Human Amniotic Fluid Research Articles

The spontaneous differentiation and chromosome loss in iPSCs of human trisomy 18 syndrome

Aneuploidy including trisomy results in developmental disabilities and is the leading cause of miscarriages in humans. Unlike trisomy 21, pathogenic mechanisms of trisomy 18 remain unclear. Here, we successfully generated induced pluripotent stem cells (iPSCs) from human amniotic fluid cells (AFCs) with trisomy 18 pregnancies. We found that trisomy 18 iPSCs (18T-iPSCs) were prone to differentiate spontaneously. Intriguingly, 18T-iPSCs lost their extra 18 chromosomes and converted to diploid cells after 10 generations. fluorescence in situ hybridization analysis showed chromosome loss was a random event that might happen in any trisomic cells. Selection undifferentiated cells for passage accelerated the recovery of euploid cells. Overall, our findings indicate the genomic instability of trisomy 18 iPSCs bearing an extra chromosome 18.

Lipidomics of equine amniotic fluid: Identification of amphiphilic (O-acyl)-ω-hydroxy-fatty acids

Amniotic fluid is essential for the growth and maturation of the fetus prior to parturition. While our knowledge of human amniotic fluid is extensive, current data for equine amniotic fluid is limited. We therefore undertook a detailed lipidomics analysis of equine amniotic fluid. Using a non-targeted high-resolution mass spectrometric lipidomics analysis of equine amniotic fluid, we were able to characterize a diverse array of individual lipids. This non-biased analytical approach detected, for the first time, the presence of (O-acyl)-ω-hydroxy-fatty acids (OAHFA) with up to 52 carbon chain lengths in amniotic fluid. The identities of these lipid amphiphiles were validated both by high-resolution mass spectrometry and by tandem mass spectrometry (<2 ppm mass error) which identified the fatty acid and hydroxy-fatty acid components of individual OAHFAs. The only previous identification of OAHFAs has been in sperm and meibomian glands, and their sebaceous secretions, suggesting that these lipids may have unique functional roles in highly specialized compartments. The amphiphilic and surfactant properties of these unique lipids could provide an interface between amniotic lipids and fetal skin and/or lungs. The potential roles of OAHFAs as well as their source in amniotic fluid remain to be explored based upon these novel lipidomics findings but our study of the developmental time course of amniotic OAHFAs suggest that they may act as lubricants in delivery and/or a role in the development of fetal lung function around parturition.

Acid Stress Response Mechanisms of Group B Streptococci.

Group B streptococcus (GBS) is a leading cause of neonatal mortality and morbidity in the United States and Europe. It is part of the vaginal microbiota in up to 30% of pregnant women and can be passed on to the newborn through perinatal transmission. GBS has the ability to survive in multiple different host niches. The pathophysiology of this bacterium reveals an outstanding ability to withstand varying pH fluctuations of the surrounding environments inside the human host. GBS host pathogen interations include colonization of the acidic vaginal mucosa, invasion of the neutral human blood or amniotic fluid, breaching of the blood brain barrier as well as survival within the acidic phagolysosomal compartment of macrophages. However, investigations on GBS responses to acid stress are limited. Technologies, such as whole genome sequencing, genome-wide transcription and proteome mapping facilitate large scale identification of genes and proteins. Mechanisms enabling GBS to cope with acid stress have mainly been studied through these techniques and are summarized in the current review

ID:2041 Animal Component Free Medium Retains the Stability of Hypervariable Region 1 (HV1) of Mitochondrial DNA of Human Full Term Amniotic Fluid Mesenchymal Stem Cells

Therapeutic potential of human amniotic fluid (hAF) cells in treating human disease is very promising. Furthermore it provides an alternative in isolating stem cells from an accessible source, where the fluid is merely discarded, thus circumventing the ethical concerns. Mesenchymal stem cell (MSC) is one of the heterogenous cell populations in the amniotic fluid (AF), however, its role and potential for stem cell therapy are yet to be discovered. One of the criteria in establishing the clinical grade of MSCs is the ability of the cells to retain their genomic stability in vitro prior to bedside applications. The microenviroment niche of the culture medium used is important to generate the safe MSCs. As mitochondria play the vital role in metabolic task such as apoptosis and cell proliferation, therefore investigating mitochondria DNA (mtDNA) is essential to mark the stability of stem cell. In this study, we aimed to detect variations of mtDNA across the passage number of cultured human amniotic fluid MSCs (hAFMSCs), cultured in animal component free (ACF) medium, specialised for human MSCs. Polymorphisms of hypervariable region 1 (HV1) in the D-loop of mtDNA were investigated in a series of cultured hAFMSCs (passage 1 and 3) from three different samples (three biological replicates) as well as in their respective fresh samples (control). Extracted DNA samples were subjected to PCR to detect the sequence variations of HV1 (nt16024-16365). The sequences were then analysed using SeqScape v2.5 by comparing the sequences with The Cambridge Reference Sequence (rCRS) for human mtDNA. The common C-to-T transition at position 16223 (C16223T) for all three samples was identified. Another common base transition was also identified for the other two samples at position 16311 (T16311C).&#x0D; However, one of the samples showed more variants; a C-to-T transition at position 16290 (C16290T), an A-to-G at position 16317 (A16317G) and a G-to-A at position 16319 (G16319A). All variants were identified within the sequence of around 341bp nucleotides and their polymorphisms frequency was consistent for all three samples regardless of their passage numbers when compared to control. This pattern of polymorphisms demonstrates the HV1 mtDNA integrity of hAFMSCs cultured in ACF medium, hence highly suggesting the ability of ACF medium in retaining the stability of stem cells mtDNA in vitro. The ACF medium may serve as a good medium in culturing hAFMSCs that is safe and stable for basic research in stem cell and therapeutic applications.

Myristic acid in amniotic fluid produces appetitive responses in human newborns

BackgroundA mixture of eight fatty acids (lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, elaidic acid, and linoleic acid) that are contained in human amniotic fluid, colostrum, and milk produces appetitive responses in newborns, suggesting the existence of a transition of sensorial cues that guide newborns to the maternal breast. ObjectiveTo explore the ability of each of these eight fatty acids individually to produce appetitive responses in newborns. MethodsThe study included 12 healthy human newborns<24h after birth. Using a longitudinal design, cotton swabs that were impregnated with each of the eight fatty acids and control substances (i.e., vehicle, saline, and vanilla) were placed approximately 1cm from the newborns' nostrils for 30s. Positive responses that were suggestive of acceptance included appetitive movements (i.e., suckling) and sniffing that were directed toward the cotton swab. Lateral movements of the head away from the swab were considered negative responses. Remaining stationary with no changes in facial expressions was considered indifference. ResultsCompared with controls (i.e., vehicle, saline, and vanilla) and the other fatty acids tested, myristic acid produced the longest duration of positive facial responses (suckling and sniffing). No significant differences in negative facial responses were observed in response to the odoriferous stimuli. No reactions that were suggestive of disgust were observed. ConclusionA complex combination of stimuli, including the odor of myristic acid, may integrate sensory cues that guide newborns to the maternal breast.

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment.

Protein and Molecular Characterization of a Clinically Compliant Amniotic Fluid Stem Cell-Derived Extracellular Vesicle Fraction Capable of Accelerating Muscle Regeneration Through Enhancement of Angiogenesis.

The secretome of human amniotic fluid stem cells (AFSCs) has great potential as a therapeutic agent in regenerative medicine. However, it must be produced in a clinically compliant manner before it can be used in humans. In this study, we developed a means of producing a biologically active secretome from AFSCs that is free of all exogenous molecules. We demonstrate that the full secretome is capable of promoting stem cell proliferation, migration, and protection of cells against senescence. Furthermore, it has significant anti-inflammatory properties. Most importantly, we show that it promotes tissue regeneration in a model of muscle damage. We then demonstrate that the secretome contains extracellular vesicles (EVs) that harbor much, but not all, of the biological activity of the whole secretome. Proteomic characterization of the EV and free secretome fraction shows the presence of numerous molecules specific to each fraction that could be key regulators of tissue regeneration. Intriguingly, we show that the EVs only contain miRNA and not mRNA. This suggests that tissue regeneration in the host is mediated by the action of EVs modifying existing, rather than imposing new, signaling pathways. The EVs harbor significant anti-inflammatory activity as well as promote angiogenesis, the latter may be the mechanistic explanation for their ability to promote muscle regeneration after cardiotoxin injury.

The attempt of human amniotic fluid colony-derived stem cells for cell therapy in animal model of ventricle injury

Objective To investigate the potential of human amniotic fluid colony-derived stem cells (hAFCSCs) transplanted into lateral ventricle injury in rats for cell therapy. Methods The second-trimester amniotic fluid samples were collected through amniocentesis by ultrasound from gestational age of 16 to 22 weeks for routine prenatal diagnosis purposes and samples were only used when cytogenetic analysis revealed no major abnormalities. Using colony poculum, hAFCSCs were isolated from second-trimester amniotic fluid. The ventricle damage model is divided into normal control group, control group and experimental group, blank control group. The hAFCSCs were injected into the ventricle of the animal model. To analyse the growth (weight, brain weight, open ears, grow hair) and sensorimotor function (foot test, reflection, limb position) change in model at several points. All statistic analysis of the data was performed with SNK to compare the difference of these groups. The protein of Nestin was detected using West blotting in the brain tissue of mode. Results There were no significant differences in body weight and brain weight of rats in each group. There was no remarkable difference in open ears between the experimental group, the control group and the blank control group, but there were notable differences between the mentioned groups and the control group. There were differences in sensorimotor function between experimental control group, blank control group and the normal control group, the experimental group; the experimental group were close to that of the normal control group. It showed that the expression of nestin after 5 days cells injection with Western blotting. Conclusion The hAFCSCs could improve the growth, sensorimotor function of the ventricle damage model rat. Key words: Human amniotic fluid colony-derived stem cells; Lateral ventricle injury; Animal model; Cell therapy

Cell therapy for stress urinary incontinence: Present‐day frontiers

Stress urinary incontinence (SUI) significantly diminishes the quality of patients' lives. Currently available surgical and nonsurgical therapies remain far from ideal. At present, advances in cellular technologies have stirred growing interest in the use of autologous cell treatments aimed to regain urinary control. The objective was to conduct a review of the literature and analyse preclinical and clinical studies dedicated to various cell therapies for SUI, assessing their effectiveness, safety, and future prospects. A systematic literature search in PubMed was conducted using the following key terms: "stem," "cell," "stress," "urinary," and "incontinence." A total of 32 preclinical studies and 15 clinical studies published between 1946 and December 2014 were included in the review. Most preclinical trials have used muscle-derived stem cells and adipose-derived stem cells. However, at present, the application of other types of cells, such as human amniotic fluid stem muscle-derived progenitor cells and bone marrow mesenchymal stromal cells, is becoming more extensive. While the evidence shows that these therapies are effective and safe, further work is required to standardize surgical techniques, as well as to identify indications for their use, doses and number of doses. Future research will have to focus on clinical applications of cell therapies; namely, it will have to determine indications for their use, doses of cells, optimal surgical techniques and methods, attractive cell sources, as well as to develop clinically relevant animal models and make inroads into understanding the mechanisms of SUI improvement by cell therapies.

Ion-exchange chromatography purification of extracellular vesicles.

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

TGF\u03b2-induced osteogenic potential of human amniotic fluid stem cells via CD73-generated adenosine production

The human amniotic fluid stem cell (hAFSC) population consists of two morphologically distinct subtypes, spindle-shaped and round-shaped cells (SS-hAFSCs and RS-hAFSCs). Whilst SS-hAFSCs are routinely expanded in mesenchymal-type (MT) conditions, we previously showed that they acquire broader differentiation potential when cultured under embryonic-type (ET) conditions. However, the effects of culture conditions on RS-hAFSCs have not been determined. Here, we show that culturing RS-hAFSCs under ET conditions confers faster proliferation and enhances the efficiency of osteogenic differentiation of the cells. We show that this occurs via TGFβ-induced activation of CD73 and the associated increase in the generation of extracellular adenosine. Our data demonstrate that culture conditions are decisive for the expansion of hAFSCs and that TGFβ present in ET conditions causes the phenotype of RS-hAFSCs to revert to an earlier state of stemness. Cultivating RS-hAFSCs in ET conditions with TGFβ may therefore increase their therapeutic potential for clinical applications.

Histone Modifications Pattern Associated With a State of Mesenchymal Stem Cell Cultures Derived From Amniotic Fluid of Normal and Fetus-Affected Gestations.

Human amniotic fluid (AF)-derived mesenchymal stem cells (MSCs) sharing embryonic and adult stem cells characteristics are interesting by their multipotency and the usage for regenerative medicine. However, the usefulness of these cells for revealing the fetal diseases still needs to be assessed. Here, we have analyzed the epigenetic environment in terms of histone modifications in cultures of MSCs derived from AF of normal pregnancies and those with fetal abnormalities. The comparison of MSCs samples from AF of normal pregnancies (N) and fetus-affected (P) revealed two distinct cultures by their proliferation potential (P I and P II). Cell populations from N and P I samples had similar growth characteristics and exhibited quite similar cell surface (CD44, CD90, CD105) and stemness markers (Oct4, Nanog, Sox2, Rex1) profile that was distinct in slower growing and faster senescent P II cultures. Those differences were associated with changes in 5-Cyt DNA methylation and alterations in the expression levels of chromatin modifiers (DNMT1, HDAC1/2), activating (H4ac, H3K4me3), and repressive (H3K9me2/me3, H3K27me3) histone marks. MSCs isolated from AF with the genetic or multifactorial fetal diseases (P II samples) were enriched with repressive histone marks and H4K16ac, H3K9ac, H3K14ac modifications. This study indicates that differential epigenetic environment reflects a state of AF-MSCs dependently on their growth, phenotype, and stemness characteristics suggesting a way for better understanding of epigenetic regulatory mechanisms in AF-MSCs cultures in normal and diseased gestation conditions. J. Cell. Biochem. 118: 3744-3755, 2017. © 2017 Wiley Periodicals, Inc.

Development of a novel method for amniotic fluid stem cell storage

Background aimsCurrent procedures for collection of human amniotic fluid stem cells (hAFSCs) indicate that cells cultured in a flask for 2 weeks can then be used for research. However, hAFSCs can be retrieved directly from a small amount of amniotic fluid that can be obtained at the time of diagnostic amniocentesis. The aim of this study was to determine whether direct freezing of amniotic fluid cells is able to maintain or improve the potential of a sub-population of stem cells. MethodsWe compared the potential of the hAFSCs regarding timing of freezing, cells obtained directly from amniotic fluid aspiration (D samples) and cells cultured in a flask before freezing (C samples). Colony-forming-unit ability, proliferation, morphology, stemness-related marker expression, senescence, apoptosis and differentiation potential of C and D samples were compared. ResultshAFSCs isolated from D samples expressed mesenchymal stem cells markers until later passages, had a good proliferation rate and exhibited differentiation capacity similar to hAFSCs of C samples. Interestingly, direct freezing induced a higher concentration of cells positive for pluripotency stem cell markers, without teratoma formation in vivo. ConclusionsThis study suggests that minimal processing may be adequate for the banking of amniotic fluid cells, avoiding in vitro passages before the storage and exposure to high oxygen concentration, which affect stem cell properties. This technique might be a cost-effective and reasonable approach to the process of Good Manufacturing Process accreditation for stem-cell banks.

Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells.

Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and β-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.

Induced Retro‐Differentiation of Human Retinal Pigment Epithelial Cells on PolyHEMA

Retinal pigment epithelium (RPE) cells represent a great potential to rescue degenerated cells of the damaged retina. Activation of the virtually plastic properties of RPE cells may aid in recovery of retinal degenerative disorders without the need for entire RPE sheet transplantation. Poly (2-hydroxyethyl methacrylate)(PolyHEMA) is one of the most important hydrogels in the biomaterials world. This hydrophobic polymer does not normally support attachment of mammalian cells. In the current study we investigated the effect of PolyHEMA as a cell culture substrate on the growth, differentiation, and plasticity of hRPE cells. hRPE cells were isolated from neonatal human globes and cultured on PolyHEMA and polystyrene substrates (as controls) in 24-well culture plates. DMEM/F12 was supplemented with 10% fetal bovine serum (FBS) and/or 30% human amniotic fluid (HAF) for cultured cells on polystyrene and PolyHEMA coated vessels. Morphology, rate of cell proliferation and cell death, MTT assay, immunocytochemistry and Real-Time RT-PCR were performed to investigate the effects of PolyHEMA on the growth and differentiation of cultured hRPE cells. Proliferation rate of the cells that had been cultured on PolyHEMA was reduced; PolyHEMA did not induce cell death in the hRPE cultures. hRPE cells cultured on PolyHEMA formed many giant spheroid colonies. The giant colonies were re-cultured and the presence of retinal progenitor markers and markers of hRPE cells were detected in cell cultures on PolyHEMA. PolyHEMA seems to be promising for both maintenance and de-differentiation of hRPE cells and expansion of the retinal progenitor cells from the cultures that are originated from hRPE cells. J. Cell. Biochem. 118: 3080-3089, 2017. © 2017 Wiley Periodicals, Inc.

Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte‑like cells.

Ischemic heart disease (IHD) is a major factor influencing worldwide mortality rates. Furthermore, IHD has become a significant health problem among the Thai population. Stem cell therapy using mesenchymal stem cells (MSCs) is an alternative therapeutic method that has been applied to improve the quality of life of patients. Amniotic fluid (AF) contains a heterogeneous cell population, including MSCs, which are multipotent stem cells that have the capability to differentiate into mesenchymal lineages. The purpose of the present study was to evaluate the MSC characteristics of human (h)AF and determine its potency regarding cardiogenic differentiation. MSC characterization following flow cytometric analysis revealed that the cells expressed MSC markers, cluster of differentiation (CD)44, CD90, human leukocyte antigen-ABC and CD73. The results of the alamar blue assay demonstrated that cell proliferation rate continuously increased from the early cultivation phase up to 5-fold during days 1 to 5 of cell culturing. The highest rate of cell proliferation was observed on day 17 with a 30-fold increase compared with that on day 1. During the cardiogenic induction stage, morphological changes were observed between day 0 and day 21, and it was revealed that the hAF derived-MSCs in the cardiogenic-induced group exhibited myotube-like morphology after 7 days of cell culturing. Following cardiogenic induction, immunohistochemistry staining was performed on day 21, and reverse transcription-quantitative polymerase chain reaction on day 7 and 21. These steps were performed to detect the protein and gene expression levels of cardiac specific proteins (GATA4, cardiac troponin T, Nkx2.5 and Connexin43). The results of the present study indicated that hAF-MSCs possess the potential to differentiate into cardiomyocyte-like cells. Thus, it was concluded that hAF may be a suitable source of MSCs for stem cell therapy and tissue engineering.

Peptidome analysis of amniotic fluid from pregnancies with preeclampsia.

Preeclampsia (PE), a life-threatening, complicated pregnancy-associated disease, has recently become a research focus in obstetrics. However, the peptidome of the amniotic fluid in PE patients has rarely been investigated. The present study used peptidomic profiling to perform a comparative analysis of human amniotic fluid between normal and PE pregnancies. Centrifugal ultrafiltration and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was combined with isotopomeric dimethyl labels to gain a deeper understanding of the role of proteins and the peptidome in the onset of PE. Following ultrafiltration and LC-MS/MS, 352 peptides were identified. Of these, 23 peptides were observed to be significantly differentially expressed (6 downregulated and 17 upregulated; P<0.05). Using Gene Ontology and Blastp analyses, the functions and biological activities of these 23 peptides were identified and revealed to include autophagy, signal transduction, receptor activity, enzymatic activity and nucleic acid binding. In addition, a bibliographic search revealed that some of the identified peptides, including Titin, are crucial to the pathogenesis underlying PE. The present study identified 23 peptides expressed at significantly different levels in the amniotic fluid of PE and normal pregnancies. A comprehensive peptidome analysis is more efficient than a simple biomarker analysis at revealing deficiencies and improving the detection rate in diseases. These analyses therefore provide a substantial advantage in applications aimed at the discovery of disease-specific biomarkers.

Endocrine disruptors: Chemical contaminants - a toxic mixture for neurodevelopment.

A mixture of chemicals commonly detected in human amniotic fluid has been found to perturb thyroid signalling, development of neurons and glia in the brain, and behavioural outcomes in offspring, when tested in a frog model of embryogenesis. The findings show the exquisite sensitivity of the developing organism to environmental contaminants.

First Characterization of Human Amniotic Fluid Stem Cell Extracellular Vesicles as a Powerful Paracrine Tool Endowed with Regenerative Potential.

Human amniotic fluid stem cells (hAFS) have shown a distinct secretory profile and significant regenerative potential in several preclinical models of disease. Nevertheless, little is known about the detailed characterization of their secretome. Herein we show for the first time that hAFS actively release extracellular vesicles (EV) endowed with significant paracrine potential and regenerative effect. c‐KIT+ hAFS were isolated from leftover samples of amniotic fluid from prenatal screening and stimulated to enhance EV release (24 hours 20% O2 versus 1% O2 preconditioning). The capacity of the c‐KIT+ hAFS‐derived EV (hAFS‐EV) to induce proliferation, survival, immunomodulation, and angiogenesis were investigated in vitro and in vivo. The hAFS‐EV regenerative potential was also assessed in a model of skeletal muscle atrophy (HSA‐Cre, Smn F7/F7 mice), in which mouse AFS transplantation was previously shown to enhance muscle strength and survival. hAFS secreted EV ranged from 50 up to 1,000 nm in size. In vitro analysis defined their role as biological mediators of regenerative, paracrine effects while their modulatory role in decreasing skeletal muscle inflammation in vivo was shown for the first time. Hypoxic preconditioning significantly induced the enrichment of exosomes endowed with regenerative microRNAs within the hAFS‐EV. In conclusion, this is the first study showing that c‐KIT+ hAFS dynamically release EV endowed with remarkable paracrine potential, thus representing an appealing tool for future regenerative therapy. Stem Cells Translational Medicine 2017;6:1340–1355

Human amniotic fluid contaminants alter thyroid hormone signalling and early brain development in Xenopus embryos

Thyroid hormones are essential for normal brain development in vertebrates. In humans, abnormal maternal thyroid hormone levels during early pregnancy are associated with decreased offspring IQ and modified brain structure. As numerous environmental chemicals disrupt thyroid hormone signalling, we questioned whether exposure to ubiquitous chemicals affects thyroid hormone responses during early neurogenesis. We established a mixture of 15 common chemicals at concentrations reported in human amniotic fluid. An in vivo larval reporter (GFP) assay served to determine integrated thyroid hormone transcriptional responses. Dose-dependent effects of short-term (72 h) exposure to single chemicals and the mixture were found. qPCR on dissected brains showed significant changes in thyroid hormone-related genes including receptors, deiodinases and neural differentiation markers. Further, exposure to mixture also modified neural proliferation as well as neuron and oligodendrocyte size. Finally, exposed tadpoles showed behavioural responses with dose-dependent reductions in mobility. In conclusion, exposure to a mixture of ubiquitous chemicals at concentrations found in human amniotic fluid affect thyroid hormone-dependent transcription, gene expression, brain development and behaviour in early embryogenesis. As thyroid hormone signalling is strongly conserved across vertebrates the results suggest that ubiquitous chemical mixtures could be exerting adverse effects on foetal human brain development.