27	Shh or SAG, RA	DMEM/F12, N2, B27, FGF2, and heparin	GDNF, BDNF, and NT3	38 days
Wada et al. [46]	ESCs	ESC medium^a and FGF2	Noggin and dorsomorphin	Neural rosette	FGF2, and EGF	Shh or SAG, RA	DMEM/F12, N2, B27, FGF2, and heparin	GDNF, BDNF, and NT3	38 days
Wichterle et al. [27]	mESCs	DFK5 medium (DMEM/F12, L-Glu, pen/strep, BME, and insulin-transferrin- selenium supplement	RA, Shh, Hedgehog agonist (Hh-Ag1.3), or hedgehog antibody (5E1)	Embryoid bodies	DMEM/F12 medium	-	DMEM/F12	GDNF, BDNF, CNTF, and NT3	~25 days
Zeng et al. [47]	iPSC (ESCs as positive control)	hESC medium^a	N2 and heparin	Neural rosette	Neural medium^b	RA, Shh, and FGF8	Neural basal medium, N2, and B27	GDNF, BDNF, and IGF1	>24 days

^aHuman embryonic stem cell (hESC) medium: Dulbecco’s modified Eagle’s medium (DMEM/F12), 20% knockout (KO) serum replacement, basal medium Eagle (BME), L-Glu, and non-essential amino acid (NEAA). ^bNeural induction medium: L-Glu, NEAA, penicillin/streptomycin (pen/strep), heparin, and N2. BDNF, brain-derived neurotrophic factor; bFGF, basic fibroblast growth factor; CNTF, ciliary neurotrophic factor; EGF, epidermal growth factor; ESC, embryonic stem cell; FGF, fibroblast growth factor; GDNF, glial cell-derived neurotrophic factor; HAG, human-specific Smo agonist; IGF1, insulin-like growth factor-1; iPSC, induced pluripotent stem cell; mESC, murine embryonic stem cell; MN, motor neuron; RA, retinoic acid; SAG, Smo agonist; Shh, Sonic hedgehog; smNPC, small-molecule neural precursor cell.