Physiologically based microenvironment for in vitro neural differentiation of adipose-derived stem cells

Table 1 Functions of growth factors on neural induction of adipose stem cells

Growth factors	Profile	Physiological activity	Effect on ASCs	Remarks
EGF	Small polypeptide of 53 amino acid residues and a molecular mass of approximately 6000 daltons[57]	Development of the oral cavity, lungs, gastrointestinal tract, epidermis, derma, eyelids and central nervous system[56]	Promotion of proliferation with delays of senescence and insurance of differentiation potency[55]	EGF and bFGF co-administration limits ASCs differentiation abilities by inducing ASCs into an ectodermal lineage rather than the mesodermal one[53]
bFGF	Non-glycosylated polypeptide of 18 kDa and 155 amino acid in length (heparin-binding growth factor family)	Stimulator of tissue repair and cellular viability released from an injured extracellular matrix[64]	Enhancement of proliferation, differentiation and hepatocyte growth factor expression ability[58]. Induction of the adipogenic[59] and chondrogenic[60] potential, with inhibition of osteogenic differentiation[61]
PDGF	Dimeric glycoprotein	Potent mitogen for cell of mesodermal lineage and stimulator of tissue repair released from activated platelets on bleeding[65]	Supporting of cell proliferation in vitro: It increases ASCs yield. Promotion of neural differentiation in an antioxidant microenvironment[48]	Receptor-β signalling is involved primarily in ASCs stimulation[62]. ASCs stimulation with autologous platelet-rich plasma reduces the cost of differentiation[48]

EGF: Epidermal growth factor; bEGF: Basic fibroblast growth factor; PDGF: Platelet-derived growth factor; ASCs: Adipose stem cells.

Table 2 Effects of chemicals-enriched microenvironment on adipose stem cells fate

Action	Chemicals/drugs	Profile	Recognized function	Effects on ASCs
Antioxidant	β-mercaptoethanol	Water-soluble thiol used as a reducing agent for disulfide bonds to protect sulfhydryl groups from oxidation	In peripheral intestinal nervous system increases the number of synapses and the vesicle population in the nerve terminals[69]	Key elements for the neural induction medium: Reduction of oxidative stress and reactive oxygen species production could support neural population
			Improve meiotic maturation in vitro cultured oocytes[70]
	Butylated hydroxyanisole	Mixture of two isomeric organic compounds	Inhibition of 17 β-estradiol(E2)-mediated oxidative stress and of oxidative DNA damage
	N-acetyl-l-cysteine	Synthetic derivative of endogenous amino acid, L-cysteine, precursor of the antioxidant enzyme Glutathione	Stimulator of glutathione synthase Activator of NMDA1 receptor	When co-administrated, reduction of ASCs-doubling time and increase of cell number compared with b-FGF alone supplementation[66]
	Ascorbic acid-2-phosphate (Vitamin C)	Water-soluble essential vitamin	Reducing agent and coenzyme in several metabolic pathways
Interference on DNA	Valproic acid	Branch-chained fatty acid, acting as a histone deacetylase inhibitor	Wide range of neuroprotection[71,72] Inhibitor of glycogen synthase kinase-3[73] Inducer of chromatin remodeling[74]	Promoter of neuron-like cells[75] In vivo, it improves homing of ASCs via overexpression of CXCR4 and CXCR6[76]
	5-azacytidine	Analog of cytidine nucleoside, acting as demethylating agent[77,78]	Inducer of cell plasticity and active molecule for cellular differentiation into multiple phenotype[79]	Stimulated-cells ameliorate neurological deficits when injected in rats after cerebral ischemia[80]
Anti-inflammation	Indomethacin	Synthetic nonsteroidal indole derivative	Inhibitor of COX1/2	Component of neural induction medium applied for two weeks[44]
Immuno modulation	Hydrocortisone	Glucocorticoid hormone	Suppressor of cell-mediate immunity	Form multi-nucleated myotubes, yielding protein markers for myocytes[9]
Energetic balance	N2 supplement	Chemically defined formulation containing insulin, transferrin, progesterone, putrescine and selenite	In vitro survival and expression of post-mitotic neurons in primary cultures from both the peripheral nervous system and the central nervous system	General promotion of neural cell survival
	B27 supplement	Mixture of vitamins (biotin, Tocopherol, Vitamin A), proteins (BSA, catalase, insulin transferrin, superoxide dismutase), Corticosterone, Galactose, Ethanolamine, Glutathione, Carnitine, Linoleic acid, linolenic acid, progesterone, putrescine, selenite, T3	Growth and maintenance of neurons. Differentiating Glial Precursor Cells into Astrocytes and Oligodendrocytes. Differentiating Neural Stem Cells into Neurons and Glial Cells

ASCs: Adipose stem cells.

Table 3 Biomaterials for neural phenotype of adipose stem cells

Biomaterials	Profile	Effect on ASCs	Test on animal	Limitation for clinical
Chitosan films	Naturally derived polysaccharide from chitin[81,82]	Spontaneous cell organization in a 3D architecture	Yes, higher cellular retention ratio of ASC spheroids after intramuscular injection in nude mouse[81]	Not declared
Chitosan and gelatin	Elastic-dominant, porous scaffold	Conditioning toward a neuron-like phenotype	Yes, better repair in a mouse model of traumatic brain injury[83]	Not declared
Chitosan and silk	Complex structural framework	Efficient as delivery vehicle for ASCs	Yes, proposed as nerve grafts in the regeneration of injured rat sciatic nerve[84]	Not declared
Collagen gel	Engineered neural tissue	Cells must be aligned to collagen fibres	Yes, supported robust neural regeneration of injured rat sciatic nerve[85]	Not declared
Albumin	Serum-derived porous scaffold	Promotion toward neurons	Yes, filler effect on the spinal cord cavity in animal models of spinal cord injury[86]	Not declared
Matrigel	Commercially available hydrogel	Good cell encapsulation and delivery[87]	Yes, mouse models of spinal cord injury	Not applicable for its isolation from the basement membrane of a mouse sarcoma
Alginate	Hydrogel	Neurospheres encapsulation and neural promotion[88,89]		Good biocompatible profile
Nanosized graphene oxide-laminin hybrid patterns	Engineered tissue	Efficient neuron-like cells differentiation[90]

ASCs: Adipose stem cells; 3D: Three-dimensional.