Human Amniotic Fluid Stem Cells Research Articles

Human amniotic fluid stem cell therapy can help regain bladder function in type 2 diabetic rats.

BACKGROUNDDiabetes mellitus (DM) is a serious and growing global health burden. It is estimated that 80% of diabetic patients have micturition problems such as poor emptying, urinary incontinence, urgency, and urgency incontinence. Patients with diabetic bladder dysfunction are often resistant to currently available therapies. It is necessary to develop new and effective treatment methods.AIMTo examine the therapeutic effect of human amniotic fluid stem cells (hAFSCs) therapy on bladder dysfunction in a type 2 diabetic rat model.METHODSSixty female Sprague-Dawley rats were divided into five groups: Group 1, normal-diet control (control); group 2, high-fat diet (HFD); group 3, HFD plus streptozotocin-induced DM (DM); group 4, DM plus insulin treatment (DM + insulin); group 5, DM plus hAFSCs injection via tail vein (DM + hAFSCs). Conscious cystometric studies were done at 4 and 12 wk after insulin or hAFSCs treatment to measure peak voiding pressure, voided volume, intercontraction interval, bladder capacity, and residual volume. Immunoreactivities and/or mRNA expression of muscarinic receptors, nerve growth factor (NGF), and sensory nerve markers in the bladder and insulin, MafA, and pancreatic-duodenal homeobox-1 (PDX-1) in pancreatic beta cells were studied.RESULTSCompared with DM rats, insulin but not hAFSCs treatment could reduce the bladder weight and improve the voided volume, intercontraction interval, bladder capacity, and residual volume (P < 0.05). However, both insulin and hAFSCs treatment could help to regain the blood glucose and bladder functions to the levels near controls (P > 0.05). The immunoreactivities and mRNA expression of M2- and M3-muscarinic receptors (M2 and M3) were increased mainly at 4 wk (P < 0.05), while the number of beta cells in islets and the immunoreactivities and/or mRNA of NGF, calcitonin gene-related peptide (CGRP), substance P, insulin, MafA, and PDX-1 were decreased in DM rats (P < 0.05). However, insulin and hAFSCs treatment could help to regain the expression of M2, M3, NGF, CGRP, substance P, MafA, and PDX-1 to near the levels of controls at 4 and/or 12 wk (P > 0.05).CONCLUSIONInsulin but not hAFSCs therapy can recover the bladder dysfunction caused by DM; however, hAFSCs and insulin therapy can help to regain bladder function to near the levels of control.

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

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

Human amniotic fluid stem cells can alleviate detrusor dysfunction caused by bladder outlet obstruction in rats

The present study examined whether bladder detrusor dysfunction due to partial bladder outlet obstruction (pBOO) could be improved after the treatment of human amniotic fluid stem cells (hAFSCs). 72 female rats were grouped into sham operation, pBOO, and pBOO with hAFSCs treatment (pBOO + hAFSCs) for in vitro and in vivo studies. Bladder weight, bladder wall thickness, the ratio of collagen to smooth muscle and the levels of positive CD11b/c and HIS48 cells was significantly increased after pBOO but improved after hAFSCs treatment. Cystometries showed impaired bladder function after pBOO. Protein and mRNA levels of hypoxia inducible factor-1α, CCL2, interleukin-1β, transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), α-smooth muscle actin, collagen I and collagen III were increased at 2 and/or 6 weeks, but proteins and mRNA expressions of protein gene product 9.5 were decreased at 2 and 6 weeks after pBOO. These abnormalities were improved after hAFSCs treatment. The expressions of TGF-β1 and CTGF in cultured detrusor cells of pBOO rats were increased but were improved after hAFSCs treatment. The present results showed hAFSCs treatment could improve bladder detrusor dysfunction in pBOO rats, which may be related to the reduction of inflammatory and pro-fibrotic markers in detrusor muscle cells.

Human amniotic fluid as a source of stem cells.

Human amniotic fluid collected during amniocentesis contains a heterogeneous population of differentiated and undifferentiated cells. Properties and number of these cells vary depending on the gestational age and the presence of potential fetal pathologies. The aim of this study was to analyze the effects of maternal, fetal, and environmental factors on the success rates of amniotic fluid stem cell cultures, the number of human amniotic fluid stem cells (hAFSC), their growth rates in primary cultures, and the number of cell passages. The study included 355 patients qualified for genetic amniocentesis at the Prenatal Genetic Unit, Department of Obstetrics, Gynecology and Oncologic Gynecology, Nicolaus Copernicus University Medical College in Bydgoszcz in 2011–2017. The mean age of the study participants was 34 ± 6.2 years, and mean gravidity amounted to 2.48 ± 1.4. Amniotic fluid sample volume turned out to be a highly significant (p < 0.01) predictor of culture success, and the relationship was particularly evident in women older than 40 years. Another highly significant predictor of culture success was the presence of two cell populations in the sample (p < 0.01). The likelihood of culture success correlated significantly (p < 0.05) with the season of the year at the time of amniocentesis. The number of cell passages differed significantly depending on the maternal age (p < 0.01). The number of passages also showed a highly significant relationship with the season of the year the sample was obtained (p < 0.01). Younger maternal age was identified as a determinant of high passage number (≥3), and another highly significant determinant of high passage number was the presence of two cell populations in the amniotic fluid sample (p < 0.01). Percentage of successfully established hAFSC cultures and the number of passages depended on amniotic fluid volume, the presence of two cell populations within the sample, and the season of the year. Individual characteristics of the donors, such as age and gravidity, did not exert a significant effect on the number of isolated hAFSCs and the rate of their growth. Patients’ place of residence, fetal karyotype, transportation time, and purity of the samples did not affect the success rates for primary cultures and the number of passages.

Contraceptive drug, Nestorone, enhances stem cell-mediated remodeling of the stroke brain by dampening inflammation and rescuing mitochondria

Ischemic stroke remains a significant unmet need causing massive mortality and morbidity due to few treatment options with limited therapeutic window. The progestin Nestorone® (segesterone acetate) displays high affinity for the progesterone receptor in exerting its potent birth control and hormone replacement therapy. Accumulating evidence implicates a new utility of Nestorone in affording neuroprotection in a variety of central nervous system diseases, including stroke. However, the mechanism of action mediating Nestorone's neuroprotection in stroke remains unknown. Here, we showed that stand-alone treatments of Nestorone or human amniotic fluid-derived stem cells (hAFSc), but more pronounced with their combined treatment, led to significant improvements in behavioral function and reductions in infarction and peri-infarct cell loss in adult rats with ischemic stroke. We detected significantly lower levels of pro-inflammatory signals (OX6 and IBA1) coupled with enhanced levels of stem cell proliferation (Ki67) and differentiation (DCX and MAP2) in both brain and spleen of stroke rats that received stand-alone or combined treatments of Nestorone and hAFSc. In concert, the in vitro oxygen-glucose deprivation stroke model revealed that neural stem cells treated with Nestorone exhibited increased stem cell proliferation and differentiation that was accompanied by rescue of the mitochondrial respiratory activity characterized by reduced mitochondrial reactive oxygen species, increased ATP, elevated mitochondrial deacetylase Sirtuin 3 (SIRT3), and a normalized ratio of acetyl-superoxide dismutase 2 (Ac-SOD2)/SOD2, suggesting the key role of mitochondrial metabolism and oxidative protection in Nestorone's therapeutic effects in stroke.

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.

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.

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.

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.

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.

Human Amniotic Fluid Stem Cells: General Characteristics and Potential Therapeutic Applications

Introduction: Amniotic fluid contains a mixture of different cell types sloughed from the fetal skin, respiratory, alimentary and urogenital tracts, as well as the amnion membrane. As amniotic fluid develops prior to the process of gastrulation, many cells found in its heterogeneous population do not undergo lineage specialization. Therefore, amniotic fluid-derived mesenchymal stem cells (AF-MSCs) may correspond to a new class of stem cells with properties of intermediate plasticity between pluripotent and adult stem cell types. Compared to mesenchymal stem cells (MSCs) from other sources, such as bone marrow, AF-MSCs have better properties for clinical applications, such as differentiation into the cells of three germ layers, high clonal capacity, ability to form embryoid bodies, expression of pluripotent markers, high self-renewal capacity (over 250 population doublings) with normal karyotype at late passages, long telomere length due to continued telomerase activity, specially non-tumorigenicity, low immunogenicity, anti-inflammatory and immunomodulatory properties. Conclusion: Such features have nominated AF-MSC for a range of clinical applications, including in regenerative medicine. In several studies, these cells have been used to regenerate nerve, lung, and heart tissues. Overall, AF-MSCs are expected to be an ideal source of stem cells for future regenerative medicine and tissue engineering.

The Fibrogenic Injury of Liver Ductal Organoids Is Rescued By Human Amniotic Fluid Stem Cells

The Fibrogenic Injury of Liver Ductal Organoids Is Rescued By Human Amniotic Fluid Stem Cells

Small Molecule Treatments Improve Differentiation Potential of Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (KLF4, OCT4, NOTCH1, SOX2, NANOG, LIN28a, CMYC), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (NRF1, HIF1α, PPARGC1A, ERRα, PKM, PDK1, LDHA, NFKB1, NFKB2, RELA, RELB, REL) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (Nestin, MAP2, TUBB3, ALDH1L1, GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs.

Administration of extracellular vesicles derived from human amniotic fluid stem cells: a new treatment for necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease. Amniotic fluid stem cells (AFSC) improve NEC injury but human translation remains difficult. We aimed to evaluate the use of extracellular vesicles (EV) derived from human AFSC. Human AFSC (hAFSC) were cultured according to the protocol (Celprogen Inc., California, U.S.A.). Conditioned medium was obtained, ultra-centrifuged, and EV were suspended in phosphate-buffered saline (PBS). C57BL/6 pups were grouped into: (1) breast-fed (Control, n = 11); (2) NEC + placebo (NEC + PBS; n = 10); and (3) NEC + treatment (NEC + EV; n = 11). NEC was induced post-natal days P5-9 by (A) gavage feeding hyperosmolar formula; (B) hypoxia for 10min; and (C) lipopolysaccharide. Intra-peritoneal injections of PBS or hAFSC-EV were given on P6-7. All animals were sacrificed on P9 and terminal ileum harvested. hAFSC-EV administration reduced intestinal injury (p = 0.0048), NEC incidence (score ≥ 2), and intestinal inflammation (IL-6 p < 0.0001; TNF-α p < 0.0001). Intestinal stem cell expression (Lgr5 +) and cellular proliferation (Ki67) were enhanced above control levels following hAFSC-EV administration (Lgr5 p = 0.0003; Ki67 p < 0.0001). hAFSC-EV administration reduced intestinal NEC injury and inflammation while increasing stem cell expression and cellular proliferation. hAFSC-EV administration may induce similar beneficial effects to exogenous stem cells.

Chondrogenesis of human amniotic fluid stem cells in Chitosan-Xanthan scaffold for cartilage tissue engineering

Articular chondral lesions, caused either by trauma or chronic cartilage diseases such as osteoarthritis, present very low ability to self-regenerate. Thus, their current management is basically symptomatic, progressing very often to invasive procedures or even arthroplasties. The use of amniotic fluid stem cells (AFSCs), due to their multipotentiality and plasticity, associated with scaffolds, is a promising alternative for the reconstruction of articular cartilage. Therefore, this study aimed to investigate the chondrogenic potential of AFSCs in a micromass system (high-density cell culture) under insulin-like growth factor 1 (IGF-1) stimuli, as well as to look at their potential to differentiate directly when cultured in a porous chitosan-xanthan (CX) scaffold. The experiments were performed with a CD117 positive cell population, with expression of markers (CD117, SSEA-4, Oct-4 and NANOG), selected from AFSCs, after immunomagnetic separation. The cells were cultured in both a micromass system and directly in the scaffold, in the presence of IGF-1. Differentiation to chondrocytes was confirmed by histology and by using immunohistochemistry. The construct cell-scaffold was also analyzed by scanning electron microscopy (SEM). The results demonstrated the chondrogenic potential of AFSCs cultivated directly in CX scaffolds and also in the micromass system. Such findings support and stimulate future studies using these constructs in osteoarthritic animal models.