Amniotic Fluid Mesenchymal Stem Cells Research Articles

DNMT1 driven by mouse amniotic fluid mesenchymal stem cell exosomes improved corneal cryoinjury via inducing microRNA-33 promoter DNA hypermethylation modification in corneal epithelium cells.

Severe corneal cryoinjury can cause permanent corneal swelling and bullous keratopathy, one of the main reason for loss of sight. Mouse amniotic fluid mesenchymal stem cells (mAF-MSCs) can repair corneal damage caused by freezing; however, whether the exosomes derived from mAF-MSCs have the same repair effect is unknown. In this study, the mAF-MSC-exosomes were transplanted into the eyeballs of corneal cryoinjured mice. Histopathological examination showed that the mAF-MSC-exosomes improved the corneal structure and status of corneal epithelial cells in corneal cryoinjured mice. RRBS-sequencing showed that compared with the control group, four genes (Rpl13-ps6, miR-33, Hymai, and Plagl1), underwent DNA hypermethylation modification after mAF-MSC-exosomes treatment. The result of FISH indicated that miR-33-3p hybridization signals were enhanced in corneal epithelial cells from mice treated with mAF-MSC-exosomes. Semi-quantitative PCR and western blotting indicated that mAF-MSC-exosomes contained high levels of DNMT1 mRNA and protein. Additionally, luciferase report assays indicated that miR-33-3p overexpression in NIH-3T3 mouse embryonic fibroblast cells inhibited the activity of luciferase carrying a sequence from the 3' untranslated region of Bcl6. Moreover, BCL6 mRNA and protein levels in corneal tissues from mice treated with mAF-MSC-exosomes were higher than those in the control group. Therefore, our results suggested that mAF-MSC-exosomes could repair corneal cryoinjury by releasing DNMT1, which induced hypermethylation of the miR-33 promoter in corneal epithelial cells. Consequent downregulated miR-33 transcription upregulated Bcl6 expression, ultimately achieving the repair of corneal cryoinjury in mice.

Evaluation of stability and safety of equine mesenchymal stem cells derived from amniotic fluid for clinical application.

Amniotic fluid mesenchymal stem cells (AF-MSCs), which can be obtained from fetal tissue, reportedly have self-renewal capacity and multi-lineage differentiation potential. The aim of this study was to identify the biological characteristics of AF-MSCs and evaluate their stability and safety in long-term culture. To confirm the biological characteristics of AF-MSCs, morphology, proliferation capacity, karyotype, differentiation capacity, gene expression level, and immunophenotype were analyzed after isolating AF-MSCs from equine amniotic fluid. AF-MSCs were differentiated into adipocytes, chondrocytes, and osteocytes. Immunophenotype analyses revealed expression levels of ≥95% and ≤ 2% of cells for a positive and negative marker, respectively. Analysis of the MSCs relative gene expression levels of AF-MSCs was approximately at least twice that of the control. The endotoxin level was measured to verify the safety of AF-MSCs and was found to be less than the standard value of 0.5 EU/ml. AF-MSCs were cultured for a long time without any evidence of abnormalities in morphology, proliferation ability, and karyotype. These results suggest that amniotic fluid is a competent source for acquiring equine MSCs and that it is valuable as a cell therapy due to its high stability.

Immunomodulatory-associated gene transcripts to multipotency of bovine amniotic fluid mesenchymal stem cells.

The adnexa fetal tissues are sources of mesenchymal stromal cells (MSCs) due to their noninvasive harvest, with all biological material discarded most of the time. MSCs are a promise regarding to their plasticity, self-renewal, differentiation potentials, immunomodulatory and anti-inflammatory properties, which have made clinical stem cell therapy a reality. The present study aimed to characterize and evaluate the immunomodulation ability of bovine mesenchymal cells collected from bovine amniotic fluid (bAFMSCs) isolated and subjected to sixth consecutive culture passages in vitro. The multilineage properties of the bAFMSCs collections confirmed the ability to undergo adipogenic, chondrogenic and osteogenic differentiation. The mesenchymal gene transcription CD106, CD73, CD29, CD90 and CD166 were detected in bAFMSCs, whereas CD34 and CD45 were not detected. Regarding cytokine mRNA expression, IL2, IL6, INFα, INFβ, INFγ, TNFα and TNFβ were downregulated, while IL10 was highly regulated in all studied passages. The present study demonstrated the immunological properties and multipotency of in vitro bAFMSCs collections, and thus, they can be tested in cattle pathological treatments or multiplication by nuclear transfer cloning.

Human Mesenchymal Stem Cell Transplantation Improved Functional Outcomes Following Spinal Cord Injury Concomitantly with Neuroblast Regeneration.

Spinal cord injury (SCI) is damage to the spinal cord that resulted in irreversible neuronal loss, glial scar formation and axonal injury. Herein, we used the human amniotic fluid mesenchymal stem cells (hAF-MSCs) and their conditioned medium (CM), to investigate their ability in neuroblast and astrocyte production as well as functional recovery following SCI. Fifty-four adult rats were randomly divided into nine groups (n=6), included: Control, SCI, (SCI + DMEM), (SCI + CM), (SCI + MSCs), (SCI + Astrocyte), (SCI + Astrocyte + DMEM), (SCI + Astrocyte + CM) and (SCI + Astrocyte + MSCs). Following laminectomy and SCI induction, DMEM, CM, MSCs, and astrocytes were injected. Western blot was performed to explore the levels of the Sox2 protein in the MSCs-CM. The immunofluorescence staining against doublecortin (DCX) and glial fibrillary acidic protein (GFAP) was done. Finally, Basso-Beattie-Brenham (BBB) locomotor test was conducted to assess the neurological outcomes. Our results showed that the MSCs increased the number of endogenous DCX-positive cells and decreased the number of GFAP-positive cells by mediating juxtacrine and paracrine mechanisms (P<0.001). Transplanted human astrocytes were converted to neuroblasts rather than astrocytes under influence of MSCs and CM in the SCI. Moreover, functional recovery indexes were promoted in those groups that received MSCs and CM. Taken together, our data indicate the MSCs via juxtacrine and paracrine pathways could direct the spinal cord endogenous neural stem cells (NSCs) to the neuroblasts lineage which indicates the capability of the MSCs in the increasing of the number of DCX-positive cells and astrocytes decline.

Extracellular Vesicles of Mesenchymal Stem Cells Are More Effectively Accessed through Polyethylene Glycol-Based Precipitation than by Ultracentrifugation.

Extracellular vesicles (EVs) have been identified as cell-cell communication agents, and EVs derived from mesenchymal stem cells (MSCs) exhibit therapeutic effects similar to those of the cells of origin. Precipitation methods have been used extensively for EV harvests, such as UC- (ultracentrifugation-) or PEG- (polyethylene glycol-) based methods, and the difference in EVs derived from MSCs by UC and PEG is not fully understood. We harvested EVs from amniotic fluid MSCs (AF-MSCs) by UC- or PEG-based precipitation methods and conducted a comparison study of those EVs derived by the two methods: output, RNA, and protein expression of EVs and EV biological reaction in a THP-1-cell model of LPS induction, which was considered an infection model. There was no difference in morphology, size, or specific marker-positive ratio of PEG-EVs and UC-EVs, but PEG obtained more EV particles, protein, and RNA than the UC method. In our THP-1 model of LPS induction, MSC-EVs did not lead to a change in protein expression but inhibited the LPS-induced increase in cytokine secretion. UC-EVs were more effective for TNF-α inhibition, and PEG-EVs were more effective for IL10 inhibition. Thus, our findings provide evidence that PEG-based precipitation is a more efficient mesenchymal stem cell-extracellular vesicle-derived method than UC.

Effect of Passage Number of Conditioned Medium Collected from Equine Amniotic Fluid Mesenchymal Stem Cells: Porcine Oocyte Maturation and Embryo Development.

Oocyte in vitro maturation (IVM) is the most important first step in in vitro embryo production. One prerequisite for the success of IVM in oocytes is to provide a rich culture microenvironment that meets the nutritional needs of developing oocytes. We applied different equine amniotic fluid mesenchymal stem cell conditioned medium (eAFMSC-CM) from passages 7, 18, and 27 to porcine oocytes during IVM to determine its effects on oocyte development and subsequent embryo development, specifically. The eAFMSC-CM from passage 7 (eAFMSC-CMp7) has a considerable impact on 9 genes: BAX, BCL2, SOD2, NRF2, TNFAIP6, PTGS2, HAS2, Cx37, and Cx43, which are associated with cumulus cell mediated oocyte maturation. GSH levels and distribution of mitochondrial and cortical granules were significantly increased in oocytes incubated with eAFMSC-CMp7. In addition, catalase and superoxide dismutase activities were high after IVM 44 h with eAFMSC-CMp7. After in vitro fertilization, blastocyst quality was significantly increased in the eAFMSC-CMp7 group compared to control. Lastly, the antioxidant effect of eAFMSC-CMp7 substantially regulated the expression of apoptosis, pluripotency related genes and decreased autophagy activity in blastocysts. Taken together, this study demonstrated that the eAFMSC-CMp7 enhanced the cytoplasmic maturation of oocytes and subsequent embryonic development by generating high antioxidant activity.

Human amniotic fluid mesenchymal stem cells attenuate pancreatic cancer cell proliferation and tumor growth in an orthotopic xenograft mouse model

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a malignant cancer and chemotherapy ineffectively treats PDAC, leading to the requirement for alternative tumor-targeted treatment. Human amniotic fluid mesenchymal stem cells (hAFMSCs) have been revealed to suppress tumor growth in various cancers and they are a strong candidate for treating PDAC.MethodsTo evaluate the effects of hAFMSCs on human pancreatic carcinoma cells (PANC1, AsPC1 and BxPC3 cell lines) and the possible mechanism involved, an in vitro cell coculture system was used. A PANC1 orthotopic xenograft mouse model was established and hAFMSCs were injected intravenously at 4 weeks post-xenograft.ResultsAn in vitro coculture assay showed that hAFMSCs inhibited PANC1 cell proliferation by inducing S phase cell cycle arrest and increased cell apoptosis in a time-dependent manner. In PANC1 cells, hAFMSCs caused the downregulation of Cyclin A and Cyclin B1 as well as the upregulation of p21 (CDKN1A) at 24 h post coculture. The upregulation of pro-apoptotic factors Caspase-3/-8 and Bax at 24 h post coculture reduced the migration and invasion ability of PANC1 cells through inhibiting the epithelial-mesenchymal transition (EMT) process. In a PANC1 orthotopic xenograft mouse model, a single injection of hAFMSCs showed significant tumor growth inhibition with evidence of the modulation of cell cycle and pro-apoptotic regulatory genes and various genes involved in matrix metallopeptidase 7 (MMP7) signaling-triggered EMT process. Histopathological staining showed lower Ki67 levels in tumors from hAFMSCs-treated mice.ConclusionsOur data demonstrated that hAFMSCs strongly inhibit PDAC cell proliferation, tumor growth and invasion, possibly by altering cell cycle arrest and MMP7 signaling-triggered EMT.

Conditioned medium from amniotic fluid mesenchymal stem cells could modulate Alzheimer's disease-like changes in human neuroblastoma cell line SY-SY5Y in a paracrine manner

BackgroundAlzheimer's disease is usually diagnosed by significant extracellular deposition of beta-amyloid and intracellular neurofibrillary tangle formation. Here, we investigated the paracrine effect of amniotic fluid-derived mesenchymal stem cells on AD changes in human SH–SY5Y cells. MethodsSH–SY5Y cells were divided into five groups: Control, 0.1 µg/ml LPS, 10 µg/ml LPS, 0.1 µg/ml LPS + conditioned medium, and 10 µg/ml LPS + conditioned medium. Cells were incubated with 0.1% and 10 µg/ml LPS for 48 h, followed by incubation with the conditioned medium of amniotic fluid-derived mesenchymal stem cells for the next 24 h. Beta-amyloid plaques were monitored by Congo-red staining. Survival and apoptosis were assessed by the MTT assay and flow cytometric analysis of Annexin-V. ELISA was used to measure the levels of neprilysin, angiotensin-converting enzyme, and Matrix Metalloproteinase–9. A PCR array was used to measure the expression of genes involved in neurogenesis. ResultsBright-field imaging showed beta-amyloid plaques in the group treated with 10 µg/ml LPS. We found minimal effects in groups receiving 0.1 µg/ml LPS. The data showed that the reduction in the levels of neprilysin, angiotensin-converting enzyme, and Matrix Metalloproteinase–9 in the LPS-treated cells was attenuated after incubation with the stem cell secretome (p < 0.05). Amniotic fluid stem cell secretome increased the viability of LPS-treated SH–SY5Y cells (p 0.05) and was associated with a decrease in apoptotic changes (p < 0.05). We found the modulation of several genes involved in neurogenesis in the 10 µg/ml LPS + conditioned medium group compared to cells treated with 10 µg/ml LPS alone. ConclusionAmniotic fluid stem cell secretion reduces AD-like pathologies in the human neuronal lineage.

Exosomes Derived from Human Amniotic Fluid Mesenchymal Stem Cells Preserve Microglia and Neuron Cells from Aβ.

Background: Neuroinflammation is involved in neuronal cell death that occurs in neurodegenerative diseases such as Alzheimer’s disease (AD). Microglia play important roles in regulating the brain amyloid beta (Aβ) levels, so immunomodulatory properties exerted by mesenchymal stem cells may be exploited to treat this pathology. The evidence suggests that the mechanism of action of human amniotic fluid stem cells (hAFSCs) is through their secretome, which includes exosomes (exo). Methods: We examined the effect of exosomes derived from human amniotic fluid stem cells (hAFSCs-exo) on activated BV-2 microglia cells by lipopolysaccharide (LPS) as a neuroinflammation model. To investigate the exo effect on the interplay between AD neurons and microglia, SH-SY5Y neuroblastoma cells treated with Aβ were exposed to a conditioned medium (CM) obtained from activated BV-2 or co-culture systems. Results: We found that the upregulation of the markers of pro-inflammatory microglia was prevented when exposed to hAFSC-exo whereas the markers of the anti-inflammatory macrophage phenotype were not affected. Interestingly, the hAFSC-exo pretreatment significantly inhibited the oxidative stress rise and apoptosis occurring in the neurons in presence of both microglia and Aβ. Conclusion: We demonstrated that hAFSC-exo mitigated an inflammatory injury caused by microglia and significantly recovered the neurotoxicity, suggesting that hAFSC-exo may be a potential therapeutic agent for inflammation-related neurological conditions, including AD.

Potential effect of amniotic fluid-derived stem cells on hyperoxia-induced pulmonary alveolar injury

BackgroundWith the widespread of Coronavirus Disease 2019 pandemic, in spite of the newly emerging vaccines, mutated strains remain a great obstacle to supportive and preventive measures. Coronavirus 19 survivors continue to face great danger of contacting the disease again. As long as no specific treatment has yet to be approved, a great percentage of patients experience real complications, including among others, lung fibrosis. High oxygen inhalation especially for prolonged periods is per se destructive to the lungs. Nevertheless, oxygen remains the first line support for such patients. In the present study we aimed at investigating the role of amniotic fluid-mesenchymal stem cells in preventing versus treating the hyperoxia-induced lung fibrosis in rats.MethodsThe study was conducted on adult albino rats; 5 pregnant female rats were used as amniotic fluid donors, and 64 male rats were randomly divided into two groups: Control group; where 10 rats were kept in normal atmospheric air then sacrificed after 2 months, and hyperoxia-induced lung fibrosis group, where 54 rats were exposed to hyperoxia (100% oxygen for 6 h/day) in air-tight glass chambers for 1 month, then randomly divided into the following 5 subgroups: Hyperoxia group, cell-free media-treated group, stem cells-prophylactic group, stem cells-treated group and untreated group. Isolation, culture and proliferation of stem cells were done till passage 3. Pulmonary function tests, histological examination of lung tissue under light and electron microscopes, biochemical assessment of oxidative stress, IL-6 and Rho-A levels, and statistical analysis of data were performed. F-test (ANOVA) was used for normally distributed quantitative variables, to compare between more than two groups, and Post Hoc test (Tukey) for pairwise comparisons.ResultsLabelled amniotic fluid-mesenchymal stem cells homed to lung tissue. Stem cells administration in the stem cells-prophylactic group succeeded to maintain pulmonary functions near the normal values with no significant difference between their values and those of the control group. Moreover, histological examination of lung tissues showed that stem cells-prophylactic group were completely protected while stem cells-treated group still showed various degrees of tissue injury, namely; thickened interalveolar septa, atelectasis and interstitial pneumonia. Biochemical studies after stem cells injection also showed decreased levels of RhoA and IL-6 in the prophylactic group and to a lesser extent in the treated group, in addition to increased total antioxidant capacity and decreased malondialdehyde in the stem cells-injected groups.ConclusionsAmniotic fluid-mesenchymal stem cells showed promising protective and therapeutic results against hyperoxia-induced lung fibrosis as evaluated physiologically, histologically and biochemically.

Apoptotic effects of human amniotic fluid mesenchymal stem cells conditioned medium on human MCF-7 breast cancer cell line.

Breast cancer, as the most common malignancy among women, is shown to have a high mortality rate and resistance to chemotherapy. Research has shown the possible inhibitory role of Mesenchymal stem cells in curing cancer. Thus, the present work used human amniotic fluid mesenchymal stem cell-conditioned medium (hAFMSCs-CM) as an apoptotic reagent on the human MCF-7 breast cancer cell line. Conditioned medium (CM) was prepared from hAFMSCs. After treating MCF-7 cells with CM, a number of analytical procedures (MTT, real-time PCR, western blot, and flow cytometry) were recruited to evaluate the cell viability, Bax and Bcl-2 gene expression, P53 protein expression, and apoptosis, respectively. Human fibroblast cells (Hu02) were used as the negative control. In addition, an integrated approach to meta-analysis was performed. The MCF-7 cells' viability was decreased significantly after 24 hours (P < 0.0001) and 72 hours (P < 0.05) of treatment. Compared with the control cells, Bax gene's mRNA expression increased and Bcl-2's mRNA expression decreased considerably after treating for 24 hours with 80% hAFMSCs-CM (P = 0.0012, P < 0.0001, respectively); an increasing pattern in P53 protein expression could also be observed. The flow cytometry analysis indicated apoptosis. Results from literature mining and the integrated meta-analysis showed that hAFMSCs-CM is able to activate a molecular network where Bcl2 downregulation stands in harmony with the upregulation of P53, EIF5A, DDB2, and Bax, leading to the activation of apoptosis. Our finding demonstrated that hAFMSCs-CM presents apoptotic effect on MCF-7 cells; therefore, the application of hAFMSCs-CM, as a therapeutic reagent, can suppress breast cancer cells' viabilities and induce apoptosis.

Mesenchymal stem cells from different sources show distinct therapeutic effects in hyperoxia-induced bronchopulmonary dysplasia in rats.

Mesenchymal stem cells (MSCs) have been shown as an effective medicinal means to treat bronchopulmonary dysplasia (BPD). The widely used MSCs were from Wharton's jelly of umbilical cord (UC‐MSCs) and bone marrow (BM‐MSCs). Amniotic fluid MSCs (AF‐MSCs) may be produced before an individual is born to treat foetal diseases by autoplastic transplantation. We evaluated intratracheal (IT) MSCs as an approach to treat an hyperoxia‐induced BPD animal model and compared the therapeutic effects between AF‐, UC‐ and BM‐MSCs. A BPD animal model was generated by exposing newborn rats to 95% O2. The continued stress lasted 21 days, and the treatment of IT MSCs was conducted for 4 days. The therapeutic effects were analysed, including lung histology, level of inflammatory cytokines, cell death ratio and state of angiogenesis, by sacrificing the experimental animal at day 21. The lasting hyperoxia stress induced BPD similar to the biological phenotype. The treatment of IT MSCs was safe without deaths and normal organ histopathology. Specifically, the treatment was effective by inhibiting the alveolar dilatation, reducing inflammatory cytokines, inducing angiogenesis and lowering the cell death ratio. AF‐MSCs had better therapeutic effects compared with UC‐MSCs in relieving the pulmonary alveoli histological changes and promoting neovascularization, and UC‐MSCs had the best immunosuppressive effect in plasma and lung lysis compared with AF‐MSCs and BM‐MSCs. This study demonstrated the therapeutic effects of AF‐, UC‐ and BM‐MSCs in BPD model. Superior treatment effect was provided by antenatal MSCs compared to BM‐MSC in a statistical comparison.

Exosomes derived from human amniotic fluid mesenchymal stem cells alleviate cardiac fibrosis via enhancing angiogenesis in vivo and in vitro

Cardiac fibrosis is a pathological process characterized by excess extracellular matrix (ECM) deposition and plays a critical role in nearly all types of heart disease. The mechanism of cardiac fibrosis is still unclear and no effective medication treatment of cardiac fibrosis. Research showed that mesenchymal stem cell (MSC) derived exosomes may play a critical role in cardiac fibrosis. The effect of human amniotic fluid MSC (hAFMSC)-derived exosomes (hAFMSCExos) on cardiac fibrosis has remained unclear. The hAFMSCExos were extracted using a sequential centrifugation approach. The effects of hAFMSCExos on angiogenesis were analyzed both in human umbilical vein endothelial cells (HUVECs) after oxygen and glucose deprivation (OGD) in vitro, and in isoproterenol (ISO) induced-cardiac fibrosis in vivo. The hAFMSCExos remarkably up-regulate the motility and migration of HUVECs after OGD compared with phosphate-buffered saline (PBS). Meanwhile, total tube length, total branching points and total loops were significantly raised in HUVECs after OGD treated with hAFMSCExos. The hAFMSCExos alleviated the cardiac fibrosis degree tested by hematoxylin-eosin (H&E) and Masson staining. The protein levels of Collagen I and α-smooth muscle actin (α-SMA) were lower in exosomes group rats than PBS group. Immunofluorescence suggested that hAFMSCExos can promote the expression of CD31 in the rats. Meanwhile, the number of regenerated microvessels was significantly enhanced in rats administrated with exosomes by quantitative analysis of microvessel density. Furthermore, the micro-CT scanning evidenced that hAFMSCExos promote angiogenesis after cardiac fibrosis. The levels of hypoxia-inducible factor 1 α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in the left ventricle accepted HUVECs were higher than PBS treatment at 7 days post-treatment by Western blot analysis. The hAFMSCExos have proangiogenic effects on endothelial cells and enhanced angiogenesis in cardiac fibrosis. The hAFMSCExos may be a promising potential treatment strategy for cardiac fibrosis.

Extracellular vesicles miRNA-21: a potential therapeutic tool in premature ovarian dysfunction.

Chemotherapy induces an irreversible premature ovarian dysfunction (POD). Amniotic fluid mesenchymal stem cells (AFMSCs) can rescue fertility; however, the notion that stem cells can rejuvenate follicles is highly controversial due to the predetermined ovarian reserve. This study aims to isolate AFMSC-derived extracellular vesicles (EVs) and investigate their abundancy for the anti-apoptotic miRNA-21 as a means of ovarian restoration. Female rats were divided into healthy controls and POD-induced groups. The POD induced groups were subdivided into three groups according to the therapies they received: placebo-treated POD, AFMSC and EVs groups. Rats were assessed for serum anti-Müllerian hormone (AMH) levels, ovarian caspase 3 and PTEN protein levels in the ovarian lysate. Total follicular counts (TFCs) were estimated from stained ovarian sections. Functional recovery was investigated through daily vaginal smears and mating trials. In vitro chemical transfection of the AFMSCs with selective miRNA-21 mimics/inhibitors followed by isolation of EVs for therapy was conducted in two additional groups. At the interval points studied, treatment with AFMSCs and EVs equally restored TFC, AMH levels, regular estrous cycles and fruitful conception, while it both diminished caspase 3 and PTEN levels. EVs carrying miRNA-21 mimics recapitulated the short-term effects. Placebo-treated POD or EVs carrying miRNA-21 inhibitors showed augmented ovarian follicular damage demonstrated the low AMH levels, TFC and high levels of PTEN and caspase 3. miRNA-21 allowed regeneration by modulating PTEN and caspase 3 apoptotic pathways. Our findings exemplify that EVs could serve as an innovative cell-free therapeutic tool functioning through their miRNA content and that miRNA-21 has a chief regenerative role through modulating PTEN and caspase 3 apoptotic pathways.

Amniotic fluid mesenchymal stem cells repair mouse corneal cold injury by promoting mRNA N4-acetylcytidine modification and ETV4/JUN/CCND2 signal axis activation

Severe corneal injury is one of the main causes of loss of visual function. Mesenchymal stem cells (MSCs) have the ability to repair damaged cells in vivo. The present study aimed to explore whether MSCs could function as a cell therapy tool to replace traditional methods to treat corneal injury. CD44 + /CD105 + mesenchymal stem cells isolated from mouse amniotic fluid (mAF-MSCs) were injected into mice after cryoinjury to induce corneal endothelial cell injury. Histopathological assays indicated that mAF-MSCs could promote the growth of corneal epithelial cells, reduce keratitis, and repair the corneal damage caused by low temperature. cDNA microarray analysis revealed that the mAF-MSCs affected the expression patterns of mRNAs related to cell proliferation and differentiation pathways in the mice after transplantation. The results of quantitative real-time PCR and western blotting revealed that NAT12, NAT10, and the ETV4/JUN/CCND2 signaling axis were elevated significantly in the mAF-MSC-transplantation group, compared with those in the phosphate-buffered saline-treated groups. High performance liquid chromatography–mass spectroscopy results revealed that mAF-MSCs could promote mRNA N4-acetylcytidine (ac4C) modification and high expression of N-acetyltransferase in the eyeballs. RNA immunoprecipitation-PCR results showed that a specific product comprising Vegfa, Klf4, Ccnd2, Jun, and Etv4 mRNA specific coding region sites could be amplified using PCR from complexes formed in mAF-MSC-transplanted samples cross-linked with anti-ac4C antibodies. Thus, mouse amniotic fluid MSCs could repair the mouse corneal cold injury by promoting the ETV4/JUN/CCND2 signal axis activation and improving its stability by stimulating N4-acetylcytidine modification of their mRNAs.

The effects of human platelet lysate versus commercial endothelial growth medium on the endothelial differentiation potential of human amniotic fluid mesenchymal stem cells

To differentiate stem cells into endothelial cells, vascular endothelia growth factors (VEGF) serve as the major signal for stimulating the cells. However, there are other cytokines or growth factors associated with endothelial cell development and differentiation. Human platelet lysate (hPL) has been a promising reagent in cell-based therapy since it is considered as a source of bioactive molecules and growth factors. The aim of this study was to investigate the in vitro differentiation of human amniotic fluid mesenchymal stem cells (hAF-MSCs) into endothelial-like cells under hPL together with VEGF or endothelial cell growth medium 2 (EGM-2), a commercially induced medium. In this study, hAF-MSCs were isolated from human amniotic fluid cells (hAFCs) using the direct adherence method. The cells expressed CD44, CD73, CD90, and HLA-ABC at high levels and expressed Oct-4 (octamer-binding transcription factor 4) at low levels. The cells were negative for CD31, CD34, CD45, CD105 and HLA-DR. This study found that hAF-MSCs induced with hPL and VEGF had the ability to differentiate into endothelial-like cells by presenting endothelial specific markers (vWF, VEGFR2 and eNOS), forming a network-like structure on Matrigel, and producing nitric oxide (NO). This outcome was similar to those of experiments involving EGM-2 induced cells. The present findings indicate that hPL + VEGF can induce hAF-MSCs to express endothelial cell characteristics. Our findings represent an important step forward in the development of a clinically compliant process for the production of endothelial cell-derived hAF-MSCs, and their subsequent testing in future clinical trials.

Differentiation of cardiomyocyte-like cells from human amniotic fluid mesenchymal stem cells by combined induction with human platelet lysate and 5-azacytidine

Human amniotic fluid mesenchymal stem cells (hAF-MSCs) have been shown to be effective in the treatment of many diseases. Platelet lysate (PL) contains multiple growth and differentiation factors; therefore, it can be used as a differentiation inducer. In this study, we attempted to evaluate the efficiency of human platelet lysate (hPL) on cell viability and the effects on cardiomyogenic differentiation of hAF-MSCs. When treating the cells with hPL, the result showed an increase in cell viability. Expressions of cardiomyogenic specific genes, including GATA4, cTnT, Cx43 and Nkx2.5, were higher in the combined treatment groups of 5-azacytidine (5-aza) and hPL than the expressions of cardiomyogenic specific genes in the control group and in the 5-aza treatment group. In terms of the results of immunofluorescence and immunoenzymatic staining, the highest expressions of cardiomyogenic specific proteins were revealed in combined treatment groups. It can be summarized that hPL may be an effective supporting cardiomyogenic supplementary factor for cardiomyogenic differentiation in hAF-MSCs.

Facial rejuvenation using stem cell conditioned media combined with skin needling: A split-face comparative study.

The use of stem cells derived growth factors is representing a novel treatment modality in facial rejuvenation. Nowadays, skin needling is considered a very famous treatment of skin aging. However, the addition of such derived products, augments its therapeutic efficacy in the management and delay of skin aging. Comparing the effect of amniotic fluid mesenchymal stem cell derived conditioned media (AF-MSC-CM) combined with skin needling versus the needling alone in the management of facial aging. Both sides of the face of ten volunteers, suffering from facial aging, were treated with five sessions of skin needling, 2weeks apart. After skin microneedling, AF-MSC-CM was added topically to the right side only. Clinical, histological, and morphometrical assessment of the treated skin was done at 1month after the last session. The percentage of improvement of aged skin increased significantly on the combined treated side (AF-MSC-CM and dermaroller [DR]), when compared with the other side (DR only) (P<.001). Remodeling of the dermal structures was observed mainly on the combined side. Meanwhile, histometry of the epidermis revealed a significant increase in the epidermal thickness on both treated sides. AF-MSC-CM combined with skin needling was more efficient in managing facial aging than skin needling alone.

Repair of rat cranial bone defect by using amniotic fluid-derived mesenchymal stem cells in polycaprolactone fibrous scaffolds and platelet-rich plasma

Introduction: Tissue regenerative medicine strategies, as a promising alternative has become of major interest to the reconstruction of critical size bone defects. This study evaluated the effects of the simultaneous application of polycaprolactone (PCL), amniotic fluid mesenchymal stem cells (AF-MSCs) and platelet-rich plasma (PRP) on the repair of rat cranial bone defects. Methods: The AF-MSCs were isolated at the end of the second week of pregnancy in rats. PRP obtained from rat blood and the random PCL fibrous scaffolds were prepared using the electrospinning method. Circular full thickness (5 mm) bone defects were developed on both sides of the parietal bones (animal number=24) and the scaffolds containing AF-MSCs and PRP were implanted in the right lesions. Thereafter, after eight weeks the histological and immunohistochemistry studies were performed to evaluate the bone formation and collagen type I expression. Results: The spindle-shaped mesenchymal stem cells were isolated and the electron microscope images indicated the preparation of a random PCL scaffold. Immunohistochemical findings showed that collagen type I was expressed by AF-MSCs cultured on the scaffold. The results of hematoxylin and eosin (H&E) staining indicated the formation of blood vessels in the presence of PRP. Additionally, immunofluorescence findings suggested that PRP had a positive effect on collagen type I expression. Conclusion: The simultaneous application of fibrous scaffold + AF-MSCs + PRP has positive effects on bone regeneration. This study showed that PRP can affect the formation of new blood vessels in the scaffold transplanted in the bone defect.

Full-Term Human Amniotic Fluid Mesenchymal Stem Cells Show Nephroprotective Effect In An Acute Kidney Injury Model

Full-Term Human Amniotic Fluid Mesenchymal Stem Cells Show Nephroprotective Effect In An Acute Kidney Injury Model