RESUMO
The small intestinal epithelium has an important role in nutrition, but also in drug absorption and metabolism. There are a few two-dimensional (2D) patient-derived induced pluripotent stem cell (iPSC)-based intestinal models enabling easy evaluation of transcellular transport. It is known that animal-derived components induce variation in the experimental outcomes. Therefore, we aimed to refine the differentiation protocol by using animal-free components. More specifically, we compared maturation of 2D-cultured iPCSs toward small intestinal epithelial cells when cultured either with or without serum, and either on Geltrex or on animal-free, recombinant laminin-based substrata. Differentiation status was characterized by qPCR, immunofluorescence imaging, and functionality assays. Our data suggest that differentiation toward definitive endoderm is more efficient without serum. Both collagen- and recombinant laminin-based coating supported differentiation of definitive endoderm, posterior definitive endoderm, and small intestinal epithelial cells from iPS-cells equally well. Small intestinal epithelial cells differentiated on recombinant laminin exhibited slightly more enterocyte specific cellular functionality than cells differentiated on Geltrex. Our data suggest that functional small intestinal epithelial cells can be generated from iPSCs in serum-free method on xeno-free substrata. This method is easily converted to an entirely xeno-free method.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mucosa Intestinal/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Células Epiteliais/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacosRESUMO
In diabetic patients, high blood glucose induces alterations in retinal function and can lead to visual impairment due to diabetic retinopathy. In immortalized retinal pigment epithelial (RPE) cultures, high glucose concentrations are shown to lead to impairment in epithelial barrier properties. For the first time, the induced pluripotent stem-cell-derived retinal pigment epithelium (hiPSC-RPE) cell lines derived from type 2 diabetics and healthy control patients were utilized to assess the effects of glucose concentration on the cellular functionality. We show that both type 2 diabetic and healthy control hiPSC-RPE lines differentiate and mature well, both in high and normal glucose concentrations, express RPE specific genes, secrete pigment epithelium derived factor, and form a polarized cell layer. Here, type 2 diabetic hiPSC-RPE cells had a decreased barrier function compared to controls. Added insulin increased the epithelial cell layer tightness in normal glucose concentrations, and the effect was more evident in type 2 diabetics than in healthy control hiPSC-RPE cells. In addition, the preliminary functionality assessments showed that type 2 diabetic hiPSC-RPE cells had attenuated autophagy detected via ubiquitin-binding protein p62/Sequestosome-1 (p62/SQSTM1) accumulation, and lowered pro- matrix metalloproteinase 2 (proMMP2) as well as increased pro-MMP9 secretion. These results suggest that the cellular ability to tolerate stress is possibly decreased in type 2 diabetic RPE cells.
Assuntos
Diabetes Mellitus Tipo 2/patologia , Retinopatia Diabética/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Epitélio Pigmentado da Retina/patologia , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Retinopatia Diabética/etiologia , Retinopatia Diabética/metabolismo , Glucose/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Permeabilidade , Epitélio Pigmentado da Retina/metabolismoRESUMO
BACKGROUND: Wound healing of retinal pigment epithelium (RPE) is a complex process that may take place in common age-related macular degeneration eye disease. The purpose of this study was to evaluate whether wounding and wound healing has an effect on Ca2+ dynamics in human embryonic stem cell (hESC)-RPEs cultured different periods of time. METHODS: The 9-day-cultured or 28-day-cultured hESC-RPEs from two different cell lines were wounded and the dynamics of spontaneous and mechanically induced intracellular Ca2+ activity was measured with live-cell Ca2+ imaging either immediately or 7 days after wounding. The healing time and speed were analyzed with time-lapse bright field microscopy. The Ca2+ activity and healing speed were analysed with image analysis. In addition the extracellular matrix deposition was assessed with confocal microscopy. RESULTS: The Ca2+ dynamics in hESC-RPE monolayers differed depending on the culture time: 9-day-cultured cells had higher number of cells with spontaneous Ca2+ activity close to freshly wounded edge compared to control areas, whereas in 28-day-cultured cells there was no difference in wounded and control areas. The 28-day-cultured, wounded and 7-day-healed hESC-RPEs produced wide-spreading intercellular Ca2+ waves upon mechanical stimulation, while in controls propagation was restricted. Most importantly, both wave spreading and spontaneous Ca2+ activity of cells within the healed area, as well as the cell morphology of 28-day-cultured, wounded and thereafter 7-day-healed areas resembled the 9-day-cultured hESC-RPEs. CONCLUSIONS: This acquired knowledge about Ca2+ dynamics of wounded hESC-RPE monolayers is important for understanding the dynamics of RPE wound healing, and could offer a reliable functionality test for RPE cells. The data presented in here suggests that assessment of Ca2+ dynamics analysed with image analysis could be used as a reliable non-invasive functionality test for RPE cells.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Epitélio Pigmentado da Retina/citologia , Cicatrização , Diferenciação Celular , HumanosRESUMO
The impairment of autophagic and proteasomal cleansing together with changes in pigmentation has been documented in retinal pigment epithelial (RPE) cell degeneration. However, the function and co-operation of these mechanisms in melanosome-containing RPE cells is still unclear. We show that inhibition of proteasomal degradation with MG-132 or autophagy with bafilomycin A1 increased the accumulation of premelanosomes and autophagic structures in human embryonic stem cell (hESC)-derived RPE cells. Consequently, upregulation of the autophagy marker p62 (also known as sequestosome-1, SQSTM1) was confirmed in Western blot and perinuclear staining. Interestingly, cells treated with the adenosine monophosphatedependent protein kinase activator, AICAR (5-Aminoimidazole-4-carboxamide ribonucleotide), decreased the proteasome inhibitor-induced accumulation of premelanosomes, increased the amount of autophagosomes and eradicated the protein expression of p62 and LC3 (microtubule-associated protein 1A/1B-light chain 3). These results revealed that autophagic machinery is functional in hESC-RPE cells and may regulate cellular pigmentation with proteasomes.
Assuntos
Autofagia/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/citologia , Leupeptinas/farmacologia , Macrolídeos/farmacologia , Pigmentação/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Linhagem Celular , Humanos , Melanossomas/efeitos dos fármacos , Melanossomas/metabolismo , Epitélio Pigmentado da Retina/citologiaRESUMO
In in vitro live-cell imaging, it would be beneficial to grow and assess human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells on thin, transparent, rigid surfaces such as cover glasses. In this study, we assessed how the silanization of glass with 3-aminopropyltriethoxysilane (APTES), 3-(trimethoxysilyl)propyl methacrylate (MAPTMS), or polymer-ceramic material Ormocomp affects the surface properties, protein binding, and maturation of hESC-RPE cells. The surface properties were studied by contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and a protein binding assay. The cell adherence and proliferation were evaluated by culturing hESCRPE cells on collagen IV-coated untreated or silanized surfaces for 42 days. The Ormocomp treatment significantly increased the hydrophobicity and roughness of glass surfaces compared to the APTES and MAPTMS treatments. The XPS results indicated that the Ormocomp treatment changes the chemical composition of the glass surface by increasing the carbon content and the number of C-O/âO bonds. The protein-binding test confirmed that the Ormocomp-treated surfaces bound more collagen IV than did APTES- or MAPTMS-treated surfaces. All of the silane treatments increased the number of cells: after 42 days of culture, Ormocomp had 0.38, APTES had 0.16, MAPTMS had 0.19, and untreated glass had only 0.062, all presented as million cells cm(-2). There were no differences in cell numbers compared to smoother to rougher Ormocomp surfaces, suggesting that the surface chemistry and, more specifically, the collagen binding in combination with Ormocomp are beneficial to hESC-RPE cell culture. This study clearly demonstrates that Ormocomp treatment combined with collagen coating significantly increases hESC-RPE cell attachment compared to commonly used silanizing agents APTES and MAPTMS. Ormocomp silanization could thus enable the use of microscopic live cell imaging methods for hESC-RPE cells.
Assuntos
Células-Tronco Embrionárias/citologia , Células Epiteliais/citologia , Vidro/química , Adesão Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Células-Tronco Embrionárias/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Espectroscopia Fotoeletrônica , Propilaminas , Silanos/química , Propriedades de SuperfícieRESUMO
BACKGROUND: Bone morphogenetic protein 4 (BMP4) belongs to the transforming growth factor ß (TGF-ß) family of proteins. BMPs regulate cell proliferation, differentiation and motility, and have also been reported to be involved in cancer pathogenesis. We have previously shown that BMP4 reduces breast cancer cell proliferation through G1 cell cycle arrest and simultaneously induces migration in a subset of these cell lines. Here we examined the effects of BMP4 in a more physiological environment, in a 3D culture system. METHODS: We used two different 3D culture systems; Matrigel, a basement membrane extract from mouse sarcoma cells, and a synthetic polyethylene glycol (PEG) gel. AlamarBlue reagent was used for cell proliferation measurements and immunofluorescence was used to determine cell polarity. Expression of cell cycle regulators was examined by Western blot and matrix metalloproteinase (MMP) expression by qRT-PCR. RESULTS: The MCF-10A normal breast epithelial cells formed round acini with correct apicobasal localization of α6 integrin in Matrigel whereas irregular structures were seen in PEG gel. The two 3D matrices also supported dissimilar morphology for the breast cancer cells. In PEG gel, BMP4 inhibited the growth of MCF-10A and the three breast cancer cell lines examined, thus closely resembling the 2D culture conditions, but in Matrigel, no growth inhibition was observed in MDA-MB-231 and MDA-MB-361 cells. Furthermore, BMP4 induced the expression of the cell cycle inhibitor p21 both in 2D and 3D culture, thereby partly explaining the growth arrest. Interestingly, MDA-MB-231 cells formed large branching, stellate structures in response to BMP4 treatment in Matrigel, suggestive of increased cell migration or invasion. This effect was reversed by Batimastat, a broad-spectrum MMP inhibitor, and subsequent analyses showed BMP4 to induce the expression of MMP3 and MMP14, that are thus likely to be responsible for the stellate phenotype. CONCLUSIONS: Taken together, our results show that Matrigel provides a more physiological environment for breast epithelial cells than PEG gel. Moreover, BMP4 partly recapitulates in 3D culture the growth suppressive abilities previously seen in 2D culture and induces an MMP-dependent migratory phenotype in MDA-MB-231 cells.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Metaloendopeptidases/metabolismo , Fenótipo , Proteína Morfogenética Óssea 4/farmacologia , Neoplasias da Mama/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Esferoides Celulares , Células Tumorais CultivadasRESUMO
PURPOSE: The production of functional retinal pigment epithelium (RPE) cells from human embryonic (hESCs) and human induced pluripotent stem cells (hiPSCs) in defined and xeno-free conditions is highly desirable, especially for their use in cell therapy for retinal diseases. In addition, differentiated RPE cells provide an individualized disease model and drug discovery tool. In this study, we report the differentiation of functional RPE-like cells from several hESC lines and one hiPSC line in culture conditions, enabling easy translation to clinical quality cell production under Good Manufacturing Practice regulations. METHODS: Pluripotent stem cells were cultured on human fibroblast feeder cells in serum-free medium. The differentiation toward RPE was induced by removing basic fibroblast growth factor and feeder cells from the serum-free conditions. RPE differentiation was also achieved using xeno-free and defined culture conditions. The RPE cell morphology and pigmentation of the cells were analyzed and the expression of genes and proteins characteristic for RPE cells was evaluated. In vitro functionality of the cells was analyzed using ELISA measurements for pigment epithelium derived factor (PEDF) secretion and phagocytosis of photoreceptor outer segments (POS). The integrity of the generated RPE layers was analyzed using transepithelial electric resistance measurements. RESULTS: We generated putative RPE cells with typical pigmented cobblestone-like morphology. The expression of RPE-specific markers was confirmed at the gene and protein level. The differentiated cells were able to phagocytose POS and secrete PEDF characteristic of native RPE cells. In addition, cultured cells formed a polarized epithelium with high integrity and exhibited excellent transepithelial electric resistance values, indicating well established, tight junctions. Moreover, we introduced an improved method to generate functional putative RPE cells without xeno-components under defined conditions. CONCLUSIONS: We have developed a progressive differentiation protocol for the production of functional RPE-like cells from hESCs and hiPSCs. Our results demonstrate that putative hESC-RPE and hiPSC-RPE express genes and proteins characteristic for RPE cells, as well as being able to phagocytose POS and secrete PEDF. Furthermore, our results show that RPE-like cells can be differentiated in xeno-free and defined culture conditions, which is mandatory for Good Manufacturing Practice-production of these cells for clinical use.
Assuntos
Diferenciação Celular , Células Epiteliais/citologia , Células-Tronco Pluripotentes/citologia , Epitélio Pigmentado da Retina/citologia , Animais , Diferenciação Celular/genética , Linhagem Celular , Forma Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/metabolismo , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Imunofluorescência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Especificidade de Órgãos/genética , Fagocitose , Pigmentação/fisiologia , Células-Tronco Pluripotentes/metabolismoRESUMO
In vitro cell culture models representing the physiological and pathological features of the outer retina are urgently needed. Artificial tissue replacements for patients suffering from degenerative retinal diseases are similarly in great demand. Here, we developed a co-culture system based solely on the use of human induced pluripotent stem cell (hiPSC)-derived cells. For the first time, hiPSC-derived retinal pigment epithelium (RPE) and endothelial cells (EC) were cultured on opposite sides of porous polylactide substrates prepared by breath figures (BF), where both surfaces had been collagen-coated by Langmuir-Schaefer (LS) technology. Small modifications of casting conditions during material preparation allowed the production of free-standing materials with distinct porosity, wettability and ion diffusion capacity. Complete pore coverage was achieved by the collagen coating procedure, resulting in a detectable nanoscale topography. Primary retinal endothelial cells (ACBRI181) and umbilical cord vein endothelial cells (hUVEC) were utilised as EC references. Mono-cultures of all ECs were prepared for comparison. All tested materials supported cell attachment and growth. In mono-culture, properties of the materials had a major effect on the growth of all ECs. In co-culture, the presence of hiPSC-RPE affected the primary ECs more significantly than hiPSC-EC. In consistency, hiPSC-RPE were also less affected by hiPSC-EC than by the primary ECs. Finally, our results show that the modulation of the porosity of the materials can promote or prevent EC migration. In short, we showed that the behaviour of the cells is highly dependent on the three main variables of the study: the presence of a second cell type in co-culture, the source of endothelial cells and the biomaterial properties. The combination of BF and LS methodologies is a powerful strategy to develop thin but stable materials enabling cell growth and modulation of cell-cell contact. STATEMENT OF SIGNIFICANCE: Artificial blood-retinal barriers (BRB), mimicking the interface at the back of the eye, are urgently needed as physiological and disease models, and for tissue transplantation targeting patients suffering from degenerative retinal diseases. Here, we developed a new co-culture model based on thin, biodegradable porous films, coated on both sides with collagen, one of the main components of the natural BRB, and cultivated endothelial and retinal pigment epithelial cells on opposite sides of the films, forming a three-layer structure. Importantly, our hiPSC-EC and hiPSC-RPE co-culture model is the first to exclusively use human induced pluripotent stem cells as cell source, which have been widely regarded as an practical candidate for therapeutic applications in regenerative medicine.
Assuntos
Colágeno/farmacologia , Células Epiteliais/citologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Epitélio Pigmentado da Retina/citologia , Adulto , Materiais Biocompatíveis/farmacologia , Técnicas de Cocultura , Impedância Elétrica , Humanos , Porosidade , ÁguaRESUMO
OBJECTIVE: In coeliac disease, small-bowel mucosal permeability is increased in response to gluten consumption. However, the signalling routes leading to such a barrier defect remain obscure. As the epidermal growth factor receptor (EGFR) pathway is up-regulated in untreated coeliac disease, and since this cascade has been related to epithelial hyperpermeability, the aim of this study was to establish whether blocking the EGFR route would restore the barrier after gliadin insult in vitro. MATERIAL AND METHODS: Epithelial barrier function was assessed by measuring transepithelial electrical resistance (TER) in Caco-2 epithelial monolayers treated with pepsin trypsin (PT)-digested gliadin with or without monoclonal antibodies against EGFR family members or by inhibitors of the EGFR pathway signalling molecules. Furthermore, tight-junctional integrity was determined by Western blotting and immunofluorescence staining of the tight-junctional protein occludin. RESULTS: PT-gliadin significantly reduced TER and the expression of occludin protein. Blocking of the EGFR signalling pathway could not prevent gliadin-triggered damage. In fact, a function-blocking monoclonal antibody against EGFR (ErbB1) actually potentiated the harmful effects of gliadin on TER. CONCLUSIONS: The epithelial barrier-disrupting properties of gliadin are independent of the EGFR signalling cascade. However, our results suggest that activation of the EGFR pathway might actually be protective against gliadin-triggered hyperpermeability. Further studies are needed to elucidate the specific gliadin-triggered signalling cascades which lead to increased epithelial permeability in coeliac disease.
Assuntos
Doença Celíaca/metabolismo , Receptores ErbB/antagonistas & inibidores , Gliadina/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Western Blotting , Células CACO-2 , Células Cultivadas , Impedância Elétrica , Humanos , Técnicas In Vitro , Mucosa Intestinal/metabolismo , Microscopia Confocal , Transdução de Sinais/efeitos dos fármacos , Estatísticas não ParamétricasRESUMO
Retinal pigment epithelium (RPE) performs important functions for the maintenance of photoreceptors and vision. Malfunctions within the RPE are implicated in several retinal diseases for which transplantations of stem cell-derived RPE are promising treatment options. Their success, however, is largely dependent on the functionality of the transplanted cells. This requires correct cellular physiology, which is highly influenced by the various ion channels of RPE, including voltage-gated Ca2+ (CaV ) channels. This study investigated the localization and functionality of CaV channels in human embryonic stem cell (hESC)-derived RPE. Whole-cell patch-clamp recordings from these cells revealed slowly inactivating L-type currents comparable to freshly isolated mouse RPE. Some hESC-RPE cells also carried fast transient T-type resembling currents. These findings were confirmed by immunostainings from both hESC- and mouse RPE that showed the presence of the L-type Ca2+ channels CaV 1.2 and CaV 1.3 as well as the T-type Ca2+ channels CaV 3.1 and CaV 3.2. The localization of the major subtype, CaV 1.3, changed during hESC-RPE maturation co-localizing with pericentrin to the base of the primary cilium before reaching more homogeneous membrane localization comparable to mouse RPE. Based on functional assessment, the L-type Ca2+ channels participated in the regulation of vascular endothelial growth factor secretion as well as in the phagocytosis of photoreceptor outer segments in hESC-RPE. Overall, this study demonstrates that a functional machinery of voltage-gated Ca2+ channels is present in mature hESC-RPE, which is promising for the success of transplantation therapies. Stem Cells Translational Medicine 2019;8:179&15.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp/métodos , Fagocitose/fisiologia , Doenças Retinianas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Diabetic retinopathy is a common diabetes complication that threatens the eyesight and may eventually lead to acquired visual impairment or blindness. While a substantial heritability has been reported for proliferative diabetic retinopathy (PDR), only a few genetic risk factors have been identified. Using genome-wide sib pair linkage analysis including 361 individuals with type 1 diabetes, we found suggestive evidence of linkage with PDR at chromosome 10p12 overlapping the CACNB2 gene (logarithm of odds = 2.73). Evidence of association between variants in CACNB2 and PDR was also found in association analysis of 4,005 individuals with type 1 diabetes with an odds ratio of 0.83 and P value of 8.6 × 10-4 for rs11014284. Sequencing of CACNB2 revealed two coding variants, R476C/rs202152674 and S502L/rs137886839. CACNB2 is abundantly expressed in retinal cells and encodes the ß2 subunit of the L-type calcium channel. Blocking vascular endothelial growth factor (VEGF) by intravitreous anti-VEGF injections is a promising clinical therapy to treat PDR. Our data show that L-type calcium channels regulate VEGF expression and secretion from retinal pigment epithelial cells (ARPE19) and support the role of CACNB2 via regulation of VEGF in the pathogenesis of PDR. However, further genetic and functional studies are necessary to consolidate the findings.
Assuntos
Canais de Cálcio Tipo L/genética , Diabetes Mellitus Tipo 1/genética , Retinopatia Diabética/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Adulto , Alelos , Estudos de Casos e Controles , Feminino , Ligação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: To study immunogenic properties of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) and to evaluate subretinal xenotransplantation of hESC-RPE on porous polyethylene terephthalate (PET) in rabbits. METHODS: Human ESC-RPE cells were characterized by morphology, transepithelial electrical resistance (TER), protein expression and photoreceptor outer segment phagocytosis in vitro. Expression of major histocompatibility complex (MHC) proteins was assessed in conventionally or xeno-free produced hESC-RPE ± interferon-gamma (IFN-γ) stimulation (n = 1). Xeno-free hESC-RPE on PET with TER < 200 Ω·cm2 > or PET alone were transplanted into 18 rabbits with short-term triamcinolone ± extended tacrolimus immunosuppression. Rabbits were monitored by spectral domain optical coherence tomography. After 4 weeks, the eyes were processed for histology and transmission electron microscopy. RESULTS: Upon in vitro IFN-γ stimulation, xeno-free hESC-RPE expressed lower level of MHC-II proteins compared to the conventional cells. Outer nuclear layer (ONL) atrophy was observed over the graft in most cases 4 weeks post-transplantation. In 3/4 animals with high TER hESC-RPE, but only in 1/3 animals with low TER hESC-RPE, ONL atrophy was observed already within 1 week. Retinal cell infiltrations were more frequent in animals with high TER hESC-RPE. However, the difference was not statistically significant. In three animals, preservation of ONL was observed. Weekly intravitreal tacrolimus did not affect ONL preservation. In all animals, hESC-RPE cells survived for 4 weeks, but without tacrolimus, enlarged vacuoles accumulated in hESC-RPE (n = 1). CONCLUSIONS: Xenografted xeno-free hESC-RPE monolayers can survive and retain some functionality for 4 weeks following short-term immunosuppression. The preliminary findings of this study suggest that further investigations to improve transplantation success of hESC-RPE xenografts in rabbits should be addressed especially toward the roles of hESC-RPE maturation stage and extended intravitreal immunosuppression.
Assuntos
Células-Tronco Embrionárias Humanas/transplante , Poliésteres , Doenças Retinianas/cirurgia , Epitélio Pigmentado da Retina/transplante , Transplante de Células-Tronco/métodos , Alicerces Teciduais , Animais , Diferenciação Celular , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Seguimentos , Células-Tronco Embrionárias Humanas/citologia , Humanos , Masculino , Fagocitose , Coelhos , Doenças Retinianas/patologia , Epitélio Pigmentado da Retina/citologia , Tomografia de Coerência Óptica , Transplante HeterólogoRESUMO
Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell therapies show tremendous potential for the treatment of retinal degenerative diseases. A tissue engineering approach, where cells are delivered to the subretinal space on a biodegradable carrier as a sheet, shows great promise for these RPE cell therapies. The aim of the present study was to assess whether a flexible, elastic and biodegradable poly(trimethylene carbonate) (PTMC) film promotes the formation of functional hESC-RPE and performs better than often used biodegradable poly(d,l-lactide) (PDLLA) film. Human ESC-RPE maturation and functionality on PTMC films was assessed by cell proliferation assays, RPE-specific gene and protein expression, phagocytic activity and growth factor secretion. It is demonstrated that the mechanical properties of PTMC films have close resemblance to those of the native Bruch's membrane and support the formation hESC-RPE monolayer in serum-free culture conditions with high degree of functionality. In contrast, use of PDLLA films did not lead to the formation of confluent monolayers of hESC-RPE cells and had unsuitable mechanical properties for retinal application. In conclusion, the present study indicates that flexible and elastic biodegradable PTMC films show potential for retinal tissue engineering applications. Copyright © 2017 John Wiley & Sons, Ltd.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Dioxanos , Células Epiteliais/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Membranas Artificiais , Polímeros , Epitélio Pigmentado da Retina/metabolismo , Plásticos Biodegradáveis/química , Plásticos Biodegradáveis/farmacologia , Proliferação de Células/efeitos dos fármacos , Dioxanos/química , Dioxanos/farmacologia , Células Epiteliais/citologia , Células-Tronco Embrionárias Humanas/citologia , Humanos , Polímeros/química , Polímeros/farmacologia , Epitélio Pigmentado da Retina/citologiaRESUMO
BACKGROUND: The TGFbeta1-induced signal transduction processes involved in growth and differentiation are only partly known. The three-dimensional epithelial differentiation model, in which T84 epithelial cells are induced to differentiate either with TGFbeta1 or IMR-90 mesenchymal cell-secreted soluble factors, is previously shown to model epithelial cell differentiation seen in intestine. That model has not been used for large scale gene expression studies, such as microarray method. Therefore the gene expression changes were studied in undifferentiated and differentiated three-dimensional T84 cultures with cDNA microarray method in order to study the molecular changes and find new players in epithelial cell differentiation. RESULTS: The expression of 372 genes out of 5188 arrayed sequences was significantly altered, and 47 of them were altered by both mediators. The data were validated and the altered genes are presented in ontology classes. For the genes tested the expressions in protein level were in accordance with the mRNA results. We also found 194 genes with no known function to be potentially important in epithelial cell differentiation. The mRNA expression changes induced by TGFbeta1 were bigger than changes induced by soluble factors secreted by IMR-90 mesenchymal cells. The gene expression data was depicted in already known signaling pathway routes. CONCLUSION: Our results reveal potential new signaling pathways and several new genes affected by TGFbeta in epithelial cell differentiation. The differentiation induced by TGFbeta1 appears to be more potent than the differentiation induced by mesenchymal cells. This study indicates that our cell culture model is a suitable tool in studying regulatory mechanisms during epithelial cell differentiation in intestine. Furthermore the present results indicate that our model is a good tool for finding new players acting in the differentiation of epithelial cells.
Assuntos
Diferenciação Celular/genética , Colo/citologia , Regulação da Expressão Gênica , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Colo/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/química , Humanos , Mesoderma/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de SinaisRESUMO
Ca2+ is a second messenger controlling vital cellular processes, including cell maturation. Changes in Ca2+ signaling during maturation of human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) have not been assessed previously. The aim of this study was to investigate maturation-dependent changes in transient intracellular Ca2+ ([Ca2+] i ) increases in hESC-RPE. For this, we developed image analysis tools to evaluate cell-specific Ca2+ signals from the entire field of view. Spontaneous and mechanically induced transient [Ca2+] i increases (STIs and MITIs) were analyzed in hESC-RPEs cultured for 9 or 28 days, altogether from more than 80,000 cells. Both cultures showed STIs: the longer culture time resulted in twofold increase of amount of cells with STIs. Mechanical stimulation induced intercellular Ca2+ waves in cells from both time points, but longer culture time reduced Ca2+ wave spreading. Depletion of intracellular Ca2+ stores decreased cell fraction with STIs and MITIs at both time points, and absence of extracellular Ca2+ had similar effect on cells with STIs. To conclude, hESC-RPE cells undergo significant Ca2+ signaling re-arrangements during a short maturation period increasing cell fraction with STIs, while decreasing coordinated cell response to mechanical stimulation. This knowledge and proposed analysis tools can be used for assessment of hESC-RPE maturation in vitro.
Assuntos
Sinalização do Cálcio/fisiologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Linhagem Celular , HumanosRESUMO
AIMS: A fast, non-invasive and observer-independent method to analyze the homogeneity and maturity of human pluripotent stem cell (hPSC) derived retinal pigment epithelial (RPE) cells is warranted to assess the suitability of hPSC-RPE cells for implantation or in vitro use. The aim of this work was to develop and validate methods to create ensembles of state-of-the-art texture descriptors and to provide a robust classification tool to separate three different maturation stages of RPE cells by using phase contrast microscopy images. The same methods were also validated on a wide variety of biological image classification problems, such as histological or virus image classification. METHODS: For image classification we used different texture descriptors, descriptor ensembles and preprocessing techniques. Also, three new methods were tested. The first approach was an ensemble of preprocessing methods, to create an additional set of images. The second was the region-based approach, where saliency detection and wavelet decomposition divide each image in two different regions, from which features were extracted through different descriptors. The third method was an ensemble of Binarized Statistical Image Features, based on different sizes and thresholds. A Support Vector Machine (SVM) was trained for each descriptor histogram and the set of SVMs combined by sum rule. The accuracy of the computer vision tool was verified in classifying the hPSC-RPE cell maturation level. DATASET AND RESULTS: The RPE dataset contains 1862 subwindows from 195 phase contrast images. The final descriptor ensemble outperformed the most recent stand-alone texture descriptors, obtaining, for the RPE dataset, an area under ROC curve (AUC) of 86.49% with the 10-fold cross validation and 91.98% with the leave-one-image-out protocol. The generality of the three proposed approaches was ascertained with 10 more biological image datasets, obtaining an average AUC greater than 97%. CONCLUSIONS: Here we showed that the developed ensembles of texture descriptors are able to classify the RPE cell maturation stage. Moreover, we proved that preprocessing and region-based decomposition improves many descriptors' accuracy in biological dataset classification. Finally, we built the first public dataset of stem cell-derived RPE cells, which is publicly available to the scientific community for classification studies. The proposed tool is available at https://www.dei.unipd.it/node/2357 and the RPE dataset at http://www.biomeditech.fi/data/RPE_dataset/. Both are available at https://figshare.com/s/d6fb591f1beb4f8efa6f.
Assuntos
Diferenciação Celular , Epitélio Pigmentado da Retina/citologia , Células-Tronco/citologia , Máquina de Vetores de Suporte , Algoritmos , Animais , Área Sob a Curva , Linhagem Celular , Conjuntos de Dados como Assunto , Feminino , Humanos , Masculino , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Computational models of calcium (Ca²âº) signaling have been constructed for several cell types. There are, however, no such models for retinal pigment epithelium (RPE). Our aim was to construct a Ca²âº signaling model for RPE based on our experimental data of mechanically induced Ca²âº wave in the in vitro model of RPE, the ARPE-19 monolayer. METHODS: We combined six essential Ca²âº signaling components into a model: stretch-sensitive Ca²âº channels (SSCCs), P2Y2 receptors, IP3 receptors, ryanodine receptors, Ca²âº pumps, and gap junctions. The cells in our epithelial model are connected to each other to enable transport of signaling molecules. Parameterization was done by tuning the above model components so that the simulated Ca²âº waves reproduced our control experimental data and data where gap junctions were blocked. RESULTS: Our model was able to explain Ca²âº signaling in ARPE-19 cells, and the basic mechanism was found to be as follows: 1) Cells near the stimulus site are likely to conduct Ca²âº through plasma membrane SSCCs and gap junctions conduct the Ca²âº and IP3 between cells further away. 2) Most likely the stimulated cell secretes ligand to the extracellular space where the ligand diffusion mediates the Ca²âº signal so that the ligand concentration decreases with distance. 3) The phosphorylation of the IP3 receptor defines the cell's sensitivity to the extracellular ligand attenuating the Ca²âº signal in the distance. CONCLUSIONS: The developed model was able to simulate an array of experimental data including drug effects. Furthermore, our simulations predict that suramin may interfere ligand binding on P2Y2 receptors or accelerate P2Y2 receptor phosphorylation, which may partially be the reason for Ca²âº wave attenuation by suramin. Being the first RPE Ca²âº signaling model created based on experimental data on ARPE-19 cell line, the model offers a platform for further modeling of native RPE functions.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Simulação por Computador , Células Epiteliais/metabolismo , Modelos Biológicos , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/metabolismo , Algoritmos , Linhagem Celular , HumanosRESUMO
Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells are currently undergoing clinical trials to treat retinal degenerative diseases. Transplantation of hESC-RPE cells in conjuction with a supportive biomaterial carrier holds great potential as a future treatment for retinal degeneration. However, there has been no such biodegradable material that could support the growth and maturation of hESC-RPE cells so far. The primary aim of this work was to create a thin porous poly (L-lactide-co-caprolactone) (PLCL) membrane that could promote attachment, proliferation, and maturation of the hESC-RPE cells in serum-free culture conditions. The PLCL membranes were modified by atmospheric pressure plasma processing and coated with collagen IV to enhance cell growth and maturation. Permeability of the membranes was analyzed with an Ussing chamber system. Analysis with scanning electron microscopy, contact angle measurement, atomic force microscopy, and X-ray photoelectron spectroscopy demonstrated that plasma surface treatment augments the surface properties of the membrane, which enhances the binding and conformation of the protein. Cell proliferation assays, reverse transcription-polymerase chain reaction, indirect immunofluoresence staining, trans-epithelial electrical resistance measurements, and in vitro phagocytosis assay clearly demonstrated that the plasma treated PLCL membranes supported the adherence, proliferation, maturation and functionality of hESC-RPE cells in serum-free culture conditions. Here, we report for the first time, how PLCL membranes can be modified with atmospheric pressure plasma processing to enable the formation of a functional hESC-RPE monolayer on a porous biodegradable substrate, which have a potential as a tissue-engineered construct for regenerative retinal repair applications.
Assuntos
Células-Tronco Embrionárias Humanas/citologia , Epitélio Pigmentado da Retina/citologia , Engenharia Tecidual/métodos , Adesão Celular/efeitos dos fármacos , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo IV/metabolismo , Imunofluorescência , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Células-Tronco Embrionárias Humanas/ultraestrutura , Humanos , Fagocitose/efeitos dos fármacos , Espectroscopia Fotoeletrônica , Gases em Plasma/farmacologia , Poliésteres/farmacologia , Segmento Externo das Células Fotorreceptoras da Retina/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
PURPOSE: In several retinal complications, such as age-dependent macular degeneration (AMD), oxidative stress is increased and cytokine level is elevated. These are shown to alter the activation and expression of matrix metalloproteinase (MMP) both in human primary and immortalized retinal pigment epithelial (RPE) cells. However, the effects on human embryonic stem cell (hESC)-derived RPE cells remain to be elucidated. METHODS: The mature hESC-RPE cells were exposed to inflammatory cytokines (IFN-γ or TNF-α) for 24 hours or oxidative stress (H2O2) for 1 hour. Effects on barrier properties were analyzed with transepithelial electrical resistance (TEER), the expression of MMP-1, MMP-2, MMP-3, MMP-9, collagen I, and collagen IV genes with quantitative RT-PCR, and the expression of MMP-1 and MMP-3 proteins with Western blot or ELISA, respectively. Also, activation and secretion of MMP-2 and -9 proteins were analyzed with zymography. RESULTS: In normal state, mature hESC-RPE cells expressed MMP-1, -2, -3, and -9 genes in low levels, respectively. Tumor necrosis factor-α increased MMP-1 and -2 gene expression, and H2O2 increased MMP-3 and -9 gene expression. Zymography revealed IFN-γ- and TNF-α-induced secretion of MMP-2 and high-molecular-weight species of MMP (HMW MMP), but H2O2 decreased their secretion. Furthermore, TNF-α and H2O2 significantly decreased barrier properties. CONCLUSIONS: Here, cytokines induced the MMP-1 and -2 gene and protein expression. Also, H2O2 induced MMP-3 and -9 gene expression, but not their protein secretion. These data propose that under oxidative stress and cytokine stimuli, mature hESC-RPE cells resemble their native counterpart in the human eye in regard to MMP secretion and expression and could be used to model retinal disorders involving alterations in MMP activity such as AMD, diabetic retinopathy, or proliferative vitreoretinopathy in vitro.
Assuntos
Citocinas/genética , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Degeneração Macular/genética , Estresse Oxidativo , RNA Mensageiro/genética , Epitélio Pigmentado da Retina/metabolismo , Western Blotting , Células Cultivadas , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Células-Tronco Embrionárias Humanas/patologia , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/patologiaRESUMO
Extracellular matrix (ECM) interactions play a vital role in cell morphology, migration, proliferation, and differentiation of cells. We investigated the role of ECM proteins on the structure and function of human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells during their differentiation and maturation from hESCs into RPE cells in adherent differentiation cultures on several human ECM proteins found in native human Bruch's membrane, namely, collagen I, collagen IV, laminin, fibronectin, and vitronectin, as well as on commercial substrates of xeno-free CELLstart™ and Matrigel™. Cell pigmentation, expression of RPE-specific proteins, fine structure, as well as the production of basal lamina by hESC-RPE on different protein coatings were evaluated after 140 days of differentiation. The integrity of hESC-RPE epithelium and barrier properties on different coatings were investigated by measuring transepithelial resistance. All coatings supported the differentiation of hESC-RPE cells as demonstrated by early onset of cell pigmentation and further maturation to RPE monolayers after enrichment. Mature RPE phenotype was verified by RPE-specific gene and protein expression, correct epithelial polarization, and phagocytic activity. Significant differences were found in the degree of RPE cell pigmentation and tightness of epithelial barrier between different coatings. Further, the thickness of self-assembled basal lamina and secretion of the key ECM proteins found in the basement membrane of the native RPE varied between hESC-RPE cultured on compared protein coatings. In conclusion, this study shows that the cell culture substrate has a major effect on the structure and basal lamina production during the differentiation and maturation of hESC-RPE potentially influencing the success of cell integrations and survival after cell transplantation.