Role of hypoxia preconditioning in therapeutic potential of mesenchymal stem-cell-derived extracellular vesicles

Table 1 Functional categories and annotations in ingenuity pathway analyses of genes regulated by hypoxia-inducible factor 1A

Categories	P value	Top five functional annotations	Number of genes
Carbohydrate Metabolism	3.50E-20	Glycolysis of cells	22
Cardiovascular System Development and Function	3.31E-15 to 4.96E-54	Angiogenesis, Vasculogenesis, Growth of blood vessel	84
Cell Cycle	7.42E-16 to 1.82E-38	Binding of DNA, Cell cycle progression, Interphase, Binding of protein binding site, Arrest in interphase	99
Cell Death and Survival	3.79E15 to 7.3E-74	Apoptosis, Necrosis, Cell death of tumor cell lines, Apoptosis of tumor cell lines, Cell viability	150
Cell Morphology	1.66E-16 to 3.21E-24	Morphology of tumor cell lines, Tubulation of cells, Transmembrane potential of mitochondria, Cell spreading, Orientation of cells	77
Cell Signaling	2.83E-19 to 1.1E-26	Cytokine and chemokine mediated signaling pathway, Quantity of Ca2+	48
Cell-To-Cell Signaling and Interaction	3.31E15 to 1.99E-36	Binding of tumor cell lines, Binding of blood cells, Adhesion of blood cells, Binding of leukocytes, Adhesion of immune cells	105
Cellular Assembly and Organization	5.56E-16 to 1.42E-17	Organization of cytoskeleton, Microtubule dynamics, Fibrogenesis	52
Cellular Development	3.47-15 to 2.85E-73	Cell proliferation of tumor cell lines, Cell proliferation of carcinoma cell lines, Cell proliferation of breast cancer cell lines, Proliferation of muscle cells, Assembly of cells	151
Cellular Function and Maintenance	7.07E-32	Cellular homeostasis	67
Cellular Growth and Proliferation	9.07E-27 to 2.12E-30	Proliferation of vascular cells, Colony formation, Proliferation of connective tissue cells, Proliferation of lymphatic system cells, Proliferation of epithelial cells	101
Cellular Movement	3.31E-15 to 3.35E-67	Cell movement, Migration of cells, Invasion of cells, Cell movement of tumor cell lines, Migration of tumor cell lines	132
Connective Tissue Development and Function	1.36E-16 to 1.73E-30	Growth of connective tissue, Quantity of connective tissue	46
DNA Replication, Recombination, and Repair	1.67E-18 to 8.06E-31	Synthesis of DNA, Metabolism of DNA, Degradation of DNA	47
Drug Metabolism, Lipid Metabolism, Small Molecule Biochemistry	2.36E-16	Synthesis of prostaglandin E2	16
Free Radical Scavenging	6.09E-16 to 5.5E-33	Synthesis of reactive oxygen species, Production of reactive oxygen species, Generation of reactive oxygen species, Quantity of reactive oxygen species	43
Gene Expression	1.373-18 to 4.01E-31	Expression of RNA, Transcription, Transcription of RNA, Transactivation, Transactivation of RNA	88
Inflammatory Response	5.48E-20 to 2.72E-26	Inflammation of absolute anatomical region, Inflammation of organ, Inflammatory response, Inflammation of body cavity, Immune response of cells	77
Lipid Metabolism	6.24E-16 to 9.37E-24	Synthesis of eicosanoid, Metabolism of eicosanoid, Fatty acid metabolism, Synthesis of fatty acid, Synthesis of prostaglandin	48
Organismal Survival	9.55E-37 to 2.76E-39	Organismal death, Survival of organism	81
Post-Translational Modification	1.86E-18	Phosphorylation of protein	34
Protein Synthesis	2.10E-15	Metabolism of protein	44
Tissue Development	1.65E-19 to 4.13E-46	Growth of epithelial tissue, Development of epithelial tissue, Growth of nervous tissue	62
Tissue Morphology	3.02E-17 to 3.06E-26	Quantity of cells, Quantity of tumor cell lines	42

Table 2 Functional categories and annotations in Ingenuity Pathway Analyses of genes regulated by hypoxia-inducible factor 1A

Categories	P value	Top five functional annotations	Number of genes
Carbohydrate Metabolism	2.45E-12 to 2.81E-17	Quantity of carbohydrate, Uptake of D-glucose, Synthesis of polysaccharide, Glycolysis, Uptake of monosaccharide	44
Cardiovascular System Development and Function	1.00E-11 to 8.41E-31	Angiogenesis, Development of vasculature, Vasculogenesis, Endothelial cell development, Proliferation of endothelial cells	68
Cell Cycle	6.82E-12 to 4.39E-19	Mitogenesis, Binding of DNA, Interphase, Arrest in interphase, Cell cycle progression	53
Cell Death and Survival	1.08E-11 to 5.12E-36	Cell death of tumor cell lines, Apoptosis of tumor cell lines, Cell viability, Cell survival, Apoptosis	88
Cell Morphology	1.11E-11 to 4.76E-19	Tubulation of cells, Morphology of tumor cell lines, Autophagy of cells, Formation of cellular protrusions, Autophagy	67
Cell Signaling	6.16E-12 to 1.73E-12	Quantity of Ca2+, Synthesis of nitric oxide	29
Cell-To-Cell Signaling and Interaction	7.17E-12 to 1.36E-26	Activation of cells, Interaction of tumor cell lines, Binding of tumor cell lines, Activation of blood cells, Binding of professional phagocytic cells	62
Cell-mediated Immune Response	4.19E-14 to 7.70E-15	T cell development, T cell homeostasis	26
Cellular Assembly and Organization	1.74E-12 to 3.51E-20	Microtubule dynamics, Organization of cytoskeleton, Organization of cytoplasm, Development of cytoplasm	52
Cellular Development	1.10E-11 to 2.98E-39	Cell proliferation of tumor cell lines, Colony formation of cells, Proliferation of smooth muscle cells, Cell proliferation of carcinoma cell lines, Cell proliferation of breast cancer cell lines	89
Cellular Function and Maintenance	4.81E-13 to 6.37E-27	Cellular homeostasis, Lymphocyte homeostasis, Function of blood cells	63
Cellular Growth and Proliferation	8.67E-13 to 3.51E-27	Colony formation, Proliferation of connective tissue cells, Proliferation of vascular cells, Proliferation of lymphatic system cells, Proliferation of epithelial cells	72
Cellular Movement	9.45E-12 to 2.99E-30	Invasion of cells, Cellular infiltration, Cell movement of myeloid cells, Migration of cells, Cell movement of tumor cell lines	72
Connective Tissue Development and Function	1.56E-16 to 1.63E-25	Growth of connective tissue, Quantity of connective tissue cells, Quantity of connective tissue, Inflammation of joint, Rheumatic Disease	50
DNA Replication, Recombination, and Repair	4.08E-22	Synthesis of DNA	29
Digestive System Development and Function	1.42E-13	Morphology of digestive system	28
Embryonic Development	1.52E-12 to 1.2E-22	Development of body trunk, Development of abdomen, Growth of embryo, Formation of lymphoid tissue, Formation of lung	60
Free Radical Scavenging	3.11E-13 to 1.77E-17	Metabolism of reactive oxygen species, Synthesis of reactive oxygen species, Production of reactive oxygen species	29
Hair and Skin Development and Function	1.33E-13	Growth of skin	17
Hematological System Development and Function	1.17E-11 to 8.08E-21	Quantity of blood cells, Quantity of leukocytes, Quantity of lymphocytes, Quantity of myeloid cells, Quantity of T lymphocytes	52
Inflammatory Response	9.18E-22 to 4E-24	Inflammation of absolute anatomical region, Inflammatory response, Inflammation of body cavity	59
Lipid Metabolism	8.00E-12 to 3.77E-16	Concentration of lipid, Synthesis of lipid	41
Lymphoid Tissue Structure and Development	3.73E-18	Quantity of lymphatic system cells	35
Molecular Transport	7.49E-15	Transport of molecule	44
Nervous System Development and Function	4.54E-12	Sensory system development	22
Organ Development, Renal and Urological System Development	8.50E-12 to 3.25E-12	Growth of kidney, Growth of renal glomerulus	11
Organ Morphology	1.78E-13	Morphology of gland	22
Organismal Development	1.92E-12 to 7.70E-25	Morphology of body cavity, Formation of vessel, Morphology of head, Development of genitourinary system, Growth of organism	73
Organismal Survival	7.68E-26 to 1.24E-28	Organismal death, Survival of organism	75
Post-Translational Modification	4.05E-12 to 1.23E-15	Phosphorylation of protein, Activation of protein	31
Skeletal and Muscular System Development and Function	1.08E-11 to 1.22E-26	Morphology of muscle, Function of muscle, Growth of smooth muscle	39
Tissue Development	1.73E-13 to 3.23E-30	Development of epithelial tissue, Growth of epithelial tissue, Growth of nervous tissue, Accumulation of cells, Formation of epithelial tissue	58
Tissue Morphology	3.33E-14 to 2.65E-24	Quantity of cells, Quantity of progenitor cells	60

Table 3 Use of extracellular vesicles, derived from hypoxic mesenchymal stem cells, in regenerative medicine

Source of MSC	Culture	Hypoxic preconditions, O2%, percentage	Time of exposition	Major findings	Ref.
Human umbilical-cord	α-Mem deprived of FBS	1	72 h	Proangiogenic effects with an increase in UPAR, angiogenin, VEGF, IGF, Tie-2/TEK and IL-6 expression	[104]
	α-Mem deprived of FBS	1	72 h	Promoted angiogenesis in vitro and in vivo	[103]
	DMEM/high glucose media with 10% Exo depleted FBS and 1% penicillin/streptomycin	1	48 h	Enhanced of miRNA-126 exerting a pro-angiogenic effect in endothelial cells thereby activating Spred 1/Ras/Erk pathway	[117]
	α-Mem 10% EV free FBS	1	Not defined	EV encapsulated in a hyaluronic acid adhesive hydrogel have angiogenic properties and nerve regeneration effects after traumatic spinal cord injury	[107]
Olfactory mucosa	DMEM supplemented with 10% EV-depleted FBS	3	48 h	Promoted angiogenesis via miR-612 transfer	[112]
Adipose tissue	α-Mem 10% EV free serum	5	48 h	Promoted vessel formation in vitro. Enhanced angiogenesis, neovascularization and graft survival in vivo. Activation of VEGF/VEGF-R	[105,106]
	EV depleted standard medium	5	72 h	Promoted angiogenesis	[32]
	RPMI medium	1	72 h	Promoted angiogenesis, inhibition of apoptosis, immunomodulation, intracellular ATP recovery and reduction of ROS	[122]
	Microvascular endothelial cell growth medium 2 media deprived of FBS with supplement of 1× serum	1	24 h	Improved diabetic wound healing. Enhanced fibroblasts proliferation and migration activating PI3K/Akt pathway	[132]
	DMEM/F12 with 10% EV-free FBS	0–20 (5 cycles)	Hypoxia 60 min–reoxygenation 30 min	miRNA-224-5p in EV decreases TXNIP expression in cardiomyocytes and protects them from hypoxia mediated injury	[128]
Bone marrow	DMEM with low glucose containing inactivated 15% FBS	—	12 h	Increased of miRNA-21. Synaptic dysfunction restoration, inactivation of STAT3 and NF-kB, reduced plaque deposition and amyloid-β. Regulation of inflammatory responses in APP/PS1 mouse model	[114]
	DMEM with 10% FBS and 1% penicillin-streptomycin	5	6 d	High HMGB1 expression. Activation of JNK pathway and induction of HIF-1α/VEGF expression promoting angiogenesis	[111]
	Exosome-depleted fetal bovine serum	1	48 h	Increased exosomal levels of miRNA-216a-5p. Inhibition of TLR4/NF-κB and activation of PI3KAKT signaling pathway shifting microglial M1/M2 polarization	[115]
	α-Mem 10% exosomes-depleted FBS	2	48 h	Promoted angiogenesis	[118]
	Mesenchymal Stem Cells Medium (Sciencell) 5% exosomes-depleted FBS	1	48 h	Alleviate intervertebral disc degeneration by delivering miR-17-5p	[119]
	DMEM/F12 10% exosomes-depleted FBS	3	48 h	promote cartilage regeneration via the miR-205–5p/PTEN/AKT pathway	[120]
	DMEM/F12 10% exosomes-depleted FBS	5	48 h	EV improved chondrocyte proliferation and migration and suppressed chondrocyte apoptosis. miRNA-18-3P/JAK/STAT or miRNA-181c-5p/MAPK signaling pathway may be involved	[121]
	DMEM low glucose 10% platelet lysate	1	48 h	EV increase angiogenesis, reduced neuronal degeneration, brain atrophy and improved neurological recovery	[116]
Murine bone	α-Mem 10% Exo-removed FBS	0.5	24 h	Significant enrichment of miRNA-210. Promoted survival and recovery of cardiac functions. Also, reduced apoptosis and fibrosis and increased the mobilization of cardiac progenitor cells	[124]
	DMEM/F12 supplemented with 10% fetal bovine serum	1	72 h	Overexpression of miR-210 regulated PI3K/AKT and p53 signaling by targeting AIFM3 reducing apoptosis and tissue death after a myocardial infarction	[125]
	α-Mem 10% Exo-removed FBS	1	72 h	Overexpression of miR-125b-5p. Ability to modify the direction of exosomes to ischemic tissue	[126]

AIFM3: Apoptosis-inducing factor, mitochondria-associated 3; AKT: Protein kinase B (PKB), named derived from kinase encoded by oncogene in transforming retrovirus from thymoma cell line AKT-8 of stock A, strain k, AKR mouse; APP: Amyloid precursor protein; ATP: Adenosine triphosphate; DMEM: Dulbecco’s modified Eagle medium; Erk: Extracellular signal-regulated kinase; Exo: Exosomes; FBS: Fetal bovine serum; GM-CSF: Granulocyte macrophage colony-stimulating factor; HMGB1: High mobility group box 1 protein; IGF: Insulin-like growth factor; IL-6: Interleukin 6; JNK: Jun N-terminal kinase; MAPK: Mitogen-activated protein kinase; NF-B: Nuclear factor kappa B; P53: Tumor protein 53 (antioncogene); PI3K: Phosphatidylinositol 3-kinase; PS1: PreSenilin 1; RKCM: Growth medium stem cell; ROS: Reactive oxygen species; RPMI: Gibco Roswell Park Memorial Institute; Spred 1: Sprouty-related EVH1 domain-containing protein 1; STAT3: Signal transducer and activator of transcription 3; TEK: Tyrosine endothelial kinase; Tie-2: Tyrosine kinase receptor 2; TLR4: Toll-like receptor 4; TXNIP: Thioredoxin-interacting protein.; UPAR: Urokinase-type plasminogen activator receptor; VEGF: Vascular endothelial growth factor.