Human Amnion Epithelial Cells Research Articles

Human Amnion Epithelial Cells Protect against White Matter Brain Injury after Repeated Endotoxin Exposure in the Preterm Ovine Fetus

Intrauterine inflammation is a significant cause of injury to the developing fetal brain. Using a preterm fetal sheep model of in utero infection, we asked whether human amnion epithelial cells (hAECs) were able to reduce inflammation-induced fetal brain injury. Surgery was undertaken on pregnant sheep at ~105 days gestation (term is 147 days) for implantation of vascular catheters. Lipopolysaccharide (LPS; 150 ng/kg bolus) or saline was administered IV at 109, 110, and 111 days. Sixty million fluorescent-labeled hAECs were administered at 110, 111, and 112 days gestation via the brachial artery catheter. Brains were collected at 114 days for histological assessment. hAECs were observed within the cortex, white matter, and hippocampus. Compared to control lambs, LPS administration was associated with significant and widespread fetal brain inflammation and injury as evidenced by increased number of activated microglia in the periventricular white matter (p = 0.02), increased pyknosis, cell degeneration (p = 0.01), and a nonsignificant trend of fewer oligodendrocytes in the subcortical and periventricular white matter. Administration of hAECs to LPS-treated animals was associated with a significant mitigation in both inflammation and injury as evidenced by fewer activated microglia (p = 0.03) and pyknotic cells (p = 0.03), significantly more oligodendrocytes in the subcortical and periventricular white matter (p = 0.01 and 0.02, respectively), and more myelin basic protein-positive cells within the periventricular white matter (p = 0.02). hAEC administration to fetal sheep exposed to multiple doses of LPS dampens the resultant fetal inflammatory response and mitigates associated brain injury.

Human amnion epithelial cells modulate the inflammatory response to ventilation in preterm lambs.

Ventilation of preterm neonates causes pulmonary inflammation that can contribute to lung injury, propagate systemically and result in long-term disease. Modulation of this initial response may reduce lung injury and its sequelae. We aimed to determine the effect of human amnion epithelial cells (hAECs) on immune activation and lung injury in preterm neonatal lambs. Preterm lambs received intratracheal hAECs (90x106) or vehicle, prior to 2 h of mechanical ventilation. Within 5 min of ventilation onset, lambs also received intravenous hAECs (90x106) or vehicle. Lung histology, bronchoalveolar lavage (BAL) cell phenotypes, and cytokine profiles were examined after 2 h of ventilation, and in unventilated controls. Histological indices of lung injury were higher than control, in vehicle-treated ventilated lambs but not in hAEC-treated ventilated lambs. Ventilation-induced pulmonary leukocyte recruitment was greater in hAEC-treated lambs than in vehicle-treated lambs. Lung IL-1β and IL-6 mRNA expression was higher in vehicle- and hAEC-treated ventilated lambs than in controls but IL-8 mRNA levels were greater than control only in vehicle-treated ventilated lambs. Numbers of CD44+ and CD21+ lymphocytes and macrophages from the lungs were altered in vehicle- and hAEC-treated ventilated lambs. Numbers of CD8+ macrophages were lower in hAEC-treated ventilated lambs than in vehicle-treated ventilated lambs. Indices of systemic inflammation were not different between vehicle- and hAEC-treated lambs. Human amnion epithelial cells modulate the pulmonary inflammatory response to ventilation in preterm lambs, and reduce acute lung injury. Immunomodulatory effects of hAECs reduce lung injury in preterm neonates and may protect against longer-term respiratory disease.

Human amnion epithelial cells rescue cell death via immunomodulation of microglia in a mouse model of perinatal brain injury

BackgroundHuman amnion epithelial cells (hAECs) are clonogenic and have been proposed to reduce inflammatory-induced tissue injury. Perturbation of the immune response is implicated in the pathogenesis of perinatal brain injury; modulating this response could thus be a novel therapy for treating or preventing such injury. The immunomodulatory properties of hAECs have been shown in other animal models, but a detailed investigation of the effects on brain immune cells following injury has not been undertaken. Here, we investigate the effects of hAECs on microglia, the first immune responders to injury within the brain.MethodsWe generated a mouse model combining neonatal inflammation and perinatal hyperoxia, both of which are risk factors associated with perinatal brain injury. On embryonic day 16 we administered lipopolysaccharide (LPS), or saline (control), intra-amniotically to C57Bl/6 J mouse pups. On postnatal day (P)0, LPS pups were placed in hyperoxia (65% oxygen) and control pups in normoxia for 14 days. Pups were given either hAECs or saline intravenously on P4.ResultsAt P14, relative to controls, LPS and hyperoxia pups had reduced body weight, increased density of apoptotic cells (TUNEL) in the cortex, striatum and white matter, astrocytes (GFAP) in the white matter and activated microglia (CD68) in the cortex and striatum, but no change in total microglia density (Iba1). hAEC administration rescued the decreased body weight and reduced apoptosis and astrocyte areal coverage in the white matter, but increased the density of total and activated microglia. We then stimulated primary microglia (CD45lowCD11b+) with LPS for 24 h, followed by co-culture with hAEC conditioned medium for 48 h. hAEC conditioned medium increased microglial phagocytic activity, decreased microglia apoptosis and decreased M1 activation markers (CD86). Stimulating hAECs for 24 h with LPS did not alter release of cytokines known to modulate microglia activity.ConclusionsThese data demonstrate that hAECs can directly immunomodulate brain microglia, probably via release of trophic factors. This observation offers promise that hAECs may afford therapeutic utility in the management of perinatal brain injury.

THU-419 - GMP-level isolation, quality assessment and biosafety distribution evaluation on human amnion epithelial cell for cell-based therapies

THU-419 - GMP-level isolation, quality assessment and biosafety distribution evaluation on human amnion epithelial cell for cell-based therapies

Feto-Maternal Trafficking of Exosomes in Murine Pregnancy Models.

Timing and initiation of labor are well-orchestrated by signals communicated between the fetal and maternal compartments; however, how these signals are communicated is not completely understood. Fetal exosomes, intercellular signaling vesicles, may play a key role in the process. The objective of this study was to evaluate exosome trafficking in vivo from fetal to maternal compartments. Pregnant CD-1 mice were intra-amniotically injected on gestational day 16 and 17 with exosomes isolated from primary human amnion epithelial cells fluorescently labeled with the lipophilic dye 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR). All our analyses were performed on samples collected on Day 18. After 24 h, mice were imaged using Bruker MS FX PRO In vivo Imager and tissues were collected. In vivo imaging of mouse showed fluorescence in the uterus, on the exosome-injected side whereas the uterine tissues from the uninjected side and saline and dye alone injected animals remained negative. Histological analysis of placenta showed exosome migration from the fetal to the maternal side of the placenta. Fluorescence released from exosomes was seen in maternal blood samples as well as in maternal uterus and kidneys. This study demonstrates that exosomal cargo can be carried through systemic route from the fetal to the maternal side of the uterine tissues during pregnancy, supporting the idea that fetal signals can be delivered via exosomes.

Serelaxin improves the therapeutic efficacy of RXFP1-expressing human amnion epithelial cells in experimental allergic airway disease

Current asthma therapies primarily target airway inflammation (AI) and suppress episodes of airway hyperresponsiveness (AHR) but fail to treat airway remodelling (AWR), which can develop independently of AI and contribute to irreversible airway obstruction. The present study compared the anti-remodelling and therapeutic efficacy of human bone marrow-derived mesenchymal stem cells (MSCs) to that of human amnion epithelial stem cells (AECs) in the setting of chronic allergic airways disease (AAD), in the absence or presence of an anti-fibrotic (serelaxin; RLX). Female Balb/c mice subjected to the 9-week model of ovalbumin (OVA)-induced chronic AAD, were either vehicle-treated (OVA alone) or treated with MSCs or AECs alone [intranasally (i.n.)-administered with 1×106 cells once weekly], RLX alone (i.n.-administered with 0.8mg/ml daily) or a combination of MSCs or AECs and RLX from weeks 9-11 (n=6/group). Measures of AI, AWR and AHR were then assessed. OVA alone exacerbated AI, epithelial damage/thickness, sub-epithelial extracellular matrix (ECM) and total collagen deposition, markers of collagen turnover and AHR compared with that in saline-treated counterparts (all P<0.01 compared with saline-treated controls). RLX or AECs (but not MSCs) alone normalized epithelial thickness and partially diminished the OVA-induced fibrosis and AHR by ∼40-50% (all P<0.05 compared with OVA alone). Furthermore, the combination treatments normalized epithelial thickness, measures of fibrosis and AHR to that in normal mice, and significantly decreased AI. Although AECs alone demonstrated greater protection against the AAD-induced AI, AWR and AHR, compared with that of MSCs alone, combining RLX with MSCs or AECs reversed airway fibrosis and AHR to an even greater extent.

Induction of Amnion Epithelial Apoptosis by Cortisol via tPA/Plasmin System.

Rupture of fetal membranes (ROM) can initiate parturition at both term and preterm birth. Apoptosis of the amnion epithelium plays a key role in structural remodeling of the membranes preceding ROM. However, the causative factors for apoptosis remain unidentified. Toward the end of gestation, a feed-forward regeneration of cortisol via 11β-hydroxysteroid dehydrogenase 1 exists in the fetal membranes. Here, we have examined whether cortisol accumulation is a causative factor for amnion cells apoptosis. By using primary human amnion epithelial and fibroblast cells, we demonstrated cortisol induced apoptosis specifically in epithelial cells but not in fibroblasts via reciprocal regulation of tissue-type plasminogen activator (tPA)/plasmin system. Cortisol increased PLAT expression, the gene encoding tPA, via glucocorticoid receptor binding to a glucocorticoid response element in PLAT promoter, thereby increasing plasmin activity in epithelial cells. Further study revealed that a Fas-mediated extrinsic apoptotic pathway was involved in the induction of epithelial cells apoptosis by cortisol, which was blocked by inhibiting either tPA or plasmin. Consistently, cortisol increased cleaved-caspase-3 and tPA abundance in amnion tissue explants. Moreover, the abundance of cortisol, cleaved-caspase-3, and tPA was significantly increased in amnion tissue after labor-initiated spontaneous rupture of membranes. In conclusion, local accumulation of cortisol is a causative factor for amnion epithelial apoptosis via activation of tPA/plasmin system toward the end of gestation. This may contribute to the ROM at both term and preterm birth.

Human amnion epithelial cells expressing HLA-G as novel cell-based treatment for liver disease

Despite routine liver transplantation and supporting medical therapies, thousands of patients currently wait for an organ and there is an unmet need for more refined and widely available regenerative strategies to treat liver diseases. Cell transplants attempt to maximize the potential for repair and/or regeneration in liver and other organs. Over 40years of laboratory pre-clinical research and 25years of clinical procedures have shown that certain liver diseases can be treated by the infusion of isolated cells (hepatocyte transplant). However, like organ transplants, hepatocyte transplant suffers from a paucity of tissues useful for cell production. Alternative sources have been investigated, yet with limited success. The tumorigenic potential of pluripotent stem cells together with their primitive level of hepatic differentiation, have limited the use of stem cell populations.Stem cell sources from human placenta, and the amnion tissue in particular are receiving renewed interest in the field of regenerative medicine. Unlike pluripotent stem cells, human amnion epithelial (AE) cells are easily available without ethical or religious concerns; they do not express telomerase and are not immortal or tumorigenic when transplanted. In addition, AE cells have been reported to express genes normally expressed in mature liver, when transplanted into the liver. Moreover, because of the possibility of an immune-privileged status related to their expression of HLA-G, it might be possible to transplant human AE cells without immunosuppression of the recipient.

Therapeutic Use of Human Amnion-Derived Products: Cell-Based Therapy for Liver Disease

Since the early 20th century, the placenta has started to regain its status as tissue with great potential in regenerative medicine. In particular, cells isolated from the fetal membrane, amnion epithelial cells (AEC), have been described for their proficient capacity to support regeneration at several organs, primarily the liver. The investigation on regenerative and corrective potential of human AEC has been conducted on relevant preclinical models, showing a satisfactory level of liver regeneration and correction of inborn error of metabolism. When sufficient numbers of proficient cells are engrafted, phenotypic correction of a disease or at least conversion of a life-threatening disease into a milder and more manageable form is attained. Human AEC transplantation offers several advantages in liver cell-based approaches: AEC can be easily isolated by a wide array of donors; isolation and cryopreservation procedures commonly result in a high viability batch of cells, potentially “off the shelf” in every major medical center worldwide; AEC showed satisfactory levels of expression for multiple hepatic features when transplanted, and have been referred to as “immune-privileged” cells since they do not elicit a strong immune response once infused. Finally, unlike other stem cells, human AEC do not have the potential to form any kind of tumor once transplanted. Future and ongoing clinical studies are fundamental to unveil the true power of these immune-privileged cells that might dramatically change regenerative medicine applications.

Isolation of Human Amnion Epithelial Cells According to Current Good Manufacturing Procedures.

Different cell types can be isolated from human placental tissues, and some have been reported to retain phenotypic plasticity and characteristics that make them a promising source of cells for regenerative medicine. Among these are human amnion epithelial cells (hAECs). Adoption of current good manufacturing practices (cGMP) and enhanced quality control is essential when isolating hAECs in order to deliver a safe and effective cellular product for clinical purposes. This unit describes a detailed protocol for selective isolation of hAECs from human term placenta with little to no contamination by other cell types. A method for characterizing the heterogeneity of the hAEC suspension is also provided. The resulting cell product will be useful for clinical as well as basic research applications. © 2016 by John Wiley & Sons, Inc.

The combined effect of PDX1, epidermal growth factor and poly-L-ornithine on human amnion epithelial cells' differentiation.

BackgroundIt has been suggested that the ectopic expression of PDX1, a dominant pancreatic transcription factor, plays a critical role in the developmental programming of the pancreas even from cells of unrelated tissues such as keratinocytes and amniotic fluid stem cells. In this study we have chosen to drive pancreatic development in human amnion epithelial cells by inducing endogenous PDX1 expression. Further, we have investigated the role of Epidermal Growth Factor (EGF) and Poly-L-Ornithine (PLO) on this differentiation process.ResultsHuman amnion epithelial cells expressed high levels of endogenous PDX1 upon transduction with an adenoviral vector expressing murine Pdx1. Other markers of various stages of pancreatic differentiation such as NKX6.1, SOX17, RFX6, FOXA2, CFTR, NEUROD1, PAX4 and PPY were also expressed upon Pdx1 transduction.Although initial expression of pancreatic progenitor markers was higher in culture conditions lacking EGF, for a sustained and increased expression EGF was required. Culture on PLO further increased the positive impact of EGF.ConclusionPancreatic marker expression subsequent to mPdx1 transduction suggests that this approach may facilitate the in vitro differentiation of hAECs into cells of the endocrine pancreas. This result may have important implications in diabetes therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0108-y) contains supplementary material, which is available to authorized users.

Aquaporin 3 Expression Induced by Salvia Miltiorrhiza via ERK1/2 Signal Pathway in the Primary Human Amnion Epithelium Cells from Isolated Oligohydramnios.

Salvia miltiorrhiza is one of the most common Chinese herbal drugs, which is effective to treat oligohydramnios. In this study, the aim was to investigate how Salvia miltiorrhiza regulate aquaporin 3 expression in the human amnion epithelial cells (hAECs) with normal amniotic fluid volume or isolated oligohydramnios, whether via extracellular signal regulated kinase1/2 (ERK1/2) signal transduction pathway or not. Primary hAECs cultures from 120 patients were incubated with Salvia miltiorrhiza or/and ERK1/2 inhibitor-- U0126. Localization of aquaporin 3 was detected by immunohistochemistry and the expression of total ERK1/2, phospho-ERK1/2 (p-ERK1/2) and aquaporin 3 was detected by Western blot. The results were: (1) In hAECs with normal amniotic fluid volume, treatment with 10 µmol/L of U0126 for 6 h resulted in the optimal inhibition of p-ERK1/2 (P<0.05). However, the expression of total ERK1/2 or aquaporin 3 did not significantly change after different concentrations or time of U0126 treatment. Salvia miltiorrhiza significantly up-regulated aquaporin 3 expression, which was not affected by U0126. (2) In hAECs with isolated oligohydramnios, treatment with 5 μmol/L of U0126 for 2 h resulted in the optimal inhibition of p-ERK1/2 and the lowest expression of aquaporin 3 (P<0.05). Moreover, Salvia miltiorrhiza significantly up-regulated aquaporin 3 expression, which was obviously blocked by U0126. These results suggest that Salvia miltiorrhiza may regulate aquaporin 3 expression in hAECs. In addition, in hAECs with isolated oligohydramnios, Salvia miltiorrhiza may regulate the expression of aquaporin 3 via the ERK1/2 signal transduction pathway, which provides a novel thread to the improved treatment for isolated oligohydramnios.

Acid sphingomyelinase mediates 50-Hz magnetic field-induced EGF receptor clustering on lipid raft

Previously, we found that exposure to a 50-Hz magnetic field (MF) could induce epidermal growth factor receptor (EGFR) clustering and phosphorylation on cell surface. In order to explore the possible mechanisms, the roles of acid sphingomyelinase (ASMase) and lipid raft in MF-induced EGFR clustering were investigated in the present study. Human amnion epithelial (FL) cells were exposed to a 50-Hz MF at 0.4 mT for different durations. Intracellular ASMase activity was detected using the Amplex® Red Sphingomyelinase Assay Kit. EGFR clustering, ASMase, and lipid rafts on cell membrane were analyzed using confocal microscopy after indirect immunofluorescence staining. Results showed that disturbing lipid rafts with nystatin could inhibit MF-induced EGFR clustering, indicating that it was dependent on intact lipid raft. Exposure of FL cells to MF significantly enhanced ASMase activity and induced ASMase translocation to membrane that co-localized with lipid rafts. Treatment with imipramine, an ASMase inhibitor, inhibited the MF-induced EGFR clustering. This inhibitory effect could be blocked by the addition of C2-ceramide in the culture medium. It suggested that ASMase mediated the 50-Hz MF-induced EGFR clustering via ceramide which was produced from hydrolyzation on lipid rafts.

Evaluating the Impact of Human Amnion Epithelial Cells on Angiogenesis.

The effects of human amnion epithelial cells (hAECs) on angiogenesis remain controversial. It is yet unknown if the presence of inflammation and/or gestational age of hAEC donors have an impact on angiogenesis. In this study, we examined the differences between term and preterm hAECs on angiogenesis in vitro and in vivo. Conditioned media from term hAECs induced the formation of longer huVEC tubules on Matrigel. Both term and preterm hAECs expressed VEGFA, PDGFB, ANGPT1, and FOXC1, which significantly increased after TNFα and IFNγ stimulation. In the presence of TNFα and IFNγ, coculture with term hAECs reduced gene transcription of Tie-2 and Foxc1 in huVECs, while coculture with preterm hAECs increased gene transcription of PDGFRα and PDGFRβ and reduced gene transcription of FOXC1 in huVECs. In vivo assessment of angiogenesis using vWF immunostaining revealed that hAEC treatment decreased angiogenesis in a bleomycin model of lung fibrosis but increased angiogenesis in a neonatal model of hyperoxia-induced lung injury. In summary, our findings suggested that the impact of hAECs on angiogenesis may be influenced by the presence of inflammation and underlying pathology.

806: Multi-well plate immunoassay for measuring mitogen-activated protein kinase signaling in human amnion epithelial cells

806: Multi-well plate immunoassay for measuring mitogen-activated protein kinase signaling in human amnion epithelial cells

The Paracrine Effect of Transplanted Human Amniotic Epithelial Cells on Ovarian Function Improvement in a Mouse Model of Chemotherapy-Induced Primary Ovarian Insufficiency.

Human amnion epithelial cells (hAECs) transplantation via tail vein has been reported to rescue ovarian function in mice with chemotherapy-induced primary ovarian insufficiency (POI). To test whether intraperitoneally transplanted hAECs could induce therapeutic effect and to characterize the paracrine effect of transplanted hAECs, we utilized a chemotherapy induced mice model of POI and investigated the ability of hAECs and conditioned medium collected from cultured hAECs (hAECs-CM) to restore ovarian function. We found that transplantation of hAECs or hAECs-CM either 24 hours or 7 days after chemotherapy could increase follicle numbers and partly restore fertility. By PCR analysis of recipient mice ovaries, the presence of SRY gene was only detected in mice transplanted with male hAECs 24 hours following chemotherapy. Further, the gene expression level of VEGFR1 and VEGFR2 in the ovaries decreased, although VEGFA increased 2 weeks after chemotherapy. After treatment with hAECs or hAEC-CM, the expression of both VEGFR1 and VEGFR2 increased, consistent with the immunohistochemical analysis. In addition, both hAECs and hAECs-CM treatment enhanced angiogenesis in the ovaries. The results suggested that hAECs-CM, like hAECs, could partly restore ovarian function, and the therapeutic function of intraperitoneally transplanted hAECs was mainly induced by paracrine-mediated ovarian protection and angiogenesis.

Telomere Fragment Induced Amnion Cell Senescence: A Contributor to Parturition?

Oxidative stress (OS)-induced senescence of the amniochorion has been associated with parturition at term. We investigated whether telomere fragments shed into the amniotic fluid (AF) correlated with labor status and tested if exogenous telomere fragments (T-oligos) could induce human and murine amnion cell senescence. In a cross-sectional clinical study, AF telomere fragment concentrations quantitated by a validated real-time PCR assay were higher in women in labor at term compared to those not in labor. In vitro treatment of primary human amnion epithelial cells with 40 μM T-oligos ([TTAGGG]2) that mimic telomere fragments, activated p38MAPK, produced senescence-associated (SA) β-gal staining and increased interleukin (IL)-6 and IL-8 production compared to cells treated with complementary DNA sequences (Cont-oligos, [AATCCC]2). T-oligos injected into the uteri of pregnant CD1 mice on day 14 of gestation, led to increased p38MAPK, SA-β-gal (SA β-gal) staining in murine amniotic sacs and higher AF IL-8 levels on day 18, compared to saline treated controls. In summary, term labor AF samples had higher telomere fragments than term not in labor AF. In vitro and in situ telomere fragments increased human and murine amnion p38MAPK, senescence and inflammatory cytokines. We propose that telomere fragments released from senescent fetal cells are indicative of fetal cell aging. Based on our data, these telomere fragments cause oxidative stress associated damages to the term amniotic sac and force them to release other DAMPS, which, in turn, provide a sterile immune response that may be one of the many inflammatory signals required to initiate parturition at term.

Human amnion epithelial cells improved pulmonary hypertension associated with bronchopulmonary dysplasia

Human amnion epithelial cells improved pulmonary hypertension associated with bronchopulmonary dysplasia

Immunosuppressive potential of human amnion epithelial cells in the treatment of experimental autoimmune encephalomyelitis.

BackgroundMultiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). In recent years, it has been found that cells such as human amnion epithelial cells (hAECs) have the ability to modulate immune responses in vitro and in vivo and can differentiate into multiple cell lineages. Accordingly, we investigated the immunoregulatory effects of hAECs as a potential therapy in an MS-like disease, EAE (experimental autoimmune encephalomyelitis), in mice.MethodsUsing flow cytometry, the phenotypic profile of hAECs from different donors was assessed. The immunomodulatory properties of hAECs were examined in vitro using antigen-specific and one-way mixed lymphocyte proliferation assays. The therapeutic efficacy of hAECs was examined using a relapsing-remitting model of EAE in NOD/Lt mice. T cell responsiveness, cytokine secretion, T regulatory, and T helper cell phenotype were determined in the peripheral lymphoid organs and CNS of these animals.ResultsIn vitro, hAECs suppressed both specific and non-specific T cell proliferation, decreased pro-inflammatory cytokine production, and inhibited the activation of stimulated T cells. Furthermore, T cells retained their naïve phenotype when co-cultured with hAECs. In vivo studies revealed that hAECs not only suppressed the development of EAE but also prevented disease relapse in these mice. T cell responses and production of the pro-inflammatory cytokine interleukin (IL)-17A were reduced in hAEC-treated mice, and this was coupled with a significant increase in the number of peripheral T regulatory cells and naïve CD4+ T cells. Furthermore, increased proportions of Th2 cells in the peripheral lymphoid organs and within the CNS were observed.ConclusionThe therapeutic effect of hAECs is in part mediated by inducing an anti-inflammatory response within the CNS, demonstrating that hAECs hold promise for the treatment of autoimmune diseases like MS.

Derivation and characterization of human embryonic stem cells on human amnion epithelial cells

Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs.