Adult human neural stem cell therapeutics: Current developmental status and prospect

Table 1 Isolation and in vitro culture methods for adult neural stem cells

Culture methods	Cell source	Dissociating method	Media composition	Plate coating	Maximal in vitro culture	Ref.
Adherent culture method	Temporal lobe	Physical Mincing and enzymatic digestion with papain	DMEM/F12 supplemented with 10 ng/mL bFGF, 20 ng/mL TGFα, 2.5 μg/mL heparin, 2% B27 (without retinoic acid), 10 mmol/L hepes, and 1% FBS			[54]
	Temporal lobe	Mechanical trituration and enzymatic dissociation using papain and DNase I	DMEM/F12 supplemented with 1% B27, 50 ng/mL EGF, 50 ng/mL bFGF, and 0.5% FBS	Poly-L-ornithine	18 passages	[18]
Neurosphere culture method	Hippocampal and lateral ventricle wall tissue	Mechanical dissociation and enzyme digestion using hyaluronic acid, kynurenic acid, and trypsin	DMEM/F12 supplemented with 10 ng/mL EGF, 20 ng/mL EGF, B27, and 2 mmol/L glutamine			[39]
	Temporal lobe	Enzymic digestion with trypsin	N2 medium supplemented with 5% FBS	Poly-2-hydroxyethyl methacrylate		[40]
	Hippocampus Amygdala Frontal cortex Temporal cortex	Enzymic digestion with hyaluronidase, kynurenic acid, and trypsin	DMEM/F12 supplemented with 0.6% glucose, 2 mmol/L glutamine, 3 mmol/L sodium bicarbonate, 5 mmol/L HEPES buffer, 25 mg/mL insulin, 10 mg/mL heparan sulfate, 100 mg/mL transferrin, 20 nmol/L progesterone, 60 mmol/L putrescine, 30 nmol/L selenium chloride, 20 ng/mL EGF, and 20 ng/mL bFGF-2			[41]
	Temporal lobe from 11-wk-old postnatal male Hippocampus, ventricular zone, motor cortex and corpus callosum from and a 27-year-old male	Enzymic digestion with papain, DNase I, and neutral protease	Initially, DMEM/F-12 supplemented with glutamine and 10% FBS After 24 h, DMEM/F12 supplemented with BIT-9500 (bovine serum albumin, transferrin, insulin, 20 ng/mL bFGF, 20 ng/mL EGF, and 20 ng/mL PDGF-AB) and 25% conditioned medium from rat stem cells that produces secretory bFGF and glycosylated form of cystatin C	Fibronectin	More than 70 population doublings in the 11-wk-old postnatal male More than 30 population doublings in the 27-year-old male	[42]
	Temporal lobe	Mechanical dissociation and enzymic digestion with DNase I and trypsin	DMEM/F12 supplemented with 1 mol/L HEPES, 2% B27, 0.1% EGF, and 0.1% bFGF		11 mo	[43]
	Temporal lobe	Enzymic digestion with Papain and DNase I	DMEM/F12 supplemented with bFGF and EGF		3–6 wk	[44,114]
	Lateral ventricular roof	Mechanical dissociation and enzyme digestion with DNase I and trypsin	DMEM/F12 supplemented with bFGF, EGF, and B27		3-7 wk	[45]
	Hippocampus containing hilus, temporal cortex, and subventricular zone including anterior horn and segmented lateral ventricle	Physical mincing and enzyme digestion with trypsin	DMEM/F12 supplemented with N2, 35 μg/mL bovine pituitary extract, 5% fetal calf serum, 40 ng/mL EGF, and 20 ng/mL bFGF		> 60 population doublings	[46]
	Biopsies from filum terminale below conus medullaris	Physical mincing and enzyme digestion with trypsin	DMEM/F12 supplemented with B27, 10 ng/mL LIF, 10 ng/mL bFGF, and 20 ng/mL EGF	Ultra-low attachment dish		[47]

TGFα: Transforming growth factor alpha; DMEM/F12: Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12; FBS: Fetal bovine serum; bFGF: Basic fibroblast growth factor; EGF: Epidermal growth factor.