Isolation and proteomic profiling of mesenchymal stem cell-derived extracellular vesicles: Unlocking new frontiers in regenerative and translational medicine

Table 1 List of 53 proteins isolated from the extracellular vesicles and their functions

No.	UniProt ID(s)	Protein/gene	Protein name	Function
1	Q7Z460	CLASP1	CLIP-associating protein 1	Stabilization and polarization of cytoplasmic microtubules
2	O75122	CLASP2	CLIP-associating protein 2	Mediator of ERBB2-dependent stabilization of microtubules at the cell cortex
3	Q86SQ0	PHLDB2	Pleckstrin homology-like domain family B member 2	Plays a role in acetyl-choline receptor aggregation in the postsynaptic membrane
4	Q32MQ0	ZNF750	Zinc finger protein 750	Transcription factor involved in epidermis differentiation
5	Q6NXG1	ESRP1	Epithelial splicing regulatory protein 1	mRNA splicing factor that regulates the formation of epithelial cell-specific isoforms, regulates splicing and expression of genes involved in inner ear development, auditory hair cell differentiation, and cell fate specification in the cochlear epithelium
6	O94880	PHF14	PHD finger protein 14	PDGFRA expression, regulates mesenchymal cell proliferation, suppresses the expression of CDKN1A/p21 by reducing the level of trimethylation of histone H3 ‘Lys-4’, leading to enhanced proliferation of germinal center B cells
7	P17844	DDX5	Probable ATP-dependent RNA helicase DDX5	Transcriptional coactivator for RUNX2 and involved in regulation of osteoblast differentiation, control of the circadian rhythms
8	Q92841	DDX17	Probable ATP-dependent RNA helicase DDX17	Involved in multiple cellular processes, including pre-mRNA splicing, alternative splicing, ribosomal RNA processing and miRNA processing, as well as transcription regulation
9	Q8WWZ7	ABCA5	Cholesterol transporter ABCA5	Plays a role in the processing of autolysosomes
10	Q6W2J9	BCOR	BCL-6 corepressor	Acts as a negative regulator of osteo-dentinogenic capacity in adult stem cells
11	Q5SW24	DACT2	Dapper homolog 2	Regulates intracellular signaling pathways during development, control of morphogenetic behavior of kidney ureteric bud cells
12	Q13127	REST	RE1-silencing transcription factor	Plays a role in repression of miR-132 expression in hippocampal neurons, thereby leading to neuronal cell death
13	Q8N163	CCAR2	Cell cycle and apoptosis regulator protein 2	Maintains genomic stability and cellular integrity following UV-induced genotoxic stress
14	Q13263	TRIM28	Transcription intermediary factor 1-beta	Maintains a transcriptionally repressive state of genes in undifferentiated embryonic stem cells, required for activated KRAS-mediated promoter hypermethylation and transcriptional silencing of tumor suppressor genes or other tumor-related genes in colorectal cancer cells
15	Q9UPY3	DICER1	Endoribonuclease dicer	Plays a central role in short dsRNA-mediated post-transcriptional gene silencing
16	Q14999	CUL7	Cullin-7	Regulates Golgi morphogenesis and dendrite patterning in brain, acts as a regulator in trophoblast cell epithelial-mesenchymal transition and placental development
17	Q8TCJ2	STT3B	Dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit STT3B	Plays a role in endoplasmic reticulum-associated degradation pathway that mediates ubiquitin-dependent degradation of misfolded endoplasmic reticulum proteins
18	Q2KJY2	KIF26B	Kinesin-like protein KIF26B	Helps in embryonic kidney development
19	Q9NSC2	SALL1	Sal-like protein 1	Transcriptional repressor involved in organogenesis. Plays an essential role in ureteric bud invasion during kidney development
20	Q7Z591	AKNA	Microtubule organization protein AKNA	Centrosomal protein that plays a key role in cell delamination by regulating microtubule organization
21	Q9Y4H2	IRS2	Insulin receptor substrate 2	Plays role in development, growth, glucose homeostasis as well as lipid metabolism
22	P35568	IRS1	Insulin receptor substrate 1	Plays important role in development, growth, glucose homeostasis as well as lipid metabolism
23	P42229	STAT5A	Signal transducer and activator of transcription 5A	Regulates the expression of milk proteins during lactation
24	P22681	CBL	E3 ubiquitin-protein ligase CBL	Participates in signal transduction in hematopoietic cells, regulates osteoblast differentiation and apoptosis
25	P42224	STAT1	Signal transducer and activator of transcription 1-alpha/beta	Signal transducer and transcription activator that mediates cellular responses to interferons, cytokine KITLG/SCF and other cytokines and other growth factors
26	Q8TDM6	DLG5	Disks large homolog 5	Acts as a regulator of the Hippo signaling pathway, regulating cell proliferation, maintenance of epithelial polarity, epithelial-mesenchymal transition, cell migration and invasion, in dendritic spine formation and synaptogenesis in cortical neurons
27	Q9UK61	TASOR	Protein TASOR	Multiprotein complex that mediates epigenetic repression, plays a crucial role in early embryonic development, in maintaining epiblast fitness or potency
28	Q9BRK4	LZTS2	Leucine zipper putative tumor suppressor 2	Required for central spindle formation and the completion of cytokinesis
29	O60293	ZFC3H1	Zinc finger C3H1 domain-containing protein	Subunit of the trimeric poly(A) tail exosome targeting complex that directs a subset of long and polyadenylated poly(A) RNAs for exosomal degradation
30	Q9H7Z3	NRDE2	Nuclear exosome regulator NRDE2	Responsible for DNA damage
31	Q13009	TIAM1	Rho guanine nucleotide exchange factor TIAM1	Guanyl-nucleotide exchange factor that activates RHO-like proteins and connects extracellular signals to cytoskeletal activities
32	Q9H4I2	ZHX3	Zinc fingers and homeoboxes protein 3	Acts as a transcriptional repressor, involved in the early stages of mesenchymal stem cell osteogenic differentiation
33	Q93052	LPP	Lipoma-preferred partner	Plays a structural role at sites of cell adhesion in maintaining cell shape and motility
34	Q7Z7 L1	SLFN11	Schlafen family member 11	Specifically abrogates the production of retroviruses such as human immunodeficiency virus 1 by acting as a specific inhibitor of the synthesis of retroviruses encoded proteins in a codon-usage-dependent manner
35	P53804	TTC3	E3 ubiquitin-protein ligase TTC3	Regulates neuronal differentiation by regulating actin remodeling and Golgi organization via a signaling cascade involving RHOA, CIT and ROCK
36	Q16363	LAMA4	Laminin subunit alpha-4	Mediates the attachment, migration and organization of cells into tissues during embryonic development
37	P26006	ITGA3	Integrin alpha-3	Participates in the adhesion, formation of invadopodia and matrix degradation processes, promoting cell invasion
38	P02452	COL1A1	Collagen alpha-1 (I) chain	Group I collagen (fibrillar forming collagen)
39	P16234	PDGFRA	Platelet-derived growth factor receptor alpha	Regulates embryonic development, cell proliferation, survival and chemotaxis, differentiation of bone marrow-derived mesenchymal stem cells
40	P46934	NEDD4	E3 ubiquitin-protein ligase NEDD4	Part of a signaling complex composed of NEDD4, RAP2A and TNIK which regulates neuronal dendrite extension and arborization during development
41	P17813	ENG	Endoglin	Regulates angiogenesis, migration of vascular endothelial cells
42	Q15678	PTPN14	Tyrosine-protein phosphatase non-receptor type 14	Regulates lymphangiogenesis, cell-cell adhesion, cell-matrix adhesion, cell migration, cell growth and also regulates TGF-beta gene expression
43	P29320	EPHA3	Ephrin type-A receptor 3	Upon activation by EFNA5, it regulates cell-cell adhesion, cytoskeletal organization and cell migration; in cardiac cells, it controls migration and differentiation and regulates the formation of the atrioventricular canal and septum
44	P54764	EPHA4	Ephrin type-A receptor 4	Plays an important role in the development of the nervous system by controlling different steps of axonal guidance; in axonal guidance, it plays a role in synaptic plasticity
45	Q9NQC3	RTN4	Reticulon-4	Induces the formation and stabilization of endoplasmic reticulum tubules, inhibits spreading, migration and sprouting of primary brain microvascular endothelial cells, stabilization of wiring and restriction of plasticity in the adult central nervous system
46	Q13464	ROCK1	Rho-associated protein kinase 1	Protein kinase that is a key regulator of the actin cytoskeleton and cell polarity, involved in osteoblast compaction, may regulate closure of the eyelids and ventral body wall
47	O75116	ROCK2	Rho-associated protein kinase 2	Protein kinase that is a key regulator of actin cytoskeleton and cell polarity. Regulates smooth muscle contraction, actin cytoskeleton organization, stress fiber and focal adhesion formation, neurite retraction, cell adhesion and mortality
48	Q9P2M7	CGN	Cingulin	Plays a role in the formation and regulation of the tight junction paracellular permeability barrier
49	Q68DE3	USF3	Basic helix-loop-helix domain-containing protein USF3	Involved in the negative regulation of epithelial-mesenchymal transition
50	Q9NUX5	POT1	Protection of telomeres protein 1	Component of the telomerase ribonucleoprotein complex that is essential for the replication of chromosome termini
51	Q6EMK4	VASN	Vasorin	Expressed at highest levels in aorta, at intermediate levels in kidney and placenta and at lowest levels in brain, heart, liver, lung and skeletal muscle
52	Q9C0F0	ASXL3	Putative polycomb group protein ASXL3	The PR-DUB complex is an epigenetic regulator of gene expression and acts as a transcriptional coactivator, affecting genes involved in development, cell communication, signaling, cell proliferation and cell viability
53	Q8IXJ9	ASXL1	Polycomb group protein ASXL1	Together with BAP1, negatively regulates epithelial-mesenchymal transition of trophoblast stem cells during placental development by regulating genes involved in epithelial cell integrity, cell adhesion and cytoskeletal organization. Probable polycomb group protein involved in transcriptional regulation mediated by ligand-bound nuclear hormone receptors, such as retinoic acid receptors and peroxisome proliferator-activated receptor gamma

PDGFRA: Platelet-derived growth factor receptor alpha; RUNX2: Runt-related transcription factor 2; miRNA: MicroRNA; dsRNA: Double-stranded RNA; TGF: Transforming growth factor.

Table 2 Common proteins related to regenerative medicine

Category	UniProt ID(s)	Gene symbol	Protein name	Regenerative function
ECM remodeling and structural support	P02452	COL1A1	Collagen type I alpha-1	Structural ECM, Wound healing, tissue strength
	P35568	FLNA	Filamin-A	Cytoskeletal remodeling, motility, tissue repair
	Q16363	ITGA6	Integrin alpha-6	Stem cell adhesion, marker of regenerative potential
	Q93052	ITGB1BP1	Integrin beta-1-binding protein 1	Integrin signaling in wound healing
Growth factor and regenerative signaling	P16234	PDGFRB	PDGF receptor beta	Angiogenesis, proliferation, repair
	P29320	SHC1	SHC-transforming protein 1	Growth factor adapter, survival, regeneration
	P37275	PPARG	Peroxisome proliferator-activated receptor gamma	Differentiation, tissue remodeling
	P42224	STAT1	STAT1	JAK/STAT repair and immune regulation
	P42229	STAT2	STAT2	Anti-viral and repair signaling
	Q13009	SMAD3	SMAD3	TGF-β pathway, fibrosis, repair
	Q13127	SMAD4	SMAD4	Central TGF-β/BMP signaling in regeneration
Chaperones and stress response	Q13464	HSP90AA1	Heat shock protein HSP90α	Protects stem cells, enhances survival in stress
Cell proliferation and stemness regulators	Q14999	NPM1	Nucleophosmin	Proliferation, ribosome biogenesis, stem cell support
Migration and tissue remodeling	P46934	IQGAP1	IQGAP1	Cell migration, cytoskeletal organization, repair
	Q92841	LRP1	LDL receptor related protein 1	Neuro regeneration, ECM clearance, repair

ECM: Extracellular matrix; PDGF: Platelet-derived growth factor; JAK: Janus kinase; STAT: Signal transducer and activator of transcription; TGF: Transforming growth factor; BMP: Bone morphogenetic protein; HSP: Heat shock protein; LDL: Low-density lipoprotein.

Table 3 Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis observed proteins and false discovery rate

Term ID	Term description	Observed protein count	False discovery rate
hsa04510	Focal adhesion	7	0.00066
hsa05205	Proteoglycans in cancer	7	0.00066
hsa04530	Tight junction	6	0.00091
hsa05206	MicroRNAs in cancer	6	0.00091
hsa05200	Pathways in cancer	8	0.0092
hsa04810	Regulation of actin cytoskeleton	5	0.0229
hsa04935	Growth hormone synthesis, secretion and action	4	0.0229

Table 4 Cellular component observed proteins and false discovery rate

Term ID	Term description	Observed protein count	False discovery rate
GO:0005622	Intracellular anatomical structure	57	0.0027
GO:0043229	Intracellular organelle	54	0.0027
GO:0045180	Basal cortex	3	0.0027
GO:0043226	Organelle	55	0.0033
GO:0043232	Intracellular non-membrane-bounded organelle	31	0.0045
GO:0005654	Nucleoplasm	26	0.014
GO:0043227	Membrane-bounded organelle	52	0.0153
GO:0030981	Cortical microtubule cytoskeleton	2	0.0261
GO:0032991	Protein-containing complex	30	0.0261
GO:0035517	PR-DUB complex	2	0.0261

Table 5 Molecular function observed proteins and false discovery rate

Term ID	Term description	Observed protein count	False discovery rate
GO:0005488	Binding	55	0.00044
GO:0019899	Enzyme binding	20	0.0027
GO:0005515	Protein binding	39	0.0041
GO:0003682	Chromatin binding	9	0.0404
GO:0019904	Protein domain specific binding	10	0.0404
GO:1990782	Protein tyrosine kinase binding	5	0.0404
GO:1990841	Promoter-specific chromatin binding	4	0.0404

Table 6 Biological component observed proteins and false discovery rate

Term ID	Term description	Observed protein count	False discovery rate
GO:0001837	Epithelial to mesenchymal transition	12	5.06E-13
GO:0048762	Mesenchymal cell differentiation	13	2.07E-11
GO:0010717	Regulation of epithelial to mesenchymal transition	11	3.01E-11
GO:0051893	Regulation of focal adhesion assembly	6	0.00002
GO:1903690	Negative regulation of wound healing, spreading of epidermal cells	3	0.00016
GO:1904261	Positive regulation of basement membrane assembly involved in embryonic body morphogenesis	3	0.00024
GO:0001656	Metanephros development	6	7.17E-05
GO:0045595	Regulation of cell differentiation	27	2.18E-11
GO:0060677	Ureteric bud elongation	3	0.00034
GO:0001822	Kidney development	10	1.04E-05