Amniotic Fluid Stem Cells Research Articles

Small Extracellular Vesicles from Human Amniotic Fluid Samples as Promising Theranostics.

Since the first evidence that stem cells can provide pro-resolving effects via paracrine secretion of soluble factors, growing interest has been addressed to define the most ideal cell source for clinical translation. Leftover or clinical waste samples of human amniotic fluid obtained following prenatal screening, clinical intervention, or during scheduled caesarean section (C-section) delivery at term have been recently considered an appealing source of mesenchymal progenitors with peculiar regenerative capacity. Human amniotic fluid stem cells (hAFSC) have been demonstrated to support tissue recovery in several preclinical models of disease by exerting paracrine proliferative, anti-inflammatory and regenerative influence. Small extracellular vesicles (EVs) concentrated from the hAFSC secretome (the total soluble trophic factors secreted in the cell-conditioned medium, hAFSC-CM) recapitulate most of the beneficial cell effects. Independent studies in preclinical models of either adult disorders or severe diseases in newborns have suggested a regenerative role of hAFSC-EVs. EVs can be eventually concentrated from amniotic fluid (hAF) to offer useful prenatal information, as recently suggested. In this review, we focus on the most significant aspects of EVs obtained from either hAFSC and hAF and consider the current challenges for their clinical translation, including isolation, characterization and quantification methods.

Differentiation of CD117+ Amniotic Fluid Stem Cells towards Nephron Progenitors.

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 pathogenesisMethods: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.

Treatment of necrotizing enterocolitis by conditioned medium derived from human amniotic fluid stem cells.

Necrotizing enterocolitis (NEC) is one of the most distressing gastrointestinal emergencies affecting neonates. Amniotic fluid stem cells (AFSC) improve intestinal injury and survival in experimental NEC but are difficult to administer. In this study, we evaluated whether conditioned medium (CM) derived from human AFSC have protective effects. Three groups of C57BL/6 mice were studied: (i) breast-fed mice as control; (ii) experimental NEC mice receiving PBS; and (iii) experimental NEC mice receiving CM. NEC was induced between post-natal days P5 through P9 via: (A) gavage feeding of hyperosmolar formula four-time a day; (B) 10 minutes hypoxia prior to feeds; and (C) lipopolysaccharide administration on P6 and P7. Intra-peritoneal injections of either PBS or CM were given on P6 and P7. All mice were sacrificed on P9 and terminal ileum were harvested for analyses. CM treatment increased survival and reduced intestinal damage, decreased mucosal inflammation (IL-6; TNF-α), neutrophil infiltration (MPO), and apoptosis (CC3), and also restored angiogenesis (VEGF) in the ileum. Additionally, CM treated mice had increased levels of epithelial proliferation (Ki67) and stem cell activity (Olfm4; Lgr5) compared to NEC+PBS mice, showing restored intestinal regeneration and recovery during NEC induction. CM proteomic analysis of CM content identified peptides that regulated immune and stem cell activity. CM derived from human AFSC administered in experimental NEC exhibited various benefits including reduced intestinal injury and inflammation, increased enterocyte proliferation, and restored intestinal stem cell activity. This study provides the scientific basis for the use of CM derived from AFSC in neonates with NEC.

A Prevascularized Polyurethane-Reinforced Fibrin Patch Improves Regenerative Remodeling in a Rat Right Ventricle Replacement Model.

Congenital heart defects (CHDs) affect 1 in 120 newborns in the United States. Surgical repair of structural heart defects often leads to arrhythmia and increased risk of heart failure. The laboratory has previously developed an acellular fibrin patch reinforced with a biodegradable poly(ether ester urethane) urea mesh that result in improved heart function when tested in a rat right ventricle wall replacement model compared to fixed pericardium. However, this patch does not drive significant neotissue formation. The patch materials are modified here and this patch is prevascularized with human umbilical vein endothelial cells and c-Kit+ human amniotic fluid stem cells. Rudimentary capillary-like networks form in the fibrin after culture of cell-encapsulated patches for 3 d in vitro. Prevascularized patches and noncell loaded patch controls are implanted onto full-thickness heart wall defects created in the right ventricle of athymic nude rats. Two months after surgery, defect repair with prevascularized patches results in improved heart function and the patched heart area exhibited greater vascularization and muscularization, less fibrosis, and increased M2 macrophage infiltration compared to acellular patches.

Human amniotic fluid stem cells can improve cerebral vascular remodelling and neurological function after focal cerebral ischaemia in diabetic rats

Diabetes causes vascular injury and carries a high risk of ischaemic stroke. Human amniotic fluid stem cells (hAFSCs) can enhance cerebral vascular remodelling and have the potential to improve neurological function after stroke in diabetic rats. Five groups of female rats were examined: (1) normal control, (2) type 1 diabetic (T1DM) rats induced by streptozotocin injection (DM), (3) non‐DM rats receiving 60‐minute middle cerebral artery occlusion (MCAO), (4) T1DM rats receiving 60‐minute MCAO (DM + MCAO) and (5) T1DM rats receiving 60‐minute MCAO and injection with 5 × 106 hAFSCs at 3 h after MCAO (DM + MCAO + hAFSCs). Neurological function was examined before, and at 1, 7, 14, 21 and 28 days, and cerebral infarction volume and haemorrhage, cerebral vascular density, angiogenesis and inflammatory were examined at 7 and 28 days after MCAO. hAFSCs treatment caused a significant improvement of neurological dysfunction, infarction volume, blood‐brain barrier leakage, cerebral arterial density, vascular density and angiogenesis and a reduction of brain haemorrhage and inflammation compared with non‐treatment. Our results showed that the effect of hAFSCs treatment against focal cerebral ischaemia may act through the recovery of vascular remodelling and angiogenesis and the reduction of inflammation in ischaemic brain.

The Restorative Effect of Human Amniotic Fluid Stem Cells on Spinal Cord Injury.

Spinal cord injury (SCI) is a debilitating condition within the neural system which is clinically manifested by sensory-motor dysfunction, leading, in some cases, to neural paralysis for the rest of the patient’s life. In the current study, mesenchymal stem cells (MSCs) were isolated from the human amniotic fluid, in order to study their juxtacrine and paracrine activities. Flow cytometry analysis was performed to identify the MSCs. A conditioned medium (CM) was collected to measure the level of BDNF, IL-1β, and IL-6 proteins using the ELISA assay. Following the SCI induction, MSCs and CM were injected into the lesion site, and also CM was infused intraperitoneally in the different groups. Two weeks after SCI induction, the spinal cord samples were examined to evaluate the expression of the doublecortin (DCX) and glial fibrillary acid protein (GFAP) markers using immunofluorescence staining. The MSCs’ phenotype was confirmed upon the expression and un-expression of the related CD markers. Our results show that MSCs increased the expression level of the DCX and decreased the level of the GFAP relative to the injury group (p < 0.001). Additionally, the CM promoted the DCX expression rate (p < 0.001) and decreased the GFAP expression rate (p < 0.01) as compared with the injury group. Noteworthily, the restorative potential of the MSCs was higher than that of the CM (p < 0.01). Large-scale meta-analysis of transcriptomic data highlighted PAK5, ST8SIA3, and NRXN1 as positively coexpressed genes with DCX. These genes are involved in neuroactive ligand–receptor interaction. Overall, our data revealed that both therapeutic interventions could promote the regeneration and restoration of the damaged neural tissue by increasing the rate of neuroblasts and decreasing the astrocytes.

Amniotic fluid stem cell administration can prevent epithelial injury from necrotizing enterocolitis.

Stem cell therapy has been proven to rescue intestinal injury and stimulate intestinal regeneration in necrotizing enterocolitis (NEC). Specifically, stem cells derived from amniotic fluid (AFSCs) and mesenchymal stem cells (MSCs) derived from bone marrow have shown promising results in the treatment of experimental NEC. This study aims to examine the effects of AFSCs and MSCs on the prevention of intestinal injury during experimental NEC. Supernatants from AFSC and MSC cultures were collected to perform proteomic analysis. Prior to NEC induction, mice received intraperitoneal injections of phosphate-buffered saline (PBS), 2 × 106 AFSCs, or 2 × 106 MSCs. We found that AFSCs grew faster than MSCs. Proteomic analysis indicated that AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. Administering AFSCs before NEC induction decreased NEC severity and mucosal inflammation. Intestinal proliferation and endogenous stem cell activation were increased after AFSC administration. However, administering MSCs before NEC induction had no beneficial effects. This study demonstrated that AFSCs and MSCs have different protein release profiles. AFSCs can potentially be used as a preventative strategy for neonates at risk of NEC, while MSCs cannot be used. AFSCs and MSCs have distinct protein secretory profiles, and AFSCs are primarily involved in cell development and growth, while MSCs are involved in immune regulation. AFSCs are unique in transiently enhancing healthy intestinal epithelial cell growth, which offers protection against the development of experimental NEC. The prevention of NEC via the administration of AFSCs should be evaluated in infants at great risk of developing NEC or in infants with early signs of NEC.

Nano-Hydroxyapatite vs. Xenografts: Synthesis, Characterization, and In Vitro Behavior.

This research focused on the synthesis of apatite, starting from a natural biogenic calcium source (egg-shells) and its chemical and morpho-structural characterization in comparison with two commercial xenografts used as a bone substitute in dentistry. The synthesis route for the hydroxyapatite powder was the microwave-assisted hydrothermal technique, starting from annealed egg-shells as the precursor for lime and di-base ammonium phosphate as the phosphate precursor. The powders were characterized by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray analysis (EDAX), transmission electron microscopy (TEM), X-ray fluorescence spectroscopy (XRF), and cytotoxicity assay in contact with amniotic fluid stem cell (AFSC) cultures. Compositional and structural similarities or differences between the powder synthesized from egg-shells (HA1) and the two commercial xenograft powders—Bio-Oss®, totally deproteinized cortical bovine bone, and Gen-Os®, partially deproteinized porcine bone—were revealed. The HA1 specimen presented a single mineral phase as polycrystalline apatite with a high crystallinity (Xc 0.92), a crystallite size of 43.73 nm, preferential growth under the c axes (002) direction, where it mineralizes in bone, a nano-rod particle morphology, and average lengths up to 77.29 nm and diameters up to 21.74 nm. The surface of the HA1 nanoparticles and internal mesopores (mean size of 3.3 ± 1.6 nm), acquired from high-pressure hydrothermal maturation, along with the precursor’s nature, could be responsible for the improved biocompatibility, biomolecule adhesion, and osteoconductive abilities in bone substitute applications. The cytotoxicity assay showed a better AFSC cell viability for HA1 powder than the commercial xenografts did, similar oxidative stress to the control sample, and improved results compared with Gen-Os. The presented preliminary biocompatibility results are promising for bone tissue regeneration applications of HA1, and the study will continue with further tests on osteoblast differentiation and mineralization.

MP22-16 DEVELOPMENT OF NON-INVASIVE CLINICALLY APPLICABLE IN VIVO TRACKING OF EXTRACELLULAR VESICLES USING MAGNETIC RESONANCE IMAGING (MRI)

You have accessJournal of UrologyImaging/Radiology: Uroradiology II (MP22)1 Sep 2021MP22-16 DEVELOPMENT OF NON-INVASIVE CLINICALLY APPLICABLE IN VIVO TRACKING OF EXTRACELLULAR VESICLES USING MAGNETIC RESONANCE IMAGING (MRI) Johnny Akers, Paola Aguiari, Hasmik Soloyan, Seda Mkhitaryan, Gevorg Karaptyan, Roger De Filippo, Mya Thu, Laura Perin, and Sargis Sedrakyan Johnny AkersJohnny Akers More articles by this author , Paola AguiariPaola Aguiari More articles by this author , Hasmik SoloyanHasmik Soloyan More articles by this author , Seda MkhitaryanSeda Mkhitaryan More articles by this author , Gevorg KaraptyanGevorg Karaptyan More articles by this author , Roger De FilippoRoger De Filippo More articles by this author , Mya ThuMya Thu More articles by this author , Laura PerinLaura Perin More articles by this author , and Sargis SedrakyanSargis Sedrakyan More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002013.16AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSC) hold great potential for the treatment of chronic kidney diseases (CKD). We have already shown that AFSC-EVs are renoprotective in a mouse model of CKD, Alport syndrome. However, there is an important unmet need for real-time in vivo monitoring of these therapeutic EVs after they are injected into a subject to understand their safety, targeting, and effectiveness. While current optical imaging solutions like bioluminescence and fluorescence are useful for EV tracking studies in animal models, there is limited utility in clinical applications. Here we present a novel in vivo tracking solution for our therapeutic EVs in Alport mice, utilizing clinically applicable MRI technology. METHODS: To generate trackable EVs, AFSC were labeled with a novel magnetic agent (VSCM). EVs secreted by the labeled AFSC were isolated by ultracentrifugation. The viability and morphology of labeled-cells were evaluated, and the in vitro MR properties of EVs were analyzed by magnetometer. Purity, potency and identity of labeled EV was compared to non-labeled EVs. In vivo biodistribution of labeled EVs was evaluated in WT and Alport mice by MRI at 10 min and 3 hr post injection, and retro-orbital and intra-cardiac routes of delivery were compared. RESULTS: The magnetic label did not affect the physiological characteristics of the cells and did not change identity, purity and potency (therapeutic effect in vivo) of EVs. MRI phantom studies confirmed the in vitro/ex vivo detectability of labeled-EVs. Importantly, as expected MRI studies showed that EV homing to the kidney injected intra-cardiacally into Alport mice was more efficient vs the retro-orbital route, and Prussian blue staining of sections confirmed EV homing to the kidney. CONCLUSIONS: We have developed a clinically applicable novel magnetic nanoparticle agent that can be used to label and track the biodistribution of EVs in the kidney and other organs using non-invasive, safe, and effective MRI technology that’s widely available. This technology is highly adaptable and can be deployed in both preclinical and clinical settings. Source of Funding: The GOFARR Fund; Research Career Development Award, TSRI, CHLA © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e397-e398 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Johnny Akers More articles by this author Paola Aguiari More articles by this author Hasmik Soloyan More articles by this author Seda Mkhitaryan More articles by this author Gevorg Karaptyan More articles by this author Roger De Filippo More articles by this author Mya Thu More articles by this author Laura Perin More articles by this author Sargis Sedrakyan More articles by this author Expand All Advertisement Loading ...

Amniotic fluid stem cells and the cell source repertoire for non-invasive prenatal testing

Cell-free fetal DNA (cffDNA)-based non-invasive prenatal testing (NIPT) is considered to be a very promising screening tool for pregnant women with an increased risk of fetal aneuploidy. Already millions of women worldwide underwent NIPT. However, due to the observed false-positive and false-negative results, this screening approach does not fulfil the criteria of a diagnostic test. Accordingly, positive results still require risk-carrying invasive prenatal testing, such as amniocentesis or chorionic villus sampling (CVS), for confirmation. Such hurdles need to be overcome before NIPT could become a diagnostic approach widely used in the general population. Here we discuss new evidence that besides the placenta amniotic fluid stem cells (AFSCs) could also represent an origin of cffDNA in the mother’s blood. A comprehensive picture of the involved cell source repertoire could pave the way to more reliable interpretations of NIPT results and ameliorate counselling of advice-seeking patients.Graphical abstract

Three-dimensional migration of human amniotic fluid stem cells involves mesenchymal and amoeboid modes and is regulated by mTORC1.

Three‐dimensional (3D) cell migration is an integral part of many physiologic processes. Although being well studied in the context of adult tissue homeostasis and cancer development, remarkably little is known about the invasive behavior of human stem cells. Using two different kinds of invasion assays, this study aimed at investigating and characterizing the 3D migratory capacity of human amniotic fluid stem cells (hAFSCs), a well‐established fetal stem cell type. Eight hAFSC lines were found to harbor pronounced potential to penetrate basement membrane (BM)‐like matrices. Morphological examination and inhibitor approaches revealed that 3D migration of hAFSCs involves both the matrix metalloprotease‐dependent mesenchymal, elongated mode and the Rho‐associated protein kinase‐dependent amoeboid, round mode. Moreover, hAFSCs could be shown to harbor transendothelial migration capacity and to exhibit a motility‐associated marker expression pattern. Finally, the potential to cross extracellular matrix was found to be induced by mTORC1‐activating growth factors and reduced by blocking mTORC1 activity. Taken together, this report provides the first demonstration that human stem cells exhibit mTORC1‐dependent invasive capacity and can concurrently make use of mesenchymal and amoeboid 3D cell migration modes, which represents an important step toward the full biological characterization of fetal human stem cells with relevance to both developmental research and stem cell‐based therapy.

Application of Modified mRNA in Somatic Reprogramming to Pluripotency and Directed Conversion of Cell Fate.

Modified mRNA (modRNA)-based somatic reprogramming is an effective and safe approach that overcomes the genomic mutation risk caused by viral integrative methods. It has improved the disadvantages of conventional mRNA and has better stability and immunogenicity. The modRNA molecules encoding multiple pluripotent factors have been applied successfully in reprogramming somatic cells such as fibroblasts, mesenchymal stem cells, and amniotic fluid stem cells to generate pluripotent stem cells (iPSCs). Moreover, it also can be directly used in the terminal differentiation of stem cells and fibroblasts into functional therapeutic cells, which exhibit great promise in disease modeling, drug screening, cell transplantation therapy, and regenerative medicine. In this review, we summarized the reprogramming applications of modified mRNA in iPSC generation and therapeutic applications of functionally differentiated cells.

The Human Fetal and Adult Stem Cell Secretome Can Exert Cardioprotective Paracrine Effects against Cardiotoxicity and Oxidative Stress from Cancer Treatment.

Simple SummaryAnthracyclines, such as doxorubicin (Dox), are an important class of chemotherapeutic drugs. However, their use is hampered by the risk of developing heart failure. The aim of this study was to assess and compare the cardioprotective effects exerted by a set of factors, collectively named secretomes, secreted by either adult or fetal human stem cells. Both secretome formulations were effective in counteracting Dox-induced apoptosis and mitochondrial impairment in cardiomyocytes and cardiac fibroblasts. In vivo experiments in a mouse model of Dox-induced cardiomyopathy (DIC) indicated that early administration of both secretomes during Dox treatment exerted beneficial long-term effects, preserving cardiac function and body mass. These findings suggest that the stem cell secretome could represent a feasible option for future paracrine cardioprotective therapy against Dox-related cardiotoxicity during cancer treatment.Cardiovascular side effects are major shortcomings of cancer treatments causing cardiotoxicity and late-onset cardiomyopathy. While doxorubicin (Dox) has been reported as an effective chemotherapy agent, unspecific impairment in cardiomyocyte mitochondria activity has been documented. We demonstrated that the human fetal amniotic fluid-stem cell (hAFS) secretome, namely the secreted paracrine factors within the hAFS-conditioned medium (hAFS-CM), exerts pro-survival effects on Dox-exposed cardiomyocytes. Here, we provide a detailed comparison of the cardioprotective potential of hAFS-CM over the secretome of mesenchymal stromal cells from adipose tissue (hMSC-CM). hAFS and hMSC were preconditioned under hypoxia to enrich their secretome. The cardioprotective effects of hAFS/hMSC-CM were evaluated on murine neonatal ventricular cardiomyocytes (mNVCM) and on their fibroblast counterpart (mNVFib), and their long-term paracrine effects were investigated in a mouse model of Dox-induced cardiomyopathy. Both secretomes significantly contributed to preserving mitochondrial metabolism within Dox-injured cardiac cells. hAFS-CM and hMSC-CM inhibited body weight loss, improved myocardial function, reduced lipid peroxidation and counteracted the impairment of mitochondrial complex I activity, oxygen consumption, and ATP synthesis induced by Dox. The hAFS and hMSC secretomes can be exploited for inhibiting cardiotoxic detrimental side effects of Dox during cancer therapy, thus ensuring cardioprotection via combinatorial paracrine therapy in association with standard oncological treatments.

Metabolic Profile and Neurogenic Potential of Human Amniotic Fluid Stem Cells From Normal vs. Fetus-Affected Gestations.

Human amniotic fluid stem cells (hAFSCs) possess some characteristics with mesenchymal stem cells (MSCs) and embryonic stem cells and have a broader differentiation potential compared to MSCs derived from other sources. Although hAFSCs are widely researched, their analysis mainly involves stem cells (SCs) obtained from normal, fetus-unaffected gestations. However, in clinical settings, knowledge about hAFSCs from normal gestations could be poorly translational, as hAFSCs from healthy and fetus-diseased gestations may differ in their differentiation and metabolic potential. Therefore, a more thorough investigation of hAFSCs derived from pathological gestations would provide researchers with the knowledge about the general characteristics of these cells that could be valuable for further scientific investigations and possible future clinical applicability. The goal of this study was to look into the neurogenic and metabolic potential of hAFSCs derived from diseased fetuses, when gestations were concomitant with polyhydramnios and compare them to hAFSCs derived from normal fetuses. Results demonstrated that these cells are similar in gene expression levels of stemness markers (SOX2, NANOG, LIN28A, etc.). However, they differ in expression of CD13, CD73, CD90, and CD105, as flow cytometry analysis revealed higher expression in hAFSCs from unaffected gestations. Furthermore, hAFSCs from “Normal” and “Pathology” groups were different in oxidative phosphorylation rate, as well as level of ATP and reactive oxygen species production. Although the secretion of neurotrophic factors BDNF and VEGF was of comparable degree, as evaluated with enzyme-linked immunosorbent assay (ELISA) test, hAFSCs from normal gestations were found to be more prone to neurogenic differentiation, compared to hAFSCs from polyhydramnios. Furthermore, hAFSCs from polyhydramnios were distinguished by higher secretion of pro-inflammatory cytokine TNFα, which was significantly downregulated in differentiated cells. Overall, these observations show that hAFSCs from pathological gestations with polyhydramnios differ in metabolic and inflammatory status and also possess lower neurogenic potential compared to hAFSCs from normal gestations. Therefore, further in vitro and in vivo studies are necessary to dissect the potential of hAFSCs from polyhydramnios in stem cell-based therapies. Future studies should also search for strategies that could improve the characteristics of hAFSCs derived from diseased fetuses in order for those cells to be successfully applied for regenerative medicine purposes.

Human Amniotic Fluid Stem Cell-Derived Exosomes as a Novel Cell-Free Therapy for Cutaneous Regeneration.

Adult wound healing often results in fibrotic scarring that is caused by myofibroblast aggregation. Human amniotic fluid stem cells (hAFSCs) exhibit significantly anti-fibrotic scarring properties during wound healing. However, it is little known whether hAFSCs directly or indirectly (paracrine) contribute to this process. Using the full-thickness skin-wounded rats, we investigated the therapeutic potential of hAFSC-derived exosomes (hAFSC-exo). Our results showed that hAFSC-exo accelerated the wound healing rate and improved the regeneration of hair follicles, nerves, and vessels, as well as increased proliferation of cutaneous cells and the natural distribution of collagen during wound healing. Additionally, hAFSC-exo suppressed the excessive aggregation of myofibroblasts and the extracellular matrix. We identified several miRNAs, including let-7-5p, miR-22-3p, miR-27a-3p, miR-21-5p, and miR-23a-3p, that were presented in hAFSC-exo. The functional analysis demonstrated that these hAFSC-exo-miRNAs contribute to the inhibition of the transforming growth factor-β (TGF-β) signaling pathway by targeting the TGF-β receptor type I (TGF-βR1) and TGF-β receptor type II (TGF-βR2). The reduction of TGF-βR1 and TGF-βR2 expression induced by hAFSC-exo was also confirmed in the healing tissue. Finally, using mimics of miRNAs, we found that hAFSC-exo-miRNAs were essential for myofibroblast suppression during the TGF-β1-induced human dermal fibroblast-to-myofibroblast transition in vitro. In summary, this study is the first to show that exosomal miRNAs used in hAFSC-based therapy inhibit myofibroblast differentiation. Our study suggests that hAFSC-exo may represent a strategic tool for suppressing fibrotic scarring during wound healing.

Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering.

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implement in vivo and introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that additional crosslinking of fibrin gels using bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) slows the degradation rate of fibrin. In this study, we aimed to further improve the longevity of these PEG-fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. Here, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels both in vitro (when treated with proteases and when seeded with amniotic fluid stem cells) and in vivo (when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications.

Prenatal transplantation of human amniotic fluid stem cell could improve clinical outcome of type III spinal muscular atrophy in mice

Spinal muscular atrophy (SMA) is a single gene disorder affecting motor function in uterus. Amniotic fluid is an alternative source of stem cell to ameliorate SMA. Therefore, this study aims to examine the therapeutic potential of Human amniotic fluid stem cell (hAFSC) for SMA. Our SMA model mice were generated by deletion of exon 7 of Smn gene and knock-in of human SMN2. A total of 16 SMA model mice were injected with 1 × 105 hAFSC in uterus, and the other 16 mice served as the negative control. Motor function was analyzed by three behavioral tests. Engraftment of hAFSC in organs were assessed by flow cytometry and RNA scope. Frequency of myocytes, neurons and innervated receptors were estimated by staining. With hAFSC transplantation, 15 fetuses survived (93.75% survival) and showed better performance in all motor function tests. Higher engraftment frequency were observed in muscle and liver. Besides, the muscle with hAFSC transplantation expressed much laminin α and PAX-7. Significantly higher frequency of myocytes, neurons and innervated receptors were observed. In our study, hAFSC engrafted on neuromuscular organs and improved cellular and behavioral outcomes of SMA model mice. This fetal therapy could preserve the time window and treat in the uterus.

Fetal lung underdevelopment is rescued by administration of amniotic fluid stem cell extracellular vesicles in rodents.

Fetal lung underdevelopment, also known as pulmonary hypoplasia, is characterized by decreased lung growth and maturation. The most common birth defect found in babies with pulmonary hypoplasia is congenital diaphragmatic hernia (CDH). Despite research and clinical advances, babies with CDH still have high morbidity and mortality rates, which are directly related to the severity of lung underdevelopment. To date, there is no effective treatment that promotes fetal lung growth and maturation. Here, we describe a stem cell-based approach in rodents that enhances fetal lung development via the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs). Using fetal rodent models of pulmonary hypoplasia (primary epithelial cells, organoids, explants, and in vivo), we demonstrated that AFSC-EV administration promoted branching morphogenesis and alveolarization, rescued tissue homeostasis, and stimulated epithelial cell and fibroblast differentiation. We confirmed this regenerative ability in in vitro models of lung injury using human material, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. Investigating EV mechanism of action, we found that AFSC-EV beneficial effects were exerted via the release of RNA cargo. MicroRNAs regulating the expression of genes involved in lung development, such as the miR17-92 cluster and its paralogs, were highly enriched in AFSC-EVs and were increased in AFSC-EV-treated primary lung epithelial cells compared to untreated cells. Our findings suggest that AFSC-EVs hold regenerative ability for underdeveloped fetal lungs, demonstrating potential for therapeutic application in patients with pulmonary hypoplasia.

Accelerated wound closure: Systematic evaluation of cellulose acetate effects on biologically active molecules release from amniotic fluid stem cells.

Despite a lot of intensive research on cell-scaffold interaction, the focus is mainly on the capacity of construct scaffolds to regulate cell mobility, migration, and cytotoxicity. The effect of the scaffold's topographical and material properties on the expression of biologically active compounds from stem cells is not well understood. In this study,the influence of cellulose acetate (CA) on the electrospinnability of gelatin and the roles of gelatin-cellulose acetate (Ge-CA) on modulating the release of biologically active compounds from amniotic fluid stem cells (AFSCs) is emphasized. It was found that the presence of a small amount of CA could provide a better microenvironment that mimics AFSCs' niche. However, a large amount of CA exhibited no significant effect on AFSCs migration and infiltration. Further study on the effect of surface topography and mechanical properties on AFSCs showed that the tailored microenvironment provided by the Ge-CA scaffolds had transduced physical cues to biomolecules released into the culture media. It was found that the AFSCs seeded on electrospun scaffolds with less CA proportions have profound effects on the secretion of metabolic compounds compared to those with higher CA contained and gelatin coating. The enhanced secretion of biologically active molecules by the AFSCs on the electrospun scaffolds was proven by the accelerated wound closure on the injured human dermal fibroblast (HDF) model. The rapid HDF cell migration could be anticipated due to a higher level of paracrine factors in AFSCs media. Our study demonstrates that the fibrous topography and mechanical properties of the scaffold are a key material property that modulates the high expression of biologically active compounds from the AFSCs. The discovery elucidates a new aspect of material functions and scaffolds material-AFSC interaction for regulating biomolecules release to promote tissue regeneration/repair. To the best of our knowledge, this is the first report describing the scaffolds material-AFSC interaction and the efficacy of scratch assays on quantifying the cell migration in response to the AFSCs metabolic products.

Transamniotic Stem Cell Therapy for Experimental Congenital Diaphragmatic Hernia: Structural, Transcriptional, and Cell Kinetics Analyses in the Nitrofen Model

Purpose: We examined select pulmonary effects and donor cell kinetics after transamniotic stem cell therapy (TRASCET) in a model of congenital diaphragmatic hernia (CDH). Methods: Pregnant dams (n = 58) received nitrofen on gestational day 9.5 (E9) to induce fetal CDH. Fetuses (n = 681) were divided into 4 groups: untreated (n = 99) and 3 groups receiving volume-matched intra-amniotic injections on E17 of either saline (n = 142), luciferase-labeled amniotic fluid-derived mesenchymal stem cells (afMSCs; n = 299), or acellular recombinant luciferase (n = 141). Pulmonary morphometry, quantitative gene expression of pulmonary vascular tone mediators, or screening for labeled afMSCs were performed at term (E22). Statistical comparisons were by Mann-Whitney U-test, nested ANOVA, and Wald test. Results: TRASCET led to significant downregulation of endothelial nitric oxide synthase and endothelin receptor-A expressions compared to both untreated and saline groups (both p < 0.001). TRASCET also led to a significant decrease in arteriole wall thickness compared to the untreated group (p < 0.001) but not the saline group (p = 0.180). Donor afMSCs were identified in the bone marrow and umbilical cord (p = 0.035 and 0.015, respectively, vs. plain luciferase controls). Conclusions: The effects of TRASCET in experimental CDH appear to be centered on the pulmonary vasculature and to derive from circulating donor cells.