RESUMO
Fetal membrane (amniochorion), the innermost lining of the intrauterine cavity, surround the fetus and enclose amniotic fluid. Unlike unidirectional blood flow, amniotic fluid subtly rocks back and forth, and thus, the innermost amnion epithelial cells are continuously exposed to low levels of shear stress from fluid undulation. Here, we tested the impact of fluid motion on amnion epithelial cells (AECs) as a bearer of force impact and their potential vulnerability to cytopathologic changes that can destabilize fetal membrane functions. A previously developed amnion membrane (AM) organ-on-chip (OOC) was utilized but with dynamic flow to culture human fetal amnion membrane cells. The applied flow was modulated to perfuse culture media back and forth for 48 h to mimic fluid motion. A static culture condition was used as a negative control, and oxidative stress (OS) condition was used as a positive control representing pathophysiological changes. The impacts of fluidic motion were evaluated by measuring cell viability, cellular transition, and inflammation. Additionally, scanning electron microscopy (SEM) imaging was performed to observe microvilli formation. The results show that regardless of the applied flow rate, AECs and AMCs maintained their viability, morphology, innate meta-state, and low production of pro-inflammatory cytokines. E-cadherin expression and microvilli formation in the AECs were upregulated in a flow rate-dependent fashion; however, this did not impact cellular morphology or cellular transition or inflammation. OS treatment induced a mesenchymal morphology, significantly higher vimentin to cytokeratin 18 (CK-18) ratio, and pro-inflammatory cytokine production in AECs, whereas AMCs did not respond in any significant manner. Fluid motion and shear stress, if any, did not impact AEC cell function and did not cause inflammation. Thus, when using an amnion membrane OOC model, the inclusion of a dynamic flow environment is not necessary to mimic in utero physiologic cellular conditions of an amnion membrane.
Assuntos
Líquido Amniótico , Membranas Extraembrionárias , Dispositivos Lab-On-A-Chip , Humanos , Líquido Amniótico/citologia , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/metabolismo , Âmnio/citologia , Âmnio/metabolismo , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Movimento (Física) , Estresse Oxidativo , Modelos Biológicos , Sistemas MicrofisiológicosRESUMO
PROBLEM: This study localized CD45+ immune cells and compared changes in their numbers between term, not in labor (TNIL) and term, labor (TL) human fetal membranes. METHOD OF STUDY: Fetal membranes (amniochorion) from normal TNIL and TL subjects were analyzed by immunohistochemistry (IHC), immunofluorescence (IF), and flow cytometry for evidence of total (CD45+ ) immune cells as well as innate immune cells (neutrophils, macrophages and NK cells) using specific markers. Fetal origin of immune cells was determined using polymerase chain reaction (PCR) for SRY gene in Y chromosome. RESULTS: CD45+ cells were localized in human fetal membranes for both TNIL and TL. A threefold increase in CD45+ cells was seen in TL fetal membranes of (7.73% ± 2.35) compared to TNIL (2.36% ± 0.78). This increase is primarily contributed by neutrophils. Macrophages and NK cells did not change in the membranes between TNIL and TL. Leukocytes of fetal origin are present in the fetal membranes. CONCLUSION: The fetal membranes without decidua contain a small proportion of immune cells. Some of these immune cells in the fetal membrane are fetal in origin. There is a moderate increase of immune cells in the fetal membranes at term labor; however, it is unclear whether this is a cause or consequence of labor. Further functional studies are needed to determine their contribution to membrane inflammation associated with parturition.
Assuntos
Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/imunologia , Antígenos Comuns de Leucócito/imunologia , Leucócitos/imunologia , Macrófagos/imunologia , Feminino , Humanos , MasculinoRESUMO
The placenta is a temporary organ that is discarded after birth and is one of the most promising sources of various cells and tissues for use in regenerative medicine and tissue engineering, both in experimental and clinical settings. The placenta has unique, intrinsic features because it plays many roles during gestation: it is formed by cells from two individuals (mother and fetus), contributes to the development and growth of an allogeneic fetus, and has two independent and interacting circulatory systems. Different stem and progenitor cell types can be isolated from the different perinatal tissues making them particularly interesting candidates for use in cell therapy and regenerative medicine. The primary source of perinatal stem cells is cord blood. Cord blood has been a well-known source of hematopoietic stem/progenitor cells since 1974. Biobanked cord blood has been used to treat different hematological and immunological disorders for over 30 years. Other perinatal tissues that are routinely discarded as medical waste contain non-hematopoietic cells with potential therapeutic value. Indeed, in advanced perinatal cell therapy trials, mesenchymal stromal cells are the most commonly used. Here, we review one by one the different perinatal tissues and the different perinatal stem cells isolated with their phenotypical characteristics and the preclinical uses of these cells in numerous pathologies. An overview of clinical applications of perinatal derived cells is also described with special emphasis on the clinical trials being carried out to treat COVID19 pneumonia. Furthermore, we describe the use of new technologies in the field of perinatal stem cells and the future directions and challenges of this fascinating and rapidly progressing field of perinatal cells and regenerative medicine.
Assuntos
COVID-19/terapia , Placenta/citologia , SARS-CoV-2 , Transplante de Células-Tronco/tendências , Células-Tronco/citologia , Líquido Amniótico/citologia , Ensaios Clínicos como Assunto , Transplante de Células-Tronco de Sangue do Cordão Umbilical/métodos , Transplante de Células-Tronco de Sangue do Cordão Umbilical/tendências , Síndrome da Liberação de Citocina/terapia , Portadores de Fármacos , Membranas Extraembrionárias/citologia , Feminino , Previsões , Células-Tronco Hematopoéticas/citologia , Humanos , Pulmão/patologia , Ativação de Macrófagos , Células-Tronco Mesenquimais/citologia , Nanopartículas , Gravidez , Preservação Biológica , Medicina Regenerativa/métodos , Transplante de Células-Tronco/métodos , Células-Tronco/imunologiaRESUMO
OBJECTIVE: Fetal membranes, a vital component that helps maintain pregnancy and contribute to parturition signaling, are often studied in segments due to its structural complexity. Transwells are traditionally used to study cell interactions; however, their usefulness is limited. To overcome these difficulties, a fetal membrane-organ-on-chip (FM-OO-C) was created to study interactive properties of amnion epithelial cells (AECs) and decidual cells compared to transwell systems. METHODS: Primary AECs and decidual cells from term, nonlaboring fetal membranes were cultured in a 2-chamber (AEC/decidual cell) FM-OO-C device and sandwiched between a semipermeable membrane. Cells were treated with cigarette smoke extract (CSE) or dioxin, and membrane permeability and cellular senescence were measured after 48 hours. The same experiments were conducted in transwells for comparisons. RESULTS: Compared to transwell cultures, FM-OO-C model produced better membrane permeability readings regardless of the side of treatment or time point. Membrane permeabilization was higher in AECs directly treated with CSE (1.6 fold) compared to similar treatment on the decidual side (1.2 fold). In FM-OO-C, treatments forced changes between cellular layers. This was evident when CSE and dioxin-induced senescence on one side of the chamber produced similar changes on the opposite side. This effect was minimal in the transwell system. CONCLUSION: The controlled environment of an FM-OO-C allows for improved signal propagation between cells by minimizing noise and highlighting the small changes between treatments that cannot be seen in conventional transwell devices. Fetal membrane-organ-on-chip provides a better interaction between cell types that can be used to study fetal-maternal signaling during pregnancy in future studies.
Assuntos
Comunicação Celular/fisiologia , Técnicas de Cultura de Células/métodos , Células Epiteliais/fisiologia , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/fisiologia , Técnicas Analíticas Microfluídicas/métodos , Âmnio/citologia , Âmnio/fisiologia , HumanosRESUMO
Large animals play important roles as model animals for biomedical sciences and translational research. The water buffalo (Bubalus bubalis) is an economically important, multipurpose livestock species. Important assisted reproduction techniques, such as in vitro fertilization, cryo-conservation of sperm and embryos, embryo transfer, somatic cell nuclear transfer, genetic engineering, and genome editing have been successfully applied to buffaloes. Recently, detailed whole genome data and transcriptome maps have been generated. In addition, rapid progress has been made in stem cell biology of the buffalo. Apart from embryonic stem cells, bubaline extra-embryonic stem cells have gained particular interest. The multipotency of non-embryonic stem cells has been revealed, and their utility in basic and applied research is currently investigated. In particular, success achieved in bubaline extra-embryonic stem cells may have important roles in experimental biology and therapeutic regenerative medicine. Progress in other farm animals in assisted reproduction techniques, stem cell biology and genetic engineering, which could be of importance for buffalo, will also be briefly summarized.
Assuntos
Búfalos/embriologia , Búfalos/genética , Membranas Extraembrionárias/citologia , Células-Tronco Mesenquimais/fisiologia , Animais , Genoma , TranscriptomaRESUMO
The pivotal role of inflammatory processes in human parturition is well known, but not completely understood. We have performed a study to examine the role of macrophage-inducible C-type lectin (Mincle) in inflammation-associated parturition. Using human samples, we show that spontaneous labour is associated with up-regulated Mincle expression in the myometrium and fetal membranes. Mincle expression was also increased in fetal membranes and myometrium in the presence of pro-labour mediators, the proinflammatory cytokines interleukin (IL)-1B and tumour necrosis factor (TNF), and Toll-like receptor (TLR) ligands fsl-1, poly(I:C), lipopolysaccharide (LPS) and flagellin. These clinical studies are supported by mouse studies, where an inflammatory challenge in a mouse model of preterm birth increased Mincle expression in the uterus. Importantly, elimination of Mincle decreased the effectiveness of proinflammatory cytokines and TLR ligands to induce the expression of pro-labour mediators; namely, proinflammatory cytokines and chemokines, contraction-associated proteins and prostaglandins, and extracellular matrix remodelling enzymes, matrix metalloproteinases. The data presented in this study suggest that Mincle is required when inflammatory activation precipitates parturition.
Assuntos
Membranas Extraembrionárias/imunologia , Lectinas Tipo C/imunologia , Miométrio/imunologia , Parto/imunologia , Receptores Imunológicos/imunologia , Animais , Quimiocinas/metabolismo , Citocinas/metabolismo , Matriz Extracelular/enzimologia , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Técnicas In Vitro , Mediadores da Inflamação/metabolismo , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/genética , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Miométrio/citologia , Miométrio/metabolismo , Parto/genética , Parto/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/genética , Receptores de Reconhecimento de Padrão/metabolismo , Regulação para CimaRESUMO
BACKGROUND: Preterm premature rupture of membranes is a leading contributor to maternal and neonatal morbidity and death. Epidemiologic and experimental studies have demonstrated that thrombin causes fetal membrane weakening and subsequently preterm premature rupture of membranes. Although blood is suspected to be the likely source of thrombin in fetal membranes and amniotic fluid of patients with preterm premature rupture of membranes, this has not been proved. Ureaplasma parvum is emerging as a pathogen involved in prematurity, which includes preterm premature rupture of membranes; however, until now, prothrombin production that has been induced directly by bacteria in fetal membranes has not been described. OBJECTIVE: This study was designed to investigate whether Ureaplasma parvum exposure can induce prothrombin production in fetal membranes cells. STUDY DESIGN: Primary fetal membrane cells (amnion epithelial, chorion trophoblast, and decidua stromal) or full-thickness fetal membrane tissue explants from elective, term, uncomplicated cesarean deliveries were harvested. Cells or tissue explants were infected with live Ureaplasma parvum (1×105, 1×106 or 1×107 colony-forming units per milliliter) or lipopolysaccharide (Escherichia coli J5, L-5014; Sigma Chemical Company, St. Louis, MO; 100 ng/mL or 1000 ng/mL) for 24 hours. Tissue explants were fixed for immunohistochemistry staining of thrombin/prothrombin. Fetal membrane cells were fixed for confocal immunofluorescent staining of the biomarkers of fetal membrane cell types and thrombin/prothrombin. Protein and messenger RNA were harvested from the cells and tissue explants for Western blot or quantitative reverse transcription polymerase chain reaction to quantify thrombin/prothrombin protein or messenger RNA production, respectively. Data are presented as mean values ± standard errors of mean. Data were analyzed using 1-way analysis of variance with post hoc Dunnett's test. RESULTS: Prothrombin production and localization were confirmed by Western blot and immunostainings in all primary fetal membrane cells and tissue explants. Immunofluorescence observations revealed a perinuclear localization of prothrombin in amnion epithelial cells. Localization of prothrombin in chorion and decidua cells was perinuclear and cytoplasmic. Prothrombin messenger RNA and protein expression in fetal membranes were increased significantly by Ureaplasma parvum, but not lipopolysaccharide, treatments in a dose-dependent manner. Specifically, Ureaplasma parvum at a dose of 1×107 colony-forming units/mL significantly increased both prothrombin messenger RNA (fold changes in amnion: 4.1±1.9; chorion: 5.7±4.2; decidua: 10.0±5.4; fetal membrane: 9.2±3.0) and protein expression (fold changes in amnion: 138.0±44.0; chorion: 139.6±15.1; decidua: 56.9±29.1; fetal membrane: 133.1±40.0) compared with untreated control subjects. Ureaplasma parvum at a dose of 1×106 colony-forming units/mL significantly up-regulated prothrombin protein expression in chorion cells (fold change: 54.9±5.3) and prothrombin messenger RNA expression in decidua cells (fold change: 4.4±1.9). CONCLUSION: Our results demonstrate that prothrombin can be produced directly by fetal membrane amnion, chorion, and decidua cells. Further, prothrombin production can be stimulated by Ureaplasma parvum exposure in fetal membranes. These findings represent a potential novel underlying mechanism of Ureaplasma parvum-induced rupture of fetal membranes.
Assuntos
Células Epiteliais/metabolismo , Membranas Extraembrionárias/metabolismo , Ruptura Prematura de Membranas Fetais/genética , Protrombina/genética , Células Estromais/metabolismo , Trombina/genética , Trofoblastos/metabolismo , Infecções por Ureaplasma/genética , Âmnio/citologia , Western Blotting , Córion/citologia , Decídua/citologia , Membranas Extraembrionárias/citologia , Feminino , Ruptura Prematura de Membranas Fetais/metabolismo , Ruptura Prematura de Membranas Fetais/microbiologia , Humanos , Técnicas In Vitro , Lipopolissacarídeos , Gravidez , Protrombina/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Trombina/metabolismo , Ureaplasma , Infecções por Ureaplasma/metabolismo , Infecções por Ureaplasma/microbiologiaRESUMO
OBJECTIVE: To compare four enzymatic protocols for mesenchymal stem cells (MSCs) isolation from amniotic (A-MSC) and chorionic (C-MSC) membranes, umbilical cord (UC-MSC) and placental decidua (D-MSC) in order to define a robust, practical and low-cost protocol for each tissue. RESULTS: A-MSCs and UC-MSCs could be isolated from all samples using trypsin/collagenase-based protocols; C-MSCs could be isolated from all samples with collagenase- and trypsin/collagenase-based protocols; D-MSCs were isolated from all samples exclusively with a collagenase-based protocol. CONCLUSIONS: The trypsin-only protocol was least efficient; the collagenase-only protocol was best for C-MSCs and D-MSCs; the combination of trypsin and collagenase was best for UC-MSCs and none of tested protocols was adequate for A-MSCs isolation.
Assuntos
Separação Celular/métodos , Membranas Extraembrionárias/citologia , Células-Tronco Mesenquimais/citologia , Placenta/citologia , Cordão Umbilical/citologia , Proliferação de Células , Células Cultivadas , Colagenases , Feminino , Humanos , Cinética , Gravidez , TripsinaRESUMO
The clinical application of the fetal membranes dates back to nearly a century. Their use has ranged from superficial skin dressings to surgical wound closure. The applications of the fetal membranes are constantly evolving, and key to this is the uncovering of multiple populations of stem and stem-like cells, each with unique properties that can be exploited for regenerative medicine. In addition to pro-angiogenic and immunomodulatory properties of the stem and stem-like cells arising from the fetal membranes, the dehydrated and/or decellularized forms of the fetal membranes have been used to support the growth and function of other cells and tissues, including adipose-derived mesenchymal stem cells. This concise review explores the biological origin of the fetal membranes, a history of their use in medicine, and recent developments in the use of fetal membranes and their derived stem and stem-like cells in regenerative medicine. Stem Cells Translational Medicine 2017;6:1767-1776.
Assuntos
Células-Tronco Embrionárias/citologia , Membranas Extraembrionárias/citologia , Medicina Regenerativa/métodos , Animais , Células-Tronco Embrionárias/metabolismo , Membranas Extraembrionárias/metabolismo , Humanos , Transplante de Células-Tronco/métodosRESUMO
Direct lineage reprogramming, including with small molecules, has emerged as a promising approach for generating desired cell types. We recently found that during chemical induction of induced pluripotent stem cells (iPSCs) from mouse fibroblasts, cells pass through an extra-embryonic endoderm (XEN)-like state. Here, we show that these chemically induced XEN-like cells can also be induced to directly reprogram into functional neurons, bypassing the pluripotent state. The induced neurons possess neuron-specific expression profiles, form functional synapses in culture, and further mature after transplantation into the adult mouse brain. Using similar principles, we were also able to induce hepatocyte-like cells from the XEN-like cells. Cells in the induced XEN-like state were readily expandable over at least 20 passages and retained genome stability and lineage specification potential. Our study therefore establishes a multifunctional route for chemical lineage reprogramming and may provide a platform for generating a diverse range of cell types via application of this expandable XEN-like state.
Assuntos
Reprogramação Celular , Endoderma/citologia , Membranas Extraembrionárias/citologia , Fibroblastos/metabolismo , Envelhecimento , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Diferenciação Celular , Linhagem da Célula , Sobrevivência Celular , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Instabilidade Genômica , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Neurônios/transplante , Transcrição GênicaRESUMO
BACKGROUND: The placenta is a very attractive source of mesenchymal stem cells (MSCs) for regenerative medicine due to readily availability, non-invasive acquisition, and avoidance of ethical issues. Isolating MSCs from parts of placenta tissue has obtained growing interest because they are assumed to exhibit different proliferation and differentiation potentials due to complex structures and functions of the placenta. The objective of this study was to isolate MSCs from different parts of the placenta and compare their characteristics. METHODS: Placenta was divided into amniotic epithelium (AE), amniotic membrane (AM), chorionic membrane (CM), chorionic villi (CV), chorionic trophoblast without villi (CT-V), decidua (DC), and whole placenta (Pla). Cells isolated from each layer were subjected to analyses for their morphology, proliferation ability, surface markers, and multi-lineage differentiation potential. MSCs were isolated from all placental layers and their characteristics were compared. FINDINGS: Surface antigen phenotype, morphology, and differentiation characteristics of cells from all layers indicated that they exhibited properties of MSCs. MSCs from different placental layers had different proliferation rates and differentiation potentials. MSCs from CM, CT-V, CV, and DC had better population doubling time and multi-lineage differentiation potentials compared to those from other layers. CONCLUSIONS: Our results indicate that MSCs with different characteristics can be isolated from all layers of term placenta. These finding suggest that it is necessary to appropriately select MSCs from different placental layers for successful and consistent outcomes in clinical applications.
Assuntos
Membranas Extraembrionárias/citologia , Células-Tronco Mesenquimais/citologia , Placenta/citologia , Diferenciação Celular , Proliferação de Células , Forma Celular , Feminino , Humanos , Gravidez , Nascimento a TermoRESUMO
BACKGROUND: The study of lipid metabolism in stem cell physiology has recently raised great interest. The role of lipids goes beyond the mere structural involvement in assembling extra- and intra-cellular compartments. Nevertheless, we are still far from understanding the impact of membrane lipidomics in stemness maintenance and differentiation patterns. In the last years, it has been reported how in vitro cell culturing can modify membrane lipidomics. The aim of the present work was to study the membrane fatty acid profile of mesenchymal stromal cells (MSCs) derived from human fetal membranes (hFM-MSCs) and to correlate this to specific biological properties by using chemically defined tailored lipid supplements (Refeed®). METHODS: Freshly isolated hFM-MSCs were characterized for their membrane fatty acid composition. hFM-MSCs were cultivated in vitro following a classical protocol and their membrane fatty acid profile at different passages was compared to the profile in vivo. A tailored Refeed® lipid supplement was developed with the aim of reducing the differences created by the in vitro cultivation and was tested on cultured hFM-MSCs. Cell morphology, viability, proliferation, angiogenic differentiation, and immunomodulatory properties after in vitro exposure to the tailored Refeed® lipid supplement were investigated. RESULTS: A significant modification of hFM-MSC membrane fatty acid composition occurred during in vitro culture. Using a tailored lipid supplement, the fatty acid composition of cultured cells remained more similar to their in vivo counterparts, being characterized by a higher polyunsaturated and omega-6 fatty acid content. These changes in membrane composition had no effect on cell morphology and viability, but were linked with increased cell proliferation rate, angiogenic differentiation, and immunomodulatory properties. In particular, Refeed®-supplemented hFM-MSCs showed greater ability to express fully functional cell membrane molecules. CONCLUSIONS: Culturing hFM-MSCs alters their fatty acid composition. A tailored lipid supplement is able to improve in vitro hFM-MSC functional properties by recreating a membrane environment more similar to the physiological counterpart. This approach should be considered in cell therapy applications in order to maintain a higher cell quality during in vitro passaging and to influence the outcome of cell-based therapeutic approaches when cells are administered to patients.
Assuntos
Antioxidantes/farmacologia , Membrana Celular/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Diferenciação Celular , Membrana Celular/química , Proliferação de Células , Suplementos Nutricionais , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/efeitos dos fármacos , Membranas Extraembrionárias/metabolismo , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/análise , Ácidos Graxos Monoinsaturados/metabolismo , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/metabolismo , Feminino , Humanos , Lipídeos de Membrana/análise , Lipídeos de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Placenta/citologia , Placenta/efeitos dos fármacos , Placenta/metabolismo , Gravidez , Cultura Primária de CélulasRESUMO
Spontaneous preterm birth remains the major cause of neonatal death and morbidity. Studies in non-gestational tissues report that optineurin (OPTN) is critical in the termination of NFKB1 activity and control of inflammation, central features of spontaneous preterm birth. The aims of the present study were to determine: (1) OPTN expression in fetal membranes and the myometrium during labour; (2) the effects of IL1B on OPTN expression in primary myometrial cells; and (3) the effects of OPTN short interference (si) RNA on IL1B-stimulated proinflammatory and prolabour mediators. OPTN mRNA and protein expression was significantly decreased with spontaneous term labour in fetal membranes and the myometrium. Although there was no effect of spontaneous preterm labour on OPTN expression in fetal membranes, there was decreased OPTN expression in membranes with chorioamnionitis and myometrial cells treated with 1ng mL-1 IL1B for 1 or 6h. In cells transfected with OPTN siRNA, significant increases were seen in IL1B-stimulated IL6, tumour necrosis factor, CXCL8 and monocyte chemoattractant protein-1 mRNA expression and release, cyclo-oxygenase-2 and prostanoid PTGFR receptor mRNA expression and the release of prostaglandin F2α. There was no change in IL1B-stimulated NFKBIA expression; however, there was increased NFKB1 p65 DNA-binding activity. The results of the present study suggest that OPTN is a negative regulator of inflammation-induced prolabour mediators.
Assuntos
Membranas Extraembrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Trabalho de Parto/metabolismo , Miométrio/metabolismo , Nascimento Prematuro/metabolismo , Interferência de RNA , Fator de Transcrição TFIIIA/antagonistas & inibidores , Proteínas de Ciclo Celular , Células Cultivadas , Corioamnionite/imunologia , Corioamnionite/metabolismo , Corioamnionite/patologia , Estudos de Coortes , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/imunologia , Membranas Extraembrionárias/patologia , Feminino , Humanos , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Trabalho de Parto/imunologia , Proteínas de Membrana Transportadoras , Miométrio/citologia , Miométrio/imunologia , Miométrio/patologia , Gravidez , Nascimento Prematuro/imunologia , Nascimento Prematuro/patologia , RNA Interferente Pequeno , Nascimento a Termo/imunologia , Nascimento a Termo/metabolismo , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismoRESUMO
BACKGROUND: Systemic inflammation after prolonged cardiopulmonary bypass (CPB) can cause serious multiorgan system dysfunction. Mesenchymal stem cells (MSCs) are reported to reduce inflammation and attenuate immune response. We have focused on fetal membrane (FM) as a source to provide a large number of MSCs (FM-MSCs). Allogeneic administration of FM-MSCs has been reported to mitigate autoimmune myocarditis or glomerulonephritis. The aim of this study was to investigate whether allogeneic FM-MSCs attenuate systemic inflammatory responses and lung injury in a rat CPB model. METHODS: Male Lewis rats (major histocompatibility complex haplotype: RT-1l) were divided randomly into 3 groups (n = 7 each): cannulation alone (sham group), CPB alone (control group), and CPB + MSC (MSC group). An experimental rat CPB model was established, and CPB was maintained for 30 minutes. In the MSC group, MSCs (1 × 106 cells) derived from the FM of ACI rats with a different major histocompatibility complex haplotype (RT-1a) were administrated intravenously before CPB initiation. RESULTS: Serum concentrations of tumor necrosis factor-α, interleukin-6, and interleukin-1ß in the MSC group were significantly lower compared with the control group after CPB. Similarly, mRNA expression of proinflammatory cytokines in the lung was lower in the MSC group. Allogeneic administration of FM-MSCs remarkably decreased the lung injury score, protected alveolar structure, inhibited neutrophil infiltration to the lung interstitium, and stimulated cytoprotective cytokine production in the lung. CONCLUSIONS: Allogeneic transplantation of FM-MSCs may be a potent strategy to prevent CPB-induced systemic inflammation and acute lung injury by suppressing the expression of inflammatory cytokines and promoting protective factors in the lung.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Ponte Cardiopulmonar/efeitos adversos , Membranas Extraembrionárias/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Lesão Pulmonar Aguda/etiologia , Animais , Modelos Animais de Doenças , Masculino , Ratos , Ratos Endogâmicos Lew , Transplante HomólogoRESUMO
In order to understand a possible etiology of adverse pregnancy outcomes associated with intrauterine influenza virus infection, we examined the effect of influenza virus infection on gene expression of matrix metalloproteinases (MMPs) in cultured amnion epithelial, amnion mesenchymal and chorion trophoblast cells prepared from human fetal membrane tissues by gelatin zymography, Western blotting and reverse transcriptase-PCR. The cells were infected with influenza A (H1N1) virus. The levels of pro-MMP-9 activity in culture supernatants of three types of cells were increased during the period of 24-48 h after the virus infection as compared to those of mock infection. Chorion trophoblast cells spontaneously released a much greater level of pro-MMP-2 activity than amnion epithelial and amnion mesenchymal cells. The cleavage of pro-MMP-2 into an active intermediate form was enhanced in chorion trophoblast cells by the virus infection. The activity levels of MMP-2 and MMP-9 in culture supernatants were consistent with their protein levels. The virus infection induced the mRNA expression of MMP-9, but not MMP-2, in three types of cells. These results suggest that influenza virus infection induces the gene expression of MMP-9 and the cleavage of pro-MMP-2 into an active intermediate form in human fetal membrane cells, resulting in weakening of the membranes through extracellular matrix degradation. Therefore, it is possible that the regulation of MMPs gene expression in fetal membrane cells by influenza virus infection is implicated in a part of the etiology of adverse pregnancy outcomes associated with intrauterine infection with the virus.
Assuntos
Membranas Extraembrionárias/citologia , Vírus da Influenza A Subtipo H1N1 , Influenza Humana/genética , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Células Cultivadas , Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Membranas Extraembrionárias/metabolismo , Membranas Extraembrionárias/virologia , Feminino , Regulação da Expressão Gênica , Humanos , Influenza Humana/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/virologia , Gravidez , Trofoblastos/metabolismo , Trofoblastos/virologiaRESUMO
Progesterone receptor membrane component 1 (PGRMC1) is an important novel mediator of progesterone (P4) function in fetal membrane cells. We demonstrated previously that PGRMC1 is differentially expressed in fetal membranes among pregnancy subjects and diminished in preterm premature rupture of membrane subjects. In the current study, we aim to elucidate whether PGRMC1 expression is regulated by P4, tumor necrosis factor α (TNF-α), and H2O2 in fetal membrane cells. Primary cultured membrane cells were serum starved for 24 hours followed by treatments of P4, 17 hydroxyprogesterone caproate, and medroxyprogesterone 17 acetate (MPA) at 10(-7) mol/L with ethanol as vehicle control; TNF-α at 10, 20, and 50 ng/mL with phosphate-buffered saline (PBS) as control; and H2O2 at 10 and 100 µmol/L with culture media as control for 24, 48, and 72 hours. The messenger RNA (mRNA) and protein expression of PGRMC1 was quantified using polymerase chain reaction and Western blotting, respectively. We found that PGRMC1 protein expression was regulated by MPA, TNF-α, and H2O2 in a dose-dependent manner. This regulation is also specific to the type of cell (amnion, chorion, or decidua). The upregulation of PGRMC1 by MPA might be mediated through glucocorticoid receptor (GR) demonstrated using amnion and chorion cells model with GR knockdown by specific small interfering RNA transfection. The mRNA expression of PGRMC1 was decreased by H2O2 (100 µmol/L) treatment in amnion cells, which might ultimately result in downregulation of PGRMC1 protein as our data demonstrated. None of other treatments changed PGRMC1 mRNA level in these cells. We conclude that these stimuli act as regulatory factors of PGRMC1 in a cell-specific manner.
Assuntos
Membranas Extraembrionárias/metabolismo , Proteínas de Membrana/metabolismo , Estresse Oxidativo , Progestinas/farmacologia , Receptores de Progesterona/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , 17-alfa-Hidroxiprogesterona/farmacologia , Membranas Extraembrionárias/citologia , Membranas Extraembrionárias/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/farmacologia , Medrogestona/farmacologia , Cultura Primária de Células , Progesterona/farmacologia , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Regulação para CimaRESUMO
OBJECTIVES: Mesenchymal stem cells (MSCs) are a valuable cell source in regenerative medicine and can be isolated from fetal membranes (FMs), particularly amniotic membranes. We investigated the effect of rat FM-derived MSCs (rFM-MSCs) and human amnion-derived MSCs (hAMSCs) on the inflammatory reaction in vitro and therapeutic effects in rats with acute and chronic pancreatitis. METHODS: Effect of rFM-MSCs or hAMSC-conditioned medium was investigated in vitro. Acute pancreatitis was induced by intraductal injection of 4% taurocholate, and rFM-MSCs were transplanted intravenously. Chronic pancreatitis was induced by intravenous injection of 5 mg/kg dibutyltin dichloride, and hAMSCs were transplanted intravenously. RESULTS: The inflammatory reaction of macrophages induced by lipopolysaccharide and trypsin was significantly suppressed by rFM-MSC coculture. Pancreatic acinar cell injury induced by cerulein was significantly ameliorated by hAMSC-conditioned medium. Pancreatic stellate cell activation induced by tumor necrosis factor-α was significantly decreased by hAMSC-conditioned medium. Transplantation of rFM-MSCs significantly reduced the histological score and infiltration of CD68-positive macrophages in the rat pancreas. The hAMSC transplantation significantly decreased the expression of MCP-1 and attenuated the downregulation of amylase expression in the pancreas. CONCLUSIONS: Transplantation of FM-MSCs and AMSCs suppressed the inflammatory reaction of acute and chronic pancreatitis in rats.
Assuntos
Membranas Extraembrionárias/citologia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Pancreatite Crônica/terapia , Pancreatite/terapia , Células Acinares/efeitos dos fármacos , Células Acinares/patologia , Doença Aguda , Amilases/genética , Amilases/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Feminino , Humanos , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Células Estreladas do Pâncreas/efeitos dos fármacos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
There have been major recent advances in the field of developmental biology due to the investigation on stem cells (SC). Stem cells are characterized by their capacity of auto-renewal and differentiation to different cellular phenotypes. Based on the developmental stage, they can be classified into two different types: embryonic SCs and adult SCs. It has been widely reported that several problems need to be resolved before their possible clinical applications. As a result, fetal membranes have been suggested as an alternative source of SCs. In the human amniotic epithelium, the presence of markers of pluripotent SC´s has been reported, and its capacity as a feeder layer for expansion of different SC types. Also, fetal membranes are a discarded product after delivery, and thus there are not any ethical issues related to its use. In conclusion, the human amniotic epithelium can be a strong candidate for regenerative medicine.
Assuntos
Âmnio/citologia , Células Epiteliais/citologia , Células-Tronco/citologia , Diferenciação Celular , Membranas Extraembrionárias/citologia , Humanos , Medicina Regenerativa/métodosRESUMO
mRNA of genes (tgfß, il6, il10, il1ß, and vegf165) involved in cell proliferation, inflammatory, anti-inflammatory, and angiogenic processes were detected and quantified in cultures of mesenchymal stromal cells of different passages derived from human extraembryonic tissues (amniotic sac, umbilical cord, chorionic villi and trophoblast of the placenta). The concentrations of IL-10, IL-6, IL-1ß, and TGFß were measured.
Assuntos
Membranas Extraembrionárias/citologia , Fatores Imunológicos/metabolismo , Células-Tronco Mesenquimais/imunologia , RNA Mensageiro/metabolismo , Trofoblastos/citologia , Cordão Umbilical/citologia , Células Cultivadas , Humanos , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Espectrofotometria , Estatísticas não Paramétricas , Fator de Crescimento Transformador beta/metabolismoRESUMO
Although intracerebral transplantation of various fibroblast(-like) cell populations has been shown feasible, little is known about the actual in vivo remodeling of these cellular grafts and their environment. In this study, we aimed to compare the in vitro and in vivo behavior of two phenotypically similar-but developmentally distinct-fibroblast-like cell populations, namely, mouse embryonic fibroblasts (mEFs) and mouse fetal membrane-derived stromal cells (mFMSCs). While both mEFs and mFMSCs are readily able to reduce TNF-α secretion by LPS/IFN-γ-activated BV-2 microglia, mFMSCs and mEFs display strikingly opposite behavior with regard to VEGF production under normal and inflammatory conditions. Whereas mFMSCs downregulate VEGF production upon coculture with LPS/IFN-γ-activated BV-2 microglia, mEFs upregulate VEGF production in the presence of LPS/IFN-γ-activated BV-2 microglia. Subsequently, in vivo grafting of mFMSCs and mEFs revealed no difference in microglial and astroglial responses toward the cellular grafts. However, mFMSC grafts displayed a lower degree of neoangiogenesis compared to mEF grafts, thereby potentially explaining the lower cell number able to survive in mFMSC grafts. In summary, our results suggest that physiological differences between fibroblast-like cell populations might lie at the basis of variations in histopathological and/or clinical outcome following cell grafting in mouse brain.