Aislación y caracterización de las células epiteliales del amnios / Isolation and characterization of amniotic ephitelial cells

RESUMEN: Las células epiteliales del amnios (hAECs) son células madre pluripotenciales; tienen capacidad de diferenciarse en células de las tres capas embrionarias. Como tales, se utilizan en algunas terapias regenerativas en medicina. Este estudio tiene por objetivo describir un protocolo de aislación de las células epiteliales del amnios (hAECs) a partir de placentas humanas de partos por cesárea, así como su caracterización y comportamiento in vitro. Se aislaron hAECs de 20 placentas de partos por cesárea con un protocolo optimizado. Se caracterizaron las células mediante citometría de flujo, microscopia óptica y de fluorescencia, y se evaluó la proliferación de las células mediante MTT a los 1, 3, 5 y 7 días con y sin β-mercaptoetanol en el medio de cultivo. El análisis histológico del amnios mostró un desprendimiento prácticamente completo de las células después de la segunda digestión del amnios. El promedio de células obtenidas fue de 10.97 millones de células por gramo de amnios. Las hAECs mostraron una proliferación limitada, la cual no fue favorecida por la adición de β-mercaptoetanol en el cultivo. Se observó un cambio de morfología espontanea de epitelial a mesenquimal después del cuarto pasaje. Las células epiteliales del amnios pueden ser aisladas con un protocolo simple y efectivo, sin embargo, presentan escasa capacidad proliferativa. Bajo las condiciones de este estudio, la adición de β-mercaptoetanol no favorece la capacidad proliferativa de las células.

SUMMARY: human amnion epithelial cells (hAECs) are pluripotent stem cells; they have the ability to differentiate into cells of the three embryonic layers, and are used in various regenerative therapies in medicine. This study aims to describe a protocol for the isolation of amnion epithelial cells (hAECs) from human placentas from cesarean delivery, as well as their characterization and culture conditions in vitro. hAECs were isolated from 20 cesarean delivery placentas with an optimized protocol. The cells were characterized by flow cytometry, light and fluorescence microscopy, and the proliferation of the cells was evaluated by MTT at 1, 3, 5 and 7 days with and without β-mercaptoethanol in the culture medium. Histological analysis of the amnion showed a practically complete detachment of the cells of the underlying membrane after the second digestion. The average number of cells obtained was 10.97 million cells per amnion. The hAECs perform a limited proliferation rate, which was not favored by the addition of β-mercaptoethanol in the culture. A spontaneous morphology change from epithelial to mesenchymal morphology is exhibited after the fourth passage. The epithelial cells of the amnion can be isolated with a simple and effective protocol, however, they present little proliferative capacity. Under the conditions of this study, the addition of β-mercaptoethanol does not favor the proliferation of the cells.

Comparação dos meios de preparação e preservação de membrana amniótica humana para uso no tratamento de doenças da superfície ocular / Comparison of the preparation and preservation techniques of amniotic membrane used in the treatment of ocular surface diseases

Resumo Atualmente a membra amniótica (MA) tem obtido importância devido à comprovada capacidade de reduzir inflamação, auxiliar a cicatrização e epitelização, possuindo propriedades antimicrobianas e antivirais, além de baixa imunogenicidade. As indicações de seu uso na oftalmologia têm aumentado muito nas duas últimas décadas. Objetivo: Descrever a estrutura básica e as propriedades biológicas da MA em relação aos componentes da sua matriz extracelular e fatores de crescimento, as consequências de diferentes técnicas empregadas na sua preservação e esterilização, métodos para remoção do epitélio e a comparação dos custos dos diferentes meios de conservação atualmente empregados. Métodos: Pesquisa nas bases de dados do Portal da Biblioteca Virtual em Saúde (BVS), Pubmed, Cochrane, Scielo e Lilacs com as palavras-chave: membrana amniótica, transplante, reconstrução da córnea, doenças da conjuntiva. Resultados: A literatura é vasta na descrição dos efeitos de diversos agentes e técnicas na preparação da MA, dentre elas sua preservação, esterilização e desepitelização. A membrana desnuda tem sido a escolha para a reconstrução da superfície ocular, pois facilita a cicatrização. Em relação aos agentes conservantes, o glicerol é o meio mais utilizado mundialmente pelo baixo custo e facilidade de manuseio. Conclusão: A comparação das diversas técnicas nos guia na elaboração de protocolos de preparo da MA para uso oftalmológico. A membrana desnuda facilita a cicatrização em relação a com células epiteliais. O glicerol é o meio de conservação mais utilizado pelo baixo custo e facilidade de manuseio.

Abstract Currently, the amniotic membrane (AM) has obtained importance due to its ability to reduce inflammation, helping in the healing and epithelialization processes, having antimicrobial and antiviral properties and low immunogenicity. Its indications in ophthalmology have increased considerably in the past two decades. Objective: To describe the basic structure and biological properties of the AM, the components of the extracellular matrix and growth factors, the consequences of different techniques used in its preservation, and sterilization methods for the epithelium removal. To compare the costs of the different preservation solutions currently employed. Study design: literature review. Methods: Research in BVS databases, PubMed, Cochrane, Scielo and Lilacs with keywords: amniotic membrane transplantation, corneal reconstruction, conjunctival diseases. Results: The literature is vast in describing the effects of different agents and techniques used in the preparation of MA, including its preservation, sterilization and desepithelization. The naked membrane is the choice to reconstruct the ocular surface, as it facilitates the healing course. Regarding the preservatives, glycerol is the most used worldwide due its low cost and easy handling. Conclusion: Comparing different techniques guides us in developing a MA preparation protocol for ophthalmic use. The naked membrane facilitates the healing process compared with the presence of epithelial cells. The glycerol is the most used preservation method because of its low cost and easy handling.

Ureaplasma urealyticum-derived lipid-associated membrane proteins introduce IL-6, IL-8, and TNF-α cytokines into human amniotic epithelial cells via Toll-like receptor 2 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#The purpose of this study was to determine the role of Ureaplasma urealyticum-derived lipid-associated membrane proteins (LAMPs) in the host innate immune system, specifically their effect on Toll-like receptors (TLRs).@*METHODS@#LAMPs were derived from U. urealyticum strains, and human amniotic epithelial cells (HAECs) were isolated from healthy full-term placentas. Cytokine concentrations were determined by enzyme-linked immunosorbent assay (ELISA) and TLR2 mRNA by real-time PCR. Expression of TLR2 was confirmed by Western blotting and immunohistochemistry.@*RESULTS@#LAMPs induced HAECs to produce inflammatory cytokines interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α. Cytokine production was reduced after blocking TLR2 using TLR2 inhibitor (anti-hTLR2-IgA).@*CONCLUSIONS@#LAMPs isolated from U. urealyticum induced TLR2-dependent up-regulation of inflammatory genes and cytokines in HAECs.

Human amnion mesenchymal cells inhibit lipopolysaccharide-induced TNF-α and IL-1ß production in THP-1 cells

BACKGROUND: Human amnion mesenchymal cells (hAMCs), isolated from the amniotic membrane of human placenta, are a unique population of mesenchymal stem cells. Recent studies demonstrated that hAMCs could inhibit the activities and functions of several immune cells. However, their effect on inflammatory macrophages is largely unknown. This study investigated the effect of hAMCs on expression of inflammatory cytokines and mitogen-activated protein kinases (MAPKs)/NF-kB pathway in human THP-1 macrophages induced by lipopolysaccharide (LPS). RESULTS: The levels of TNF-α and IL-1ß secreted by LPS- stimulated THP-1 cells were increased significantly compared with those in the control group. After co-culture with different numbers of hAMCs, the levels of TNF-α and IL-1ß in LPS-stimulated THP-1 cells were significantly reduced compared with the LPS group. The mRNA expression of TNF-α and IL-1ß were also markedly inhibited. Moreover, treating LPS-stimulated THP-1 cells with hAMCs supernatants could also suppress TNF-α and IL-1ß production in THP-1 cells. Important signaling pathways involved in the production of TNF-α and IL-1ß were affected by hAMCs co-culture: hAMCs remarkably suppressed NF-kB activation and down-regulated the phosphorylation of ERK and JNK in LPS- stimulated THP-1 cells. CONCLUSIONS: Human amnion mesenchymal cells inhibited the production of TNF-α and IL-1ß secreted by LPS-stimulated THP-1 cells, partly through the suppression of NF-kB activation and ERK and JNK phosphorylation.

Effects of conditioned media from human amniotic epithelial cells on corneal alkali injuries in rabbits

This study was performed to evaluate the effects of conditioned media (CM) from human amniotic epithelial cells (HAECs) on the corneal wound healing process. Eighteen rabbits (36 eyes) were used and randomly assigned to three groups according treatment: CM from HAECs (group 1), vehicle alone (group 2), and saline (group 3). Corneal alkali injuries were induced with 1 N sodium hydroxide. Each reagent used for treatment evaluation was injected into the dorsal bulbar subconjunctiva and the area of the corneal epithelial defect was measured every other day. Two animals from each group were euthanized at a time on days 3, 7, and 15, and the cornea was removed for histological examination. The sum of the epithelial defect areas measured on day 0 to day 6 as well as day 0 to day 14 in group 1 was significantly smaller than those of other groups. Histological examination revealed that the group 1 corneas had less inflammatory cell infiltration and showed more intact epithelial features compared to the other groups. These results suggest that CM from HAECs promote corneal wound healing in rabbits.

Isolation and characterization of equine amniotic membrane-derived mesenchymal stem cells

Recent studies have shown that mesenchymal stem cells (MSCs) are able to differentiate into multi-lineage cells such as adipocytes, chondroblasts, and osteoblasts. Amniotic membrane from whole placenta is a good source of stem cells in humans. This membrane can potentially be used for wound healing and corneal surface reconstruction. Moreover, it can be easily obtained after delivery and is usually discarded as classified waste. In the present study, we successfully isolated and characterized equine amniotic membrane-derived mesenchymal stem cells (eAM-MSCs) that were cultured and maintained in low glucose Dulbecco's modified Eagle's medium. The proliferation of eAM-MSCs was measured based on the cumulative population doubling level (CPDL). Immunophenotyping of eAM-MSCs by flow cytometry showed that the major population was of mesenchymal origin. To confirm differentiation potential, a multi-lineage differentiation assay was conducted. We found that under appropriate conditions, eAM-MSCs are capable of multi-lineage differentiation. Our results indicated that eAM-MSCs may be a good source of stem cells, making them potentially useful for veterinary regenerative medicine and cell-based therapy.

Immunomodulatory effects of human amniotic membrane-derived mesenchymal stem cells

Human amniotic membrane-derived mesenchymal stem cells (hAM-MSCs) are capable of differentiating into several lineages and possess immunomodulatory properties. In this study, we investigated the soluble factor-mediated immunomodulatory effects of hAM-MSCs. Mitogen-induced peripheral blood mononuclear cell (PBMC) proliferation was suppressed by hAM-MSCs in a dose-dependent manner as well as hAM-MSC culture supernatant. Moreover, interferon-gamma and interleukin (IL)-17 production significantly decreased from PBMC, whereas IL-10 from PBMCs and transforming growth factor beta (TGF-beta) production from hAM-MSCs significantly increased in co-cultures of hAM-MSCs and PBMCs. Production of several MSC factors, including hepatocyte growth factor (HGF), TGF-beta, prostaglandin E2 (PGE2), and indoleamine 2, 3 dioxygenase (IDO), increased significantly in hAM-MSCs co-cultured with PBMCs. These results indicate that the immunomodulatory effects of hAM-MSCs may be associated with soluble factors (TGF-beta, HGF, PGE2, and IDO), suggesting that hAM-MSCs may have potential clinical use in regenerative medicine.

[ effects of FGF8 and Shh on expression of dopaminergic markers from human amniotic epithelial cells]

Recently, interest has increased in the potential employment of embryonic stem cells for the treatment of Parkinson's disease, which has been considered as an alternative therapeutic strategy. Due to their pluripotent differentiation potential, the finding that they do not induce carcinoma and the fact that they do not raise the ethical concerns connected with human embryonic stem cells, human amniotic epithelial cells are considered to be a very promising cell source. The aim of this study was to investigate the effects of FGF8 and Shh on the expression of dopaminergic markers from human amniotic epithelial cells. In this study, we examined the differentiation of dopaminergic neurons in vitro from AECs using the expression of several markers including TH, DAT and D beta H. For dopaminergic differentiation, sonic hedgehog [Shh] and FGF8 were added to cultures and the cultures were allowed to differentiate for 21 days. Analysis of AECs derived dopaminergic neurons was performed at the TH, DAT, beta-tubulin III and D beta H expression levels by immunocitochemistry. The significance of the data was tested by Student's t-test [between two groups] and one-way analysis of variance [ANOVA] followed by Tukey post-test. [p<0/01, p<0/05]. Combination of Shh and FGF8 showed the higher level of TH in comparison to control group or these factors alone. Moreover, Shh is more effective than FGF8 on DAT expression in comparison to expression of D beta H. These results show the capability of AECs to express dopaminergic neural markers and this ability is affected by Shh and FGF8

Novel neurotrophic factor secreted by amniotic epithelial cells

By virtue of expressions of glial and neural surface markers and capability of neurotransmitter metabolism, amniotic epithelial cells are considered as candidate cell type for transplantation strategies to treat neurological disorders. Previously, we have reported neurotrophism exhibited by human amniotic epithelial cells when transplanted after spinal cord injury in bonnet monkeys. Amniotic epithelial cells were believed to secrete an "Epidermal Growth Factor (EGF)-like" factor and exact identification was not made. At this juncture, through the present study it was found that, chicken neural retinal cells when grown alone failed to survive and contrarily when either co-cultured with chicken amniotic epithelial cells/cultured in amniotic epithelial cell conditioned medium not only survived but also showed extensive differentiation. Fibroblast Growth Factor-2 (FGF-2) plays a critical role in retinal development especially in chicken neural retinal development. However, immunoassay using western blot did not revealed the presence of any already known isoforms of FGF-2 in the medium. It is interesting to note that while factor secreted by amniotic epithelial cells resembles EGF and/or FGF-2 in its biological action, known isoforms of them were not detected. Considering the biological closeness between EGF and FGF-2, results indicate the possibility of a novel isoform of these growth factors secreted by amniotic epithelial cells. Further studies will establish the nature of this novel factor which will enhance the application of this interesting cell type for neural transplantations.