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BACKGROUND: Smoking during pregnancy has been linked to adverse health outcomes in offspring, but the underlying mechanisms are not fully understood. To date, the effect of maternal smoking has been tested in primary tissues and animal models, but the scarcity of human tissues limits experimental studies. Evidence regarding smoking-related molecular alteration and gene expression profiles in stem cells is still lacking. METHODS: We developed a cell culture model of human amniotic fluid stem cells (hAFSCs) of nicotine (NIC) exposure to examine the impact of maternal smoking on epigenetic alterations of the fetus. RESULTS: NIC 0.1 µM(equivalent to "light" smoking, i.e., 5 cigarettes/day) did not significantly affect cell viability; however, significant alterations in DNA methylation and N6-methyladenosine (m6A) RNA methylation in hAFSCs occurred. These epigenetic changes may influence the gene expression and function of hAFSCs. Furthermore, NIC exposure caused time-dependent alterations of the expression of pluripotency genes and cell surface markers, suggesting enhanced cell stemness and impaired differentiation potential. Furthermore, NICtreated cells showed reduced mRNA levels of key adipogenic markers and hypomethylation of the promoter region of the imprinted gene H19 during adipogenic differentiation, potentially suppressing adipo/lipogenesis. Differential expression of 16 miRNAs, with predicted target genes involved in various metabolic pathways and linked to pathological conditions, including cognitive delay and fetal growth retardation, has been detected. CONCLUSIONS: Our findings highlight multi-level effects of NIC on hAFSCs, including epigenetic modifications, altered gene expression, and impaired cellular differentiation, which may contribute to long-term consequences of smoking in pregnancy and its potential impact on offspring health and development.

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Dendritic cells (DCs) are essential orchestrators of immune responses and represent potential targets for immunomodulation in autoimmune diseases. Human amniotic fluid secretome is abundant in immunoregulatory factors, with extracellular vesicles (EVs) being a significant component. However, the impact of these EVs on dendritic cells subsets remain unexplored. In this study, we investigated the interaction between highly purified dendritic cell subsets and EVs derived from amniotic fluid stem cell lines (HAFSC-EVs). Our results suggest that HAFSC-EVs are preferentially taken up by conventional dendritic cell type 2 (cDC2) through CD29 receptor-mediated internalization, resulting in a tolerogenic DC phenotype characterized by reduced expression and production of pro-inflammatory mediators. Furthermore, treatment of cDC2 cells with HAFSC-EVs in coculture systems resulted in a higher proportion of T cells expressing the regulatory T cell marker Foxp3 compared to vehicle-treated control cells. Moreover, transfer of HAFSC-EV-treated cDC2s into an EAE mouse model resulted in the suppression of autoimmune responses and clinical improvement. These results suggest that HAFSC-EVs may serve as a promising tool for reprogramming inflammatory cDC2s towards a tolerogenic phenotype and for controlling autoimmune responses in the central nervous system, representing a potential platform for the study of the effects of EVs in DC subsets.

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Prenatal surgery for the treatment of spina bifida (myelomeningocele, MMC) significantly enhances the neurological prognosis of the patient. To ensure better protection of the spinal cord by large defects, the application of skin grafts produced with cells gained from the amniotic fluid is presently studied. In order to determine the most appropriate cells for this purpose, we tried to shed light on the extremely complex amniotic fluid cellular composition in healthy and MMC pregnancies. We exploited the potential of micro-Raman spectroscopy to analyse and characterize human amniotic fluid cells in total and putative (cKit/CD117-positive) stem cells of fetuses with MMC in comparison with amniotic fluid cells from healthy individuals, human fetal dermal fibroblasts and adult adipose derived stem cells. We found that (i) the differences between healthy and MMC amniocytes can be attributed to specific spectral regions involving collagen, lipids, sugars, tryptophan, aspartate, glutamate, and carotenoids, (ii) MMC amniotic fluid contains two particular cell populations which are absent or reduced in normal pregnancies, (iii) the cKit-negative healthy amniocyte subpopulation shares molecular features with human fetal fibroblasts. On the one hand we demonstrate a different amniotic fluid cellular composition in healthy and MMC pregnancies, on the other our work confirms micro-Raman spectroscopy to be a valuable tool for discriminating cell populations in unknown mixtures of cells.

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This review aims to encapsulate the current knowledge in extracellular vesicles extracted from amniotic fluid and amniotic fluid derived stem/stromal cells. Amniotic fluid (AF) bathes the developing fetus, providing nutrients and protection from biological and mechanical dangers. In addition to containing a myriad of proteins, immunoglobulins and growth factors, AF is a rich source of extracellular vesicles (EVs). These vesicles originate from cells in the fetoplacental unit. They are biological messengers carrying an active cargo enveloped within the lipid bilayer. EVs in reproduction are known to play key roles in all stages of pregnancy, starting from fertilisation through to parturition. The intriguing biology of AF-derived EVs (AF-EVs) in pregnancy and their untapped potential as biomarkers is currently gaining attention. EV studies in numerous animal and human disease models have raised expectations of their utility as therapeutics. Amniotic fluid stem cell and mesenchymal stromal cell-derived EVs (AFSC-EVs) provide an established supply of laboratory-made EVs. This cell-free mode of therapy is popular as an alternative to stem cell therapy, revealing similar, if not better therapeutic outcomes. Research has demonstrated the successful application of AF-EVs and AFSC-EVs in therapy, harnessing their anti-inflammatory, angiogenic and regenerative properties. This review provides an overview of such studies and discusses concerns in this emerging field of research.

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Using stem cells is one of the most important determining factors in repairing lesions using regenerative medicine. Obtaining adult stem cells from patients is a perfect choice, but it is worth noting that their differentiation and proliferation potential decreases as the patient ages. For this reason, the use of amniotic fluid stem cells can be one of the excellent alternatives. This research aimed to investigate the osteogenic differentiation potential of the amniotic fluid stem cells while cultured on the polycaprolactone/poly L-lactic acid nanofibrous scaffold. Scaffolds were qualitatively evaluated by a scanning electron microscope, and their hydrophilicity and mechanical properties were studied using contact angle and tensile test, respectively. The biocompatibility and non-toxicity of the nanofibers were also evaluated using viability assay. The osteo-supportive capacity of the nanofibers was examined using alizarin red staining, alkaline phosphatase activity, and calcium release measurement. Finally, the expression level of four important bone-related genes was determined quantitatively. The results demonstrated that the mineralization rate, alkaline phosphatase activity, intracellular calcium, and bone-related genes increased significantly in the cells cultured on the polycaprolactone/poly L-lactic acid scaffold compared to the cells cultured on the tissue culture plate as a control. According to the results, it can be concluded that the polycaprolactone/poly L-lactic acid nanofibrous scaffold surprisingly improved the osteogenic differentiation potential of the amniotic fluid stem cells and, in combination with polycaprolactone/poly L-lactic acid nanofibers could be a promising candidate as bone implants.

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The present study investigates the impact of two endocrine disruptors, namely Bisphenols (BPs) and Perfluoroalkyls (PFs), on human stem cells. These chemicals leach from plastic, and when ingested through contaminated food and water, they interfere with endogenous hormone signaling, causing various diseases. While the ability of BPs and PFs to cross the placental barrier and accumulate in fetal serum has been documented, the exact consequences for human development require further elucidation. The present research work explored the effects of combined exposure to BPs (BPA or BPS) and PFs (PFOS and PFOA) on human placenta (fetal membrane mesenchymal stromal cells, hFM-MSCs) and amniotic fluid (hAFSCs)-derived stem cells. The effects of the xenobiotics were assessed by analyzing cell proliferation, mitochondrial functionality, and the expression of genes involved in pluripotency and epigenetic regulation, which are crucial for early human development. Our findings demonstrate that antenatal exposure to BPs and/or PFs may alter the biological characteristics of perinatal stem cells and fetal epigenome, with potential implications for health outcomes at birth and in adulthood. Further research is necessary to comprehend the full extent of these effects and their long-term consequences.

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Although human pluripotent stem cells (hPSCs)-derived cardiomyocytes (hPSC-CMs) can remuscularize infarcted hearts and restore post-infarct cardiac function, post-transplant rejection resulting from human leukocyte antigen (HLA) mismatching is an enormous obstacle. It is crucial to identify hypoimmunogenic hPSCs for allogeneic cell therapy. This study is conducted to demonstrate the immune privilege of HLA-Ehigh /HLA-Ghigh /HLA-IIlow human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs). Ischemia-reperfusion surgery is done to create transmural myocardial infarction in rats. At post-infarct 4 days, hPSC-CMs (1.0×107 cells per kg), including human embryonic stem cell-derived cardiomyocytes (hESC-CMs), HLA-Elow/HLA-Glow/HLA-IIhigh hiPSC-CMs, and HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs, are injected into the infarcted myocardium. Under the treatment of very low dose cyclosporine A (CsA), only HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs survive in vivo and improved post-infarct cardiac function with infarct size reduction. HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs activate the SHP-1 signaling pathway of natural killer (NK) cells and cytotoxic T cells to evade attack by NK cells and cytotoxic T cells. Herein, it is demonstrated that using a clinically relevant CsA dose, HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSC-CMs repair the infarcted myocardium and restore the post-infarct heart function. HLA-Ehigh /HLA-Ghigh /HLA-IIlow hiPSCs are less immunogenic and may serve as platforms for regeneration medicine.

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This study aimed to develop an exosome-coated polydatin (PD) nanoparticles (exo-PD) for improving the water solubility and bioavailability of polydatin and explore its salutary effects on intestinal radiation injury. Exosomes (exo) were extracted from the medium of human amniotic fluid stem cells (hAFSc). Mice were divided into control group, irradiation (IR) group, irradiation+PD (IR+PD) group, irradiation+exo (IR+exo) group and irradiation+exo-PD (IR+exo-PD) group. The results of characterization of protein markers, particle size, morphology and cellular uptake ability confirmed that exosomes were effectively isolated using ultracentrifugation. Compared with the IR group, exo-PD improved cell viability, prolonged survival of mice, improved leukocyte count and reduced diarrhea rate. Histological results showed that the exo-PD group had significant improvements in small intestinal villus length and crypt number and less crypt cell damage. exo-PD could reduce IL-1α and IL-6 levels, reduced γ-H2AX expression, increased mitochondrial membrane potential, enhanced oxidative phosphorylation, and delayed cellular senescence. exo-PD could alleviate intestinal injury by improving mitochondrial function through PI3K-AKT pathway. The exo-PD was able to reduce radiation damage to intestinal cells and could be a potential candidate for salvage of intestinal radiation damage.

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Amniotic fluid has been proposed as an easily available source of cells for numerous applications in regenerative medicine and tissue engineering. The use of amniotic fluid cells in biomedical applications necessitates their unequivocal characterization; however, the exact cellular composition of amniotic fluid and the precise tissue origins of these cells remain largely unclear. Using cells cultured from the human amniotic fluid of fetuses with spina bifida aperta and of a healthy fetus, we performed single-cell RNA sequencing to characterize the tissue origin and marker expression of cultured amniotic fluid cells at the single-cell level. Our analysis revealed nine different cell types of stromal, epithelial and immune cell phenotypes, and from various fetal tissue origins, demonstrating the heterogeneity of the cultured amniotic fluid cell population at a single-cell resolution. It also identified cell types of neural origin in amniotic fluid from fetuses with spina bifida aperta. Our data provide a comprehensive list of markers for the characterization of the various progenitor and terminally differentiated cell types in cultured amniotic fluid. This study highlights the relevance of single-cell analysis approaches for the characterization of amniotic fluid cells in order to harness their full potential in biomedical research and clinical applications.

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Neuromuscular junctions (NMJs) are specialized synapses, crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the crosstalk between the different cell populations fails, and the regenerative ability of the entire tissue is hampered. How skeletal muscle sends retrograde signals to MNs through NMJs represents an intriguing field of research, and the role of oxidative stress and its sources remain poorly understood. Recent works demonstrate the myofiber regeneration potential of stem cells, including amniotic fluid stem cells (AFSC), and secreted extracellular vesicles (EVs) as cell-free therapy. To study NMJ perturbations during muscle atrophy, we generated an MN/myotube co-culture system through XonaTM microfluidic devices, and muscle atrophy was induced in vitro by Dexamethasone (Dexa). After atrophy induction, we treated muscle and MN compartments with AFSC-derived EVs (AFSC-EVs) to investigate their regenerative and anti-oxidative potential in counteracting NMJ alterations. We found that the presence of EVs reduced morphological and functional in vitro defects induced by Dexa. Interestingly, oxidative stress, occurring in atrophic myotubes and thus involving neurites as well, was prevented by EV treatment. Here, we provided and validated a fluidically isolated system represented by microfluidic devices for studying human MN and myotube interactions in healthy and Dexa-induced atrophic conditions-allowing the isolation of subcellular compartments for region-specific analyses-and demonstrated the efficacy of AFSC-EVs in counteracting NMJ perturbations.

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Human amniotic fluid cells (hAFSCs) are a fascinating foetal cell-type that have important stem cell characteristics; however, they are a heterogeneous population that ranges from totally differentiated or progenitor cells to highly multipotent stem cells. There is no single approach to isolating the stem cell component, but the selection of a subpopulation of hAFSCs expressing c-Kit is widely employed, while a deep characterization of the two populations is still lacking. Here we performed single-cell and bulk RNAseq analysis to compare the gene expression profiles of adherent amniotic fluid cells and their subpopulation c-Kit+. Information deriving from this high throughput technology on the transcriptome was then confirmed for specific targets with protein expression experiments and functional analysis. In particular, transcriptome profiling identified changes in cellular distribution among the different clusters that correlated with significant differential expression in pathways related to stemness, proliferation, and cell cycle checkpoints. These differences were validated by RT-PCR, immunofluorescence, WB, and cell cycle assays. Interestingly, the two populations produced secretomes with different immune-modulating and pro-regenerative potentials. Indeed, the presence of TGFß, HGF, IDO was higher in EVs deriving from c-Kit+ cells, unlike IL-6. These results suggest the existence of deep intra-population differences that can influence the stemness profile of hAFSCs. This study represents a proof-of-concept of the importance of selecting c-Kit positive fractions with higher potential in regenerative medicine applications.

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Human amniotic fluid stem cells (hAFSCs) are known for their advantageous properties when compared to somatic stem cells from other sources. Recently hAFSCs have gained attention for their neurogenic potential and secretory profile. However, hAFSCs in three-dimensional (3D) cultures remain poorly investigated. Therefore, we aimed to evaluate cellular properties, neural differentiation, and gene and protein expression in 3D spheroid cultures of hAFSCs in comparison to traditional two-dimensional (2D) monolayer cultures. For this purpose, hAFSCs were obtained from amniotic fluid of healthy pregnancies and cultivated in vitro, either in 2D, or 3D under untreated or neuro-differentiated conditions. We observed upregulated expression of pluripotency genes OCT4, NANOG, and MSI1 as well as augmentation in gene expression of NF-κB-TNFα pathway genes (NFKB2, RELA and TNFR2), associated miRNAs (miR103a-5p, miR199a-3p and miR223-3p), and NF-κB p65 protein levels in untreated hAFSC 3D cultures. Additionally, MS analysis of the 3D hAFSCs secretome revealed protein upregulation of IGFs signaling the cascade and downregulation of extracellular matrix proteins, whereas neural differentiation of hAFSC spheroids increased the expression of SOX2, miR223-3p, and MSI1. Summarizing, our study provides novel insights into how 3D culture affects neurogenic potential and signaling pathways of hAFSCs, especially NF-κB, although further studies are needed to elucidate the benefits of 3D cultures more thoroughly.

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In view of the devastating impact of neonatal necrotizing enterocolitis (NEC) on newborns, the research on its intervention is particularly important. Although exosomes from human amniotic fluid stem cells (AFSC) and human breast milk (HBM) can protect against NEC, their mechanisms remain unclear. Here, we intend to compare the intervention effects of two types of exosomes on NEC mouse model and reveal their respective regulatory mechanisms. In general, both AFSC-derived exosomes (AFSC-exos) and HBM-derived exosomes (HBM- exos) can alleviate NEC- associated intestinal injury, significantly reduce NEC score, and reduce systemic and ileal inflammation and NEC related brain injury during experimental NEC. However, the mode and mechanism of action of the two sources of exosomes were not identical. In vivo, the number of ileal crypts was more significantly restored after HBM-exos intervention than AFSC-exos, and in vitro, HBM-exos preferentially inhibited the inflammatory response of intestinal epithelial cells (IECs), whereas AFSC-exos preferentially regulated the migration of IECs. Mechanistically, GO and KEGG analyses revealed the different therapeutic mechanisms of AFSC-exos and HBM-exos in NEC. Taken together, our results illustrate that AFSC-exos and HBM-exos reduce the severity of experimental NEC and intestinal damage through different mechanisms, supporting the potential of cell-free or breast milk free exosome therapy for NEC.

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In the last two decades, fetal amniotic fluid stem cells progressively attracted attention in the context of both basic research and the development of innovative therapeutic concepts. They exhibit broadly multipotent plasticity with the ability to differentiate into cells of all three embryonic germ layers and low immunogenicity. They are convenient to maintain, highly proliferative, genomically stable, non-tumorigenic, perfectly amenable to genetic modifications, and do not raise ethical concerns. However, it is important to note that among the various fetal amniotic fluid cells, only c-Kit+ amniotic fluid stem cells represent a distinct entity showing the full spectrum of these features. Since amniotic fluid additionally contains numerous terminally differentiated cells and progenitor cells with more limited differentiation potentials, it is of highest relevance to always precisely describe the isolation procedure and characteristics of the used amniotic fluid-derived cell type. It is of obvious interest for scientists, clinicians, and patients alike to be able to rely on up-todate and concisely separated pictures of the utilities as well as the limitations of terminally differentiated amniotic fluid cells, amniotic fluid-derived progenitor cells, and c-Kit+ amniotic fluid stem cells, to drive these distinct cellular models towards as many individual clinical applications as possible.

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AIM: The study aims to investigate the immunomodulatory effect of Amniotic fluid stem (AFS) cells to Th2-skewed allergic rhinitis (AR) on T-lymphocyte proliferation, viability, activation and cytokine production. BACKGROUND: AFS cells can suppress peripheral blood mononuclear cells (PBMCs) proliferation and display immunomodulatory properties, but AFS cells' immunoregulation on AR has not been defined. METHODS: Human AFS cells were derived from magnetic cell sorting and co-cultured with PBMCs from AR patients stimulated by phytohemagglutinin (PHA). The AFS cells-associated suppressive proliferation was analyzed using CellTrace™ Violet assay; the T lymphocytes proliferation, viability, activation and the Foxp3+ Treg cells were determined by flow cytometry; cytokine levels were measured using an enzyme- linked immunosorbent assay. RESULTS: We determined that AFS cells significantly inhibited PHA-induced CD3+ T lymphocyte proliferation at the ratio higher than 1:50 (AFS cells: PBMCs) (P<0.05); AFS cells obviously increased the T lymphocytes viability (P<0.01), inhibited the apoptosis of T lymphocytes (P<0.001), compared to PBMCs alone; AFS cells suppressed CD3+CD25+ T lymphocytes activated by PHA (P<0.05); AFS cells significantly promote Treg cells expansion in house dust mite (HDM)-stimulated PBMCs from AR patients (P<0.05). Compared with HDM-stimulated PBMCs, AFS cell co-culture predominantly decreased IL-4 level (P<0.05), but increased IFN-γ and IL-10 levels (P<0.01). CONCLUSION: AFS cells modulate the T-cells' immune imbalance towards Th2 suppression in AR, which can be used as a new cell banking for allergic airway diseases.

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Ascending inflammation from the vagina is a major cause of preterm birth. Currently, this condition-especially when uncontrolled-has no effective treatment. Human amniotic fluid stem cells (hAFSCs) are mesenchymal stem cells known to exert potent anti-inflammatory effects in animal models of perinatal diseases, such as periventricular leukomalacia, myelomeningocele, and neonatal sepsis. However, hAFSC therapy for inflammation-induced preterm birth has not been tested. In order to determine the therapeutic effect of hAFSC transplantation, we employed a preterm mouse model of ascending infection; this model was constructed by administering lipopolysaccharide to pregnant mice. We investigated the preterm birth rate and evaluated the inflammation of tissues, which is related to progressive infections, such as those involving the cervix, placenta, and lavage cells, using real-time qPCR. Further, we tracked the fluorescence of fluorescently labeled hAFSCs using an in vivo imaging system, and hAFSC aggregation was evaluated using immunohistochemistry analysis. We also investigated the presence of multiple types of peritoneal macrophages via flow cytometry analysis. Finally, we performed sphere culturing and co-culturing to determine the therapeutic effects of hAFSCs, such as their anti-inflammatory effects and their potential to alter macrophage polarization. We found that hAFSC administration to the peritoneal cavity significantly reduced inflammation-induced preterm birth in the mouse model. The treatment also significantly suppressed inflammation of the placenta and cervix. Transplanted hAFSCs may have aggregated with peritoneal macrophages, switching them from an inflammatory to an anti-inflammatory type. This property has been reported in vivo previously, but here, we examined the effect in vitro. Our findings support the hypothesis that hAFSCs suppress inflammation and reduce preterm birth by switching macrophage polarity. This study is the first to demonstrate that hAFSCs are effective in the treatment and prevention of inflammation-induced preterm birth.

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Necrotizing enterocolitis (NEC) is a gastrointestinal disease frequently prevalent in premature neonates. Despite advances in research, there is a lack of accurate, early diagnoses of NEC and the current therapeutic approaches remain exhausted and disappointing. In this review, we have taken a close look at the regenerative medical literature available in the context of NEC treatment. Stem cells from amniotic fluid (AFSC) administration may have the greatest protective and restorative effects on NEC. This review summarizes the potential protection and restoration AFSCs have on NEC-induced intestinal injury while comparing various components within AFSCs like conditioned medium (CM) and extracellular vesicles (EVs). In addition to therapeutic interventions that focus on targeting intestinal epithelial damage and regeneration, a novel discovery that AFSCs act in a Wnt-dependent manner provides insight into this mechanism of protection. Finally, we have highlighted the most important aspects that remain unknown that should be considered to guide future research on the translational application of AFSC-based therapy. We hope that this will be a beneficial frame of reference for the guidance of future studies and towards the clinical application of AFSC and/or its derivatives as a treatment against NEC.

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Human amniotic fluids stem cells (hAFSCs) can be easily isolated from the amniotic fluid during routinely scheduled amniocentesis. Unlike hiPSCs or hESC, they are neither tumorigenic nor immunogenic and their use does not rise ethical or safety issues: for these reasons they may represent a good candidate for the regenerative medicine. hAFSCs are generally considered multipotent and committed towards the mesodermal lineages; however, they express many pluripotent markers and share some epigenetic features with hiPSCs. Hence, we hypothesized that hAFSCs may overcome their mesodermal commitment differentiating into to ectodermal lineages. Here we demonstrated that by the sequential exposure to specific factors, hAFSCs can give rise to spinal motor neurons (MNs), as evidenced by the gradual gene and protein upregulation of early and late MN markers (PAX6, ISL1, HB9, NF-L, vAChT). When co-cultured with myotubes, hAFSCs-derived MNs were able to create functional neuromuscular junctions that induced robust skeletal muscle contractions. These data demonstrated the hAFSCs are not restricted to mesodermal commitment and can generate functional MNs thus outlining an ethically acceptable strategy for the study and treatment of the neurodegenerative diseases.

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Objective: The study aimed at isolation of CD117+ stem cells from amniotic fluid samples followed by their invitro differentiation towards nephron progenitors that can be potentially used for regenerative medicine studies and to further understand pathways involved in renal pathogenesis. Methods: This experimental study was conducted at Dow Research Institute of Biotechnology and Biomedical Sciences (DRIBBS), Dow University of Health Sciences, OJHA Campus Karachi from November 2019 to December 2020. After patient consent, a Pfannenstiel incision was performed by the gynecologist through abdominal and uterine muscles without cutting into Amniotic Membrane. Using a needle of 5CC syringe connected to sterile Redivac bottle, a blunt end insertion was passed through the membrane and the amniotic fluid was aseptically sucked into Redivac bottle, the ice bag was used for transporting amniotic fluid from hospital to the lab and samples were processed within 60 minutes after collection. Amniotic fluid was centrifuged at 4º C for 20 minutes at 1400xg. After centrifugation the cell pellet was treated for analysis of CD117+ cells using flowcytometry, once small percentage of CD117+ cells were identified the cells were prepared for differentiation and that was carried out using specific growth factors including BMP4, BMP7, FGF2, and retinoic acid, providing the niche to the stem cells for differentiation towards nephron progenitors which was confirmed by protein expression of Wilms Tumor-1 (WT1) using immunofluorescence analysis. The sample size for this invitro work was n=3. Results: We successfully isolated small percentage of CD117+ cells in amniotic fluid followed by in vitro expansion and differentiation towards nephron progenitor cells (NPCs) using well defined media and growth factors, initially differentiated cells were spindle shaped and showed fibroblastic appearance later at stage of nephron progenitors it attained the shape of rounded big clusters, differentiated cells stained positive for WT1 and negative for cluster of differentiation (CD117). Therefore, confirming the successful isolation and differentiation of amniotic fluid stem cells towards nephron progenitors. Conclusion: To the best of our knowledge this is the first study from the country on the use of Amniotic fluid stem cells and their differentiation towards nephron progenitors that can be used as substitution source of cell therapy for exploration of renal diseases at cellular and molecular level and potential regenerative medicine applications.

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Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid-derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of R26pFUCCI2 mice with cell cycle fluorescent tagging by mutually exclusive nuclear signal. mNVCM were stimulated by fetal versus perinatal hAFSC-CM and hAFSC-EVs to identify the most promising formulation for in vivo assessment in a R26pFUCCI2 neonatal mouse model of myocardial infarction (MI) via intraperitoneal delivery. While the perinatal hAFSC secretome did not provide any significant cardiogenic effect, fetal hAFSC-EVs significantly sustained mNVCM transition from S to M phase by 2-fold, while triggering cytokinesis by 4.5-fold over vehicle-treated cells. Treated mNVCM showed disorganized expression of cardiac alpha-actinin, suggesting cytoskeletal re-arrangements prior to cell renewal, with a 40% significant downregulation of Cofilin-2 and a positive trend of polymerized F-Actin. Fetal hAFSC-EVs increased cardiomyocyte cell cycle progression by 1.8-fold in the 4-day-old neonatal left ventricle myocardium short term after MI; however, such effect was lost at the later stage. Fetal hAFSC-EVs were enriched with a short isoform of Agrin, a mediator of neonatal heart regeneration acting by YAP-related signaling; yet in vitro application of YAP inhibitor verteporfin partially affected EV paracrine stimulation on mNVCM. EVs secreted by developmentally juvenile fetal hAFSC can support cardiomyocyte renewal to some extension, via intercellular conveyance of candidates possibly involving Agrin in combination with other factors. These perinatal derivative promising cardiogenic effects need further investigation to define their specific mechanism of action and enhance their potential translation into therapeutic opportunity.