Table 2.
In vitro studies demonstrating the paracrine effect of hAECs.
| Cells | Preparation | Significant findings | References |
|---|---|---|---|
| HK-2 cells | ECV | - Protection from hypoxia reoxygenation injury-induced apoptosis (Western blot: cleaved Caspase 3). - Improved their proliferation (mRNA: PCNA). |
(15) |
| Mouse BM macrophages | ECV | - Increase in M2 markers (CD206, CD163, IL4rα, and Arg1 mRNA) and downregulation of M1 markers (CD86, IFNγ, TNFα, and iNOS mRNA) after 7 days of culture. | (15) |
| ECV, CM, or EVDM | - Increased M2 marker (CD206, FC) in the M1 induction group, while no significant influence was noted in the M2 induction group or naïve macrophages group. | (17) | |
| CM | - Exposure CM, with or without pre-stimulation with LPS, reduced the proportion of CD86+ cells and increased the proportion of CD206+ cells (Flow cytometry). - qPCR showed a modest increase in M2-specific genes after LPS exposure, which was further increased when CM as compared to LPS-exposed control. - Reduced chemotaxis of macrophages toward rMIP-2 and increased their phagocytic ability. - No significant effect on macrophage proliferation, regardless of exposure to LPS. |
(33) | |
| ECV | - Treatment with 5 μg hAEC-ECV showed double the phagocytic activity in comparison to control (effect of HLF- was remarkably lower than hAEC-ECV but not significant). - Higher percentage of CD206+ (M2 marker) and lower CD86+ (M1 marker) cells in comparison to control and HLF-ECV. |
(16) | |
| CM | - Increased M2 marker (CD206) and decreased M1 marker (iNos) in macrophages treated with TNF-α + INF-γ. | (26) | |
| Human endometrial MSCs damaged by H2O2 | Trans-well hAEC coculture | - Decreased p62 protein level (WB), an indicator of autophagic flux inhibition. - Increased the LC3-II/LC3-I ratio (WB). - Restored the levels of estrogen receptor (ER, WB). |
(19) |
| Myometrial, decidual, and placental cells | ECV from oxidatively stressed hAEC | - ECV from normal or stressed hAEC increased the secretion of IL-6, IL-8, and PGE2 (ELISA) in myometrial and decidual cells. - Increased activation of NF-κβ (WB) in myometrial and decidual cells. - No effect was observed on the level of IL-6, IL-8, PGE2, IL-1β, or TNF-α in placental cells. |
(34) |
| hUVECs, hGL, and hFB | hAEC coculture using Boyden chamber | - Several features of chemotherapy damage of hGL cells were reversed coculture including improved cell count (CCK-8 assay), decreased Annexin-V (+)/PI (+) cells, and decreased cleavage of Caspase 3 expression. - Cocultured hGL showed higher expression of P-SMAD2 and P-SMAD3 proteins (WB) in comparison to chemotherapy-treated group. - Coculture increased the mean tube length of hUVECs. |
(6) |
| CM | - Increased hUVEC proliferations (CCK8) and migration (trans-well assay). | (26) | |
| ECV | - Promoted the proliferation of hUVECs and hFB. - Increased the migration of hFB (scratch assay and trans-well assay). - Increased the number of capillary-like structures were formed by hUVECs (tube-formation assay). - LY294002 to selectively block the PI3K-AKT-mTOR pathway significantly weakened the previous effects. |
(23) | |
| KGN cells | ECV | - Inhibited chemotherapy-induced granulosa cell apoptosis; shown by decreasing Bax and cleaved Caspase 3 and increased anti-apoptotic genes, Bad and Bcl2, expression (WB). - Similar reversal of chemotherapy-induced cleaved Caspase 3 elevation was seen when granulosa cells were transfected with miR-1246 or miR-21-5p mimic. |
(19) |
| Mouse RAW264.7 macrophages | CM | - CM inhibited the pro-inflammatory markers TNF-α, IL-6, and iNOS-2 and increased expression of IL-10 and CD206 genes after LPS treatment. - In the absence of LPS, no significant effect of CM on these markers was noted. - CM inhibited the migration of RAW264.7 cells that was induced by LPS in a macrophage migration assay. |
(7) |
| HSCs | CM | - Reduction of myofibroblast markers α-SMA (qPCR), TGF-β1 expression (ELISA), and collagen production ([3H] proline incorporation). - 2.74-fold increase in matrix metalloproteinase-9 (MMP-9) gene expression. - CM had a negative influence on HSC proliferation (BrdU and platelet-derived growth factor-β expression) and increased their apoptosis by (11.8%, Annexin V:PI FC). |
(31) |
| LX2 | ECV or CM | - Collagen production ([3H] proline incorporation) decreased when treated with ECV or CM. | (17) |
| LPC | CM | - Increased BrdU incorporation in liver progenitor cell (LPC) cultures after 3 days of treatment. - After 8 days of culture, CM induced differentiation toward hepatocytes as demonstrated by increased AFP and albumin expression. - Activity of tyrosine aminotransferase promoter was only observed in hAEC-CM treated LPCs - Urea production in CM-treated group was higher than untreated and hepatocyte-differentiation-medium-treated cultures after 16 days. - KEGG pathway analysis of microarray showed that genes associated with drug, glutathione, and arachidonic acid metabolism, steroid biosynthesis, and bile acid secretion were upregulated in LPC cultures, while genes associated with cell proliferation and phosphoinositide 3-kinase signaling were downregulated. - Gene Ontology (GO) pathway analysis showed that the upregulated genes were significantly enriched in multiple metabolic processes, negative regulation of cell growth, and apoptosis, while downregulated genes were enriched in pathways related to cell proliferation. |
(5) |
| Human dermal fibroblasts | CM | - α-SMA, Col-I, and Col-III induced by TGF-β1 (qPCR and WB) were significantly repressed in the TGFβ1+ CM co-treatment group compared to TGF-β1 alone. - CM showed similar results in fibroblasts derived from keloid - After TGF-β1 stimulation of human dermal fibroblasts, CM significantly increased MMP-1, and TIMP-1 (qPCR and WB). - CM attenuated the TGF-β1-induced contraction of F-actin bundles and decreased the number of bundles. - CM addition to keloid fibroblasts also resulted in a reduction in supernatant collagen. - MMP-1, MMP-2, and TIMP1 were upregulated in keloid fibroblasts by CM. |
(35) |
| ECV | - Enhanced the proliferation of fibroblasts. - Remarkably increased their migration in a scratch assay in a dose-dependent manner. - Col-I and Col-III were repressed by 100 μg/mL ECV. - Collagen in the supernatant (Sircol collagen assay) was reduced by 100 μg/mL ECV. - MMP-1 mRNA was upregulated |
(36) | |
| ECV ± PROse or RNase | - Compared to the control or RNase-treated ECV, PROse-treated ECV enhanced the migration of fibroblasts after 24 h treatment. - ECV or PROse-treated ECV increased cell index value of fibroblasts. RNase-treated ECV lacked this effect. |
(37) | |
| CM | - After damage of hDFs by H2O2, hAEC-, and hAMSC-CM improved proliferation. - The conc. of hAEC-, but not hAMSC-, CM also improved proliferation. - All test groups markedly improved cell viability, with the hAEC-CM showing the strongest effect. - Both CMs improved normal hDFs migration in a scratch assay at 24 h, while only hAMSC-CM improved migration at 12 h. - hAEC-CM, but not hAMSC-CM, increased the percentage of cells in the S phase in comparison to damaged control. - Both CMs decreased the percentage of senescent cells in comparison to damaged controls (SA-β-gal). - Both CMs reduced reactive oxygen species production (ROS), increased activity of superoxide dismutase and catalase and decreased malondialdehyde and 8-OHdG levels (ELISA) in comparison to damaged hDFs. - Both CMs decreased γ-H2AX/H2AX ratio compared to damaged cells and lower ratio was achieved with hAEC-CM compared to hAMSC-CM. - mRNA levels of p21 and protein levels of p21 and p16 were reduced after either CM treatments compared to damaged control. |
(38) | |
| Keratinocytes | CM | - Increased the migration in a wound scratch assay at 6 h, with complete healing at 18 h. - Increased the phosphorylation of ERK, JNK, and AKT 15 min after treatment, which peaked at 60 min. |
|
| - Keratinocyte migration was completely blocked when cells were pre-treated with mitomycin C and ERK inhibitor (PD98059) or JNK inhibitor (SP600125), while it was not completely inhibited by AKT inhibitor (LY294002). Incubation with mitomycin C was to exclude their effect on cell proliferation. - Coculture with hAEC increased the expression of Cyclin D1, Cyclin D3, and Mdm2 as shown by Western blotting. - Coculture hAEC resulted in a higher S-phase fraction. - Compared with CM from fibroblasts, caused higher expression of the genes EGF, KGF, PDGF, CXCL-5, and SDF1, and lower expression of TGF-β1, while compared with CM from hAMSC, resulted in higher expression of the genes EGF, KGF, and CXCL-5 and lower expression of the genes PDGF, TGF-β1, and SDF-1. |
(39) | ||
| Mouse BM-Neutrophils | ECV | - Treatment with either 1 or 5 μg of hAEC-ECV or 1 μg of hLF-ECV resulted in lower myeloperoxidase activity and higher cell death than controls. | (16) |
| Mouse T-lymphocytes (from spleen) | - Proliferation was suppressed by hAEC-ECV in comparison to hLF-ECV and control. - Using Foxp3-GFP knock-in CD4+cells and labeled exosomes with exo-red dye showed that uptake of hAEC-ECV corresponded to the maturation of CD4+ T cells into Foxp3-expressing cells, suggesting their Treg nature. |
||
| Mouse BASCs | ECV | - Differentiated to alveolar, bronchiolar, and bronchioalveolar colonies after 21 days. - More and larger-sized bronchiolar and alveolar colonies resulted compared to medium-only control. - Higher expression of club cell marker (CC10) in bronchiolar colonies, and ciliogenesis marker Foxj1 in mixed colonies with ECV (5 and 10 μg) compared to control. |
(16) |
| Human lung fibroblasts | ECV | - Exposure of HLF treated with TGF-β to ECV decreased myofibroblast differentiation in a dose-dependent way. | (16) |
| Mouse mesencephalic neuronal-glial cells | CM | - Pro-survival effect on lesioned neurons (treated with MPP+) in the form of increased longest neurite length and number of branching points per dopaminergic neuron. - Neutralizing antibodies against BDNF, CNTF, GMCSF, OSM, or NrCAM reduced the survival of dopaminergic neurons, the neurite outgrowth, the longest neurite length, and branching points per neuron, while neutralizing antibody against IL-1ra reduced the survival and branching points per neuron in CM-treated lesioned neurons. |
(25) |
| Rat Embryonic (E18) cortical neurons | CM | - Remarkably Improved survival (91.9%). | (28) |
| Rat retinal ganglion cells | CM | - Enhanced their survival (52.3 ± 14.4%). The effect was superior to BDNF, CNTF, and NT-3. - This effect was retained only in the low-sized (<30 kDa) fraction of the CM. |
(40) |
| Human umbilical cord blood-derived MSCs | CM | - Increased the number of DAT- and TH- positive cells. DAT and TH are considered dopaminergic neuron-specific markers. - Addition of K252a (Trk receptor inhibitor of BDNF and NGF) incompletely, but significantly, reversed this effect. |
(29) |
| Primary mouse microglia | CM | - Decreased the CD86+ M1 subtype after LPS stimulation for 24 h as shown by FACs. - Decreased the number of apoptotic microglia and increased their phagocytic activity. |
(41) |
| Human peripheral blood monocytes | Trans-well and CM | - Alteration of monocyte differentiation toward dendritic cells, resulting in cells that might be at an intermediate state of differentiation/maturation. The suppressive effect was less prominent compared to hAMSC. | (42) |
| Mouse peritoneal macrophages | CM | - Inhibited macrophage migration toward MIP-2. - Did not induce apoptosis (Caspase-3) or toxicity (trypan blue exclusion). |
(43) |
| Mouse peritoneal neutrophils | CM | - Inhibited neutrophil migration toward MIP-2. - Did not induce apoptosis (Caspase-3) or toxicity (trypan blue exclusion). |
(43) |
| Mouse lymphocytes | CM | - Decreased mouse T and B lymphocyte proliferation after mitogenic stimulation, but not without stimulation. - Induced apoptosis of T and B lymphocytes (Caspase-3 assay), 50% inhibition of this apoptosis was achieved with anit-FasL antibody. |
(43) |
| Human CD4+ T-lymphocytes | ECV | - Inhibited lymphocyte proliferation (CSFE) with a more evident inhibition in case of small-sized ECV compared to large-sized ECV. | (44) |
| Human PBL | CM | - Inhibited lymphocyte proliferation after their exposure to either human pancreatic islets or PHA (mitogen). | (45) |
| Human fetal osteoblast cell line (hFOB1.19) | CM | - Increased proliferation, migration, and osteogenic differentiation (ALP, OCN, OPN, and RUNX2). - Neutralizing TGFβ1 antibody treatment attenuated the positive effect of CM on migration and osteogenic differentiation. - miR-34a-5p inhibitor transfection to hAEC decreased the hAEC-CM pro-differentiation effect. |
(46) |
| Human aortic endothelial cells | CM | - Increased cell migration in scratch and trans-well migration assays. - Increased number of branching points at 6 and 48 h in Matrigel network formation assay. |
(47) |
BASCs, broncho-alveolar stem cells; BM, bone marrow; BrdU, 5-bromo-2′-deoxyuridine; CM, conditioned medium; DAT, dopamine transporter; ECV, extracellular vesicle; EVDM, ECV-depleted conditioned medium; GO, gene ontology; hAEC, human amniotic epithelial cells; hAMSC, human amniotic mesenchymal stem cells; hFB, human fibroblasts; hGL, human granulosa-lutein cells; hLF, human lung fibroblast; HSCs, hepatic stellate cells; hUVECs, human umbilical vein endothelial cells; KEGG, Kyoto Encyclopedia of Genes and Genomes; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells; MIP2, macrophage inflammatory protein-2; PROse, proteinase K; SMA, smooth muscle actin; TH, tyrosine hydroxylase. Cell lines: HK-2, human proximal tubular cell line; KGN, human granulosa-like tumor cell line; LX2, human hepatic stellate cell line.