Table 1.

In vitro differentiation protocols for per neural lineage phenotype and their application in models of neurological diseases.

Phenotypes	Source of NSCs	Differentiation Protocol	Differentiation factors	Phenotypic markers (% cells) in vitro/vivo	Models	Functional outcome	Reference
DAergic neurons	Human fetal VM tissue	Chemical-defined system	BDNF, AA, low oxygen	40–50% MAP2+ 15% TH+/MAP2+	NA	NA	[55]
				NA
DAergic neurons	Human fetal VM tissue (passage 2)	Chemical-defined system	WNT5 (SHH, FGF8, FGF2 for proliferation)	35%TH+	NA	NA	[62]
				NA
DAergic neurons	Rat embryonic VM tissue	Transfected by electroporation	Nurr1, Brn4	NA	6-OHDA PD rats	Increased DA level; Improved rotational behavior	[67]
				18%TH+ 14%DAT+
DAergic neurons	Rat embryonic VM tissue	Transfected by lentivirus	TH, Brn4	65.71 ± 5.18%TH+ 32.28 ± 4.39% DAT+	NA	NA	[66]
				NA
DAergic neurons	Rat embryonic VM tissue	Chemical-defined system and transfected by lipofectamine	SHH, FGF8 and Wnt5a	a 20-fold TH+ cells increase	6-OHDA PD mice	Increased DA level, improved rotational behavior	[60]
				9.5% TH+
DAergic neurons	Rodents embryonic cortical tissue	Transfected by retroviruses	Foxa2, Nurr1	37.1% TH+ 55.1% PITX3+/TH+ >78% VMAT2+/TH+	6-OHDA PD rats	Exhibited a mature midbrain DAergic neuronal morphology, improved rotational behavior	[73]
				about 14-fold TH+ cells increase
DAergic neurons	Rats embryonic cortical tissue	Transfected by retroviruses with appropriate vectors and promoters	Foxa2, Nurr1, ca-PKA	60% TH+/TUJ1+ 80–90% PITX3+/TH+ VMAT2+/TH+ DAT+/TH+	6-OHDA PD rats	Exhibited an extremely mature midbrain DAergic neuronal morphology, no rotational behavior improvement	[74]
				few TH+ cells <100 cells
DAergic neurons	Primate ESCs (Co-culture with PA6)	Chemical-defined system	NA	25 ± 6% TUJ1+ 35 ± 6% TH+/TUJ1+	6-OHDA PD mice	NA	[43]
				0.7% TH+
DAergic neurons	Human ESCs (Co-culture with PA6)	Chemical-defined system	SHH, FGF8	46 ± 8% MAP+ 80 ± 11% TH+/MAP+ 32% TH+	NA	NA	[75]
DAergic neurons	Mouse ESCs (Co-culture with MS5)	Chemical-defined system	SHH, FGF8	50 ± 10% TH+/TUJ1+	6-OHDA PD mice	improved rotational behavior	[42]
				10–20% TH+
DAergic neurons	Human ESCs (EB)	Chemical-defined system	SHH, FGF8	50–60% TH+/TUJ1+ 31.8 ± 3.1% TH+	NA	NA	[77, 79]
				NA
DAergic neurons	Human PESCs (EB/Dual SMAD inhibition)	Chemical-defined system	SHH C25II, FGF8, PUR and CHIR99021	60–80%/70-100% TUJ1+ 20–40%/30-40% TH+	MPTP PD primates	Increased DA level, improved rotational behavior	[76]
				5.2–8.1% TH+
DAergic neurons	Human iPSC (EB)	Chemical-defined system	SHH, FGF8	30 ± 5% TH+ 100% GIRK2+/TH+	6-OHDA PD rats	Improved rotational behavior	[78]
				~2% TH+
DAergic neurons	Human ESCs/iPSCs (Dual SMAD- inhibition)	Chemical-defined system	CHIR99021, FGF8, PUR and SHH-C25II	±75% TH+ ±50% NURR1+ ±80% FOXA2+ ±60% LMX1A+	6-OHDA PD mice/rats MPTP PD primates	Exhibited excellent DA neuron survival, improved motor deficits.	[84]
				6% TH+ (rats)
DAergic neurons	Human ESCs (Dual SMAD inhibition with EB)	Chemical-defined system	CHIR99021, SHH- C24II	NA	6-OHDA PD rats	Increased DA level, improved motor deficits, showed similar efficacy and potency to fetal DAergic neurons	[82, 178]
				54.2 ± 2.5% TH+ 81% LMX1A+/FOXA2+
DAergic neurons	Human/primate ESCs/iPSCs (Dual SMAD- inhibition)	Chemical-defined system	CHIR99021, FGF8b and SHH- C25II	43.6 ± 6.2% TH+ 95.3 ± 2.4% NURR1+/TH+ 96.7 ± 1.8% FOXA2+/TH+ 96.5 ± 2.3% LMX1A+/TH+ 56.3 ± 6.7% GIRK2+/TH+	NA	NA	[83]
DAergic neurons	Human iPSCs (Dual SMAD- inhibition)	Chemical-defined system	CHIR99021, FGF8, and PUR	42 ± 4.4% TH+ 19.9 ± 6.9% NURR1+ 70–75% FOXA2+	6-OHDA PD rats /MPTP PD primates	Improved rotational behavior(rats) increased spontaneous movement, extended dense neurites into the host striatum, increased DA synthesis	[81, 85]
				±17% TH+ ±28%TH+/NEUN+(rats) 33.3 ± 24.4% TH+(primates)
DAergic neurons	Human ESCs (Dual SMAD- inhibition)	Chemical-defined system	CHIR99021, FGF8b, SHH- C25II and SAG	69% TH+ 84% TH+/TUJ1+ >85% GIRK2+/ TH+	6-OHDA PD mice	Displayed A9 characteristics, restored functionality of the reconstructed nigrostriatal circuit, improved motor deficits.	[179]
				68% TH+/survived
DAergic neurons	Human iPSC (Dual SMAD- Inhibition with EB) Human iNSC	Chemical-defined system	CHIR 99021, FGF8, PUR, BMP5 and BMP7	30–50% TH+/TUJ1+	NA	NA	[86]
				NA
DAergic neurons	Human ESCs/iPSC (dual SMAD- Inhibition)	Chemical-defined system (3D)	CHIR99021, FGF8b and PUR	47% TH+	Fischer rats	NA	[87]
				8.12% TH+/transplanted 46.7% FOXA2/ TH+
DAergic neurons	INSCs reprogrammed from PBMNCs	Chemical-defined system	SAG1, FGF8	57.23% TH+ 62.87% TH+/FOXA2 58.69% TH+/NURR1+ 13.84% TH+ 86.78% FOXA2+/TH+ 91.72% NURR1+/TH+ 98.77% GIRK2+/TH+	6-OHDA PD mice	Improved rotational behavior	[88]
GABAergic neurons	Immortalized striatal human NSC line (STROC05)	Chemical-defined system	PUR	6.3% DARPP-32+ 46% TUJ+ 27%+ MAP2+	NA	NA	[99]
GABAergic neurons	Immortalized striatal human NSC line (ST14A)	Chemical-defined system	RA, KCl	74% GABA+	QA HD rats	maintained neuronal GABAergic phenotype, established pre- and postsynaptic contacts with endogenous striatal cells, improved motor deficits	[100]
GABAergic neurons	Immortalized human NSC line (ReNcell VM)	Chemical-defined system	VPA	68 ± 4% MAP2+ 90% GABA+/MAP2+ 54% CALB1+/MAP2+	NA	NA	[101]
			DKK1, SHH	63 ± 4% MAP2+ 96% GABA+/MAP2+ 84% CALB1+/MAP2+
GABAergic neurons	Human ESCs (EB)	Chemical-defined system	SHH/PUR	90.2 ± 4.2% GABA+/TUJ1+ 89.7 ± 8.3% DARPP32+/TUJ1+	QA HD mice	Projected to the anterior substantia nigra and potentially form connections with DAergic neurons, improved motor deficits	[102]
				62.8 ± 2.6% GABA+ 58.6 ± 3% DARPP-32+/ GABA+
GABAergic neurons	Human iPSCs (Co-culture with PA6)	Chemical-defined system	BDNF	34.1 ± 4.5% DLX2 27.0 ± 1.7%DARPP-32+ 19.1 ± 2.1% CALB1+	QA HD rat	Improved motor deficits	[41]
GABAergic neurons	Human ESCs/iPSC (Dual SMAD- Inhibition)	Chemical-defined system	DKK1, SHH-C25II	±51% MAP2+ ±78% GABA+/MAP2+ ±60.3% CTIP2+/MAP2+ ±86% GABA+/CTIP2+/MAP2+ ±53% CALB1+/MAP2+ ±70.6% CTIP2+/CALB1+/MAP2+	QA HD rat	Improved rotational behavior	[103]
GABAergic neurons	Human ESCs (Dual SMAD- Inhibition with EB)	Chemical-defined system	XAV939, SAG	±87% DARPP32+/MAP2+ ±89.5% GABA+/TUJ1+ 80–100% DARPP-32+/GABA+ 80–100% CALB1+/TUJ1+	QA HD mice	Improved motor deficits	[104]
				48.7 ± 2.8% DARPP32+/hN+
GABAergic neurons	Human ESCs/iPSC (Dual SMAD- Inhibition)	Chemical-defined system (3D)	PUR, DKK1	78%MAP2+ 61% GABA+/MAP2+ 55%DARPP-32+/MAP2+ 70%CTIP2+/MAP2+ 46%CALB1+/MAP2+ 100%CTIP2+/DARPP-32+	R6/2 HD mice	Innervated substantia nigra, improved motor deficits.	[105]
GABAergic neurons	Human ESCs/iPSCs (Dual SMAD- Inhibition)	Chemical-defined system	Activin A	20–50%DARPP-32+	QA HD rats	no motor improvement	[106]
				49 ± 5% DARPP-32+/hN+ 86 ± 4.6%GABA+/hN+ 35 ± 8%CALB1+/hN+
GABAergic neurons	Human ESCs/iPSCs (Dual SMAD- Inhibition)	Chemical-defined system	IWR1	±6%DARPP-32+/Map2b+ ±6%DARPP-32+/CTIP2+ ±60 %CTIP2+	NA	NA	[107]
				NA
Cholinergic motor neurons	Human fetal cortical NSCs	Chemical-defined system	FGF2	61% HB9+ 50% H9+/ChAT+	NA	NA	[114]
				NA
Cholinergic motor neurons	HB1.F3 human NSC line	Chemical-defined system and transfected by vector	Olig2, SHH	NA	SOD1G93A mutant mice	Migrated into ventral horn, and replaced lost host motor neurons, delayed clinical onset and extended life span.	[233]
Cholinergic motor neurons	Mouse ESCs (Co-culture with MS5)	Chemical-defined system	SHH, RA and FGF2	±60%HB9+/TUJ1	NA	NA	[42]
				NA
Cholinergic motor neurons	Human ESCs, primate ESCs (Co-culture with MS5)	Chemical-defined system	SHH, RA	20% HB9+(human) 43% HB9+(primate)	NA	NA	[116]
				NA
Cholinergic motor neurons	Human ESCs (EB)	Chemical-defined system	FGF2, RA and SHH	>50% ISL1+/TUJ1+/MAP2+ ±50% HB9+/ISL1/2+ ±21% HB9+	NA	NA	[120]
				NA
Cholinergic motor neurons	Human iPSCs (EB)	Chemical-defined system	PUR, RA	±60%OLIG2+/SOX3+ ±30%ISL1+/TUJ1+	NA	NA	[118]
				NA
Cholinergic motor neurons	Human iPSCs (EB)	Chemical-defined system	RA, SHH agonist	20%HB9+ >90%ISL1/2+/HB9+ >50%ChAT+/ISL1/2+/HB9+	NA	NA	[119]
				NA
Cholinergic motor neurons	Human ESCs and iPSCs (EB)	Chemical-defined system	PUR, RA and SAG,	83 ± 1% TUJ1+ 30 ± 6% ISL1+ 16 ± 5% HB9+ 37 ± 2% ISL1+and HB9+	NA	NA	[124]
				NA
Cholinergic motor neurons	Human ESCs and iPSCs (Dual SMAD Inhibition with EB)	Chemical-defined system	BIO, PUR and RA	40–50%HB9+	NA	NA	[122]
				NA
Cholinergic motor neurons	Human ESCs and iPSCs	Chemical-defined system (Dual SMAD inhibition)	SAG, RA and CHIR99021	74% HB9+/ISL1+	NA	NA	[125]
				NA
Cholinergic motor neurons	Human iPSCs (Dual SMAD inhibition)	Chemical-defined system	CHIR99021, PUR and RA	90 ± 9% MNX1 + 95 ± 3% ISL1+ 91 ± 6%ChAT+/MAP2+	NA	NA	[126]
				NA
Cholinergic motor neurons	Human iPSCs (Dual SMAD inhibition)	Transfected by lentivirus	NGN2, ISL1, LHX3	88.2 ± 3.5% HB9+ 86.5 ± 4.1%ChAT+	NA	NA	[121]
				NA
Cholinergic motor neurons	Human iNSCs (Reprogrammed from PBMNCs)	Chemical-defined system	RA, SAG1	14.80 ± 0.90% HB9+ 14.40 ± 1.29% ISL1+	NA	NA	[130]
				NA
Cholinergic motor neurons	Rat iNSCs (Reprogrammed from astrocytes)	Chemical-defined system	RA, SHH	34.1% ± 2.9% HB9+	NA	NA	[131]
				NA
oligodendrocytes	Human fetal diencephalic/telencephalic tissue	Chemical-defined system	FGF2, NT3 and PDGF-AA	15–20% O4+ 15–20%GalC+	Lysolecithin MS mice	Showed limited myelinating capacity	[141]
				NA
oligodendrocytes	Human fetal brain tissue	Chemical-defined system	FGF2, NT3 and PDGF-AA	80.5 ± 2.1%A2B5+ 85.4 ± 3.9%O4+ 90%GalC+	NA	NA	[140]
				NA
oligodendrocytes	Human ESCs (EB)	Chemical-defined system	RA, SHH, FGF2, NT3, PDGF-AA and IGF1	83.95% PDGFRα+ 91.3%NGN2+	Shiverer MS mice	expressed MBP and formed myelin sheaths around nerve fibers	[135, 142]
				NA
oligodendrocytes	Human ESCs (EB)	Chemical-defined system	RA, PUR/SAG, FGF2, PDGF-AA, T3, low oxygen	Spinal cord 77 ± 13% NGN2+ 38.5 ± 9.0%O4+ 29.9 ± 5.5%MBP+/O4+ Ventral forebrain 91% ± 7% NGN2+ 43% ± 5% O4+ 29.9 ± 5.5%MBP+/O4+	NA	NA	[143]
				NA
oligodendrocytes	Human ESCs and iPSCs (Dual SMAD inhibition)	Chemical-defined system	RA, SAG, NT3, PDGF-AA and T3	44–70% O4 +	Shiverer MS mice	Achieved mature oligodendrocyte differentiation and formed dense compact myelin.	[145]
				NA
oligodendrocytes	Human iPSCs (Dual SMAD inhibition)	Transfected by lentivirus	SOX10, OLIG2, NKX6.2	62.1 ± 9.5%-79.0 ± 14.8% O4 + 30.37 ± 7.87% MBP+/O4 +	Shiverer MS mice	myelinated the forebrain, remyelinated the demyelinated spinal cord	[146]
oligodendrocytes	Human iPSCs (Dual SMAD inhibition)	Transfected by lentivirus	SOX10	50–65% O4 +	Shiverer MS mice	myelinated neurons	[147, 234]
				48.13 ± 4.15%MBP+
oligodendrocytes	Human ESCs and iPSCs (Dual SMAD inhibition)	Chemical-defined system	XAV939, PUR, PDGFRα, IGF-1, cAMP and T3	35% O4+	NA	NA	[149]
				NA
oligodendrocytes	Human ESCs	Transfected by lentivirus	SOX10, OLIG2	19.24 ± 3.18% O4+ 81.58 ± 3.94% FOXG1+/O4+			[148]
Cortical glutamatergic neurons	Human ESCs and iPSCs (Monolayer)	Chemical-defined system	Noggin	<65% TUJ1+ ±60% VGLUT1+/TUJ1+ <75% TBR1+/TUJ1+ <72% CTIP2+/TUJ1+ <18% CTIP2+/TBR1+/TUJ1+	NA	NA	[155]
				NA
Cortical glutamatergic neurons	Human ESCs and iPSCs (Dual SMAD inhibition with monolayer)	Chemical-defined system	FGF2, Vitamin A	22–29% TBR1+ 25–30% CTIP2+ 28–36% BRN2+	NA	NA	[164, 165]
				NA
Cortical glutamatergic neurons	Human iPSCs (EB)	Chemical-defined system	BMP4, WNT3A and cyclopamine	62.2 ± 2.1% TBR1+ ±80% VGLUT1+/TUJ1+	MCAO rats	Alleviated sensorimotor deficits, differentiated to glutamatergic neurons and form excitatory, glutamatergic synapses	[166, 168, 169]
				2.5 ± 0.3% TBR1+
Cortical glutamatergic neurons	Human ESCs and iPSCs (EB)	Chemical-defined system (3D)	None	30-40% TBR1+ ±30% CTIP2+ ±10%SATB2	NA	NA	[170]
				NA

The phenotypes of neural lineages, sources of neural stem cells, differentiation protocols, drivers of differentiation, representative phenotypic markers (in vitro) for evaluating the differentiation efficiency and culture homogeneity, expression of representative phenotypic markers after transplantation into corresponding neurological disease model, and improvement of functional outcomes after transplantation are broadly reviewed.

+ represents the percentage of cells stained positive for a specific marker in the differentiation system (in vitro) or in the transplanted population.

AA ascorbic acid, BDNF brain derived neurotrophic factor, BIO GSK3β inhibitor 6-bromoindirubin-3′-oxime, BMP5 bone morphogenic protein 5, BMP7 bone morphogenic protein 7, BRN2 brain-specific homeobox/POU domain protein 2 (POU3F2), Brn4 brain-specific homeobox/POU domain protein 4, CALB1 calbindin 1, Ca-PKA constitutively active protein kinase A, CHAT choline acetyltransferase, CHIR99021 GSK3β inhibitor, CTIP2 b-cell CLL/lymphoma 11b(BCL11B)/COUP-TF-interacting protein 2 (COUP-TFII), 3D three-dimensional, DA dopamine, DARPP-32 dopamine and cAMP-regulated neuronal phosphoprotein 32, DAT dopamine transporter, DKK1 dickkopf-1, DLX2 distal-less homeobox 2, ESCs embryonic stem cells, EB embryoid body, EGF epidermal growth factor, FGF2 fibroblast growth factor 2/basic fibroblast growth factor (bFGF), FGF8 fibroblast growth factor 8, FGF8b fibroblast growth factor 8 isoform b, FOXA2 forkhead box protein A2, FOXG1 forkhead box protein G1, GABA γ-aminobutyric acid, GalC Galactocerebrosides, GIRK2 G protein-activated inward rectifier potassium channel 2 (KCNJ6), HB9 homeobox HB9/motor neuron and pancrease homeobox 1 (MNX1), HD Huntington’s disease, hN human nucleus, IGF-1 insulin-like growth factor 1, iNSC induced neural stem cells, iPSCs induced pluripotent stem cells, IWR1 a tankyrase/Wnt inhibitor, ISL1 ISL LIM homeobox 1, ISL1/2 ISL LIM homeobox 1/2, LHX3 LIM homeobox 3, MAP2 microtubule-associated protein 2, MBP myelin basic protein, MPTP 1-methyl-4-phenyl-1236-tetrahydropyridine, MS multiple sclerosis, MS-5 stromal cell line derived from irradiated murine bone marrow cultures, NGN2 neurogenin 2, NKX6-2 NK6 homeobox 2, NSCs neural stem cells, NURR1 nuclear receptor related 1 protein, NT3 neurotrophin-3, 6-OHDA 6-hydroxydopamine, OLIG2 oligodendrocyte transcription factor 2, PA6 stromal cell line derived from newborn calvaria tissue of the C57BL/6 mice, PBMNCs peripheral blood mononuclear cells, PD Parkinson’s disease, PGDF-AA platelet-derived growth factor AA, PGDFα platelet-derived growth factor -alpha receptor, PUR purmorphamine, PITX3 paired-like homeodomain 3, QA quinolinic acid, RA retinoic acid, SAG smoothened agonist, SATB2 special AT-rich sequence-binding protein 2, SHH sonic hedgehog, SHH-C24II recombinant human SHH, SHH-C25II recombinant mouse SHH, SMAD transcription factor and member of the BMP and TGF-β signaling pathways, T3 triiodothyronine, TBR1 T-box brain 1, SOX3 SRY box 3, SOX10 SRY box 10, TH tyrosine hydroxylase, TUJ1 neuron-specific class III beta-tubulin (TUBB3), VGLUT vesicular glutamate transporter, VM ventral midbrain, VPA valproic acid, VMAT2 vesicular monoamine transporter 2, WNT5 wingless-type MMTV integration site family 5, WNT5a wingless-type MMTV integration site family 5a, XAV939 WNT/β-catenin inhibitor.