RESUMEN
Introduction: The retinal pigment epithelium (RPE) plays essential roles in maintaining retinal functions as well as choroidal capillaries and can lead to visual disorders if dysfunctional. Transplantation of human-induced pluripotent stem cell-derived RPE (hiPSC-RPE) is a promising therapy for such RPE impaired conditions including age-related macular degeneration. The challenge with cell suspension transplantation is targeted delivery of graft cells and undesired cell reflux. Gelatin hydrolysate, a soluble variant with specific molecular weight distribution, is examined in this study for its potential use in hiPSC-RPE suspension transplantation, particularly in reducing cell reflux and enhancing RPE engraftment. Methods: A retinal bleb model was created using polydimethylsiloxane (PDMS) soft lithography to quantify cellular reflux. We examined the effects of gelatin hydrolysate on the hiPSC-RPE of various aspects of cell behavior and performance such as cell viability, hypoxia reaction, morphology, induction of inflammation and immune responses. Results: Gelatin hydrolysate at 5 % concentration effectively mitigated cell reflux in vitro mimic, improved cell viability, reduced cell aggregation, and had an inhibitory effect on hypoxic reactions due to cell deposition with hiPSC-RPE. Additionally, gelatin hydrolysate did not affect cell adhesion and morphology, and decreased the expression of major histocompatibility complex class II molecules, which suggests reduced immunogenicity of hiPSC-RPE. Conclusion: Gelatin hydrolysate is considered a valuable and useful candidate for future regenerative therapies in hiPSC-RPE suspension transplantation.
RESUMEN
Retinal pigment epithelium (RPE) cells is the outermost layer of the retina and RPE dysfunction is a key factor in the disease pathogenesis of age-related macular degeneration (AMD). Transplantation therapy using induced pluripotent stem cell (iPSC)-derived RPEs has recently received much attention as a treatment for AMD. Preserving these cells under the best possible conditions is important, and preservation methods using Y-27632 have been reported. Rho-associated coiled-coil containing kinase (ROCK) inhibitors are known to inhibit cell death, emerging as important drug candidates for stem cell differentiation and regenerative medicine. However, it has recently been shown that ROCK inhibitors may have a vasodilatory effect on human retinal arterioles, a side effect that should ideally be avoided in RPE transplantation. Although ROCK inhibitors hold great potential, optimizing efficacy while minimizing adverse reactions is critical for translation into a clinical treatment. We examined the effect of transient exposure of RPE cells to ROCK inhibitor Y-27632 to determine whether the extracellular presence of the drug is necessary for ongoing Rho/ROCK downregulation. Human RPE cells were subcultured as a suspension for 4 h in drug-free medium following exposure to Y-27632 for 2 h. A Y-27632 concentration of >10 µM improved cell survival beyond 4 h and cell proliferation in recovery culture medium. ROCK2 expression levels were specifically downregulated by Y-27632 in the Rho/ROCK signaling pathway. In conclusion, we demonstrated that the effect of Y-27632 is not dependent on its extracellular availability and can last beyond the 2 h of exposure. The lasting Rho/ROCK signaling pathway downregulation by Y-27632 suggests that RPE cell transplantation with ROCK inhibitor-free media is possible, which can minimize side effects to host tissue and have wider implications for transplantation methods requiring ROCK inhibition.
RESUMEN
The transplantation of adipose tissue-derived stem cells (ADSCs) improves cardiac contractility after myocardial infarction (MI); however, little is known about the electrophysiological consequences of transplantation. The purpose of this study was to clarify whether the transplantation of ADSCs increases or decreases the incidence of ventricular tachyarrhythmias (VT) in a rat model of MI. MI was induced experimentally by permanent occlusion of the left anterior descending artery of Lewis rats. ADSCs were harvested from GFP-transgenic rats, and were cultured until passage four. ADSCs (10×10(6)) resuspended in 100µL saline or pro-survival cocktail (PSC), which enhances cardiac graft survival, were injected directly into syngeneic rat hearts 1week after MI. The recipients of ADSCs suspended in PSC had a larger graft area compared with those receiving ASDCs suspended in saline at 1week post-transplantation (number of graft cells/section: 148.7±10.6 vs. 22.4±3.4, p<0.05, n=5/group). Thereafter, all ADSC recipients were transplanted with ASDCs in PSC. ADSCs were transplanted into infarcted hearts, and the mechanical and electrophysiological functions were assessed. Echocardiography revealed that ADSC recipients had improved contractile function compared with those receiving PSC vehicle (fractional shortening: 21.1±0.9 vs. 14.1±1.2, p<0.05, n≥12/group). Four weeks post-transplantation, VT was induced via in vivo programmed electrical stimulation. The recipients of ADSCs showed a significantly lower incidence of induced VT compared with the control (31.3% vs. 83.3%, p<0.05, n≥12/group). To understand the electrical activity following transplantation, we performed ex vivo optical mapping using a voltage sensitive dye, and found that ADSC transplantation decreased conduction velocity and its dispersion in the peri-infarct area. These results suggest that ADSC transplantation improved cardiac mechanical and electrophysiological functions in subacute MI.
Asunto(s)
Adipocitos/fisiología , Infarto del Miocardio/terapia , Trasplante de Células Madre , Células Madre/fisiología , Taquicardia/terapia , Adipocitos/citología , Animales , Diferenciación Celular , Proliferación Celular , Vasos Coronarios/patología , Modelos Animales de Enfermedad , Estimulación Eléctrica , Sistema de Conducción Cardíaco , Masculino , Contracción Miocárdica , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Miocardio/patología , Ratas , Ratas Endogámicas Lew , Células Madre/citología , Taquicardia/patología , Taquicardia/fisiopatología , Trasplante IsogénicoRESUMEN
To investigate the effects of the Rho-dependent protein kinase (ROCK) inhibitor Y-27632 on the kinetics of E-cadherin, F-actin, and Oct3/4 distributions in dissociated human embryonic stem (hES) cells and to analyze their interactions morphologically, Y-27632-treated [R(i) (+)] and untreated [R(i) (-)] cells were immunohistochemically stained for E-cadherin and Oct3/4 within 24h of dissociation and also for F-actin. Furthermore, the gene expression of E-cadherin, Oct3/4, and RhoA was confirmed by quantitative real-time RT-PCR. E-cadherin expression intensified linearly along the membranes of R(i) (+) cells or intercellular junctions in cell clusters. F-actin accumulated along the periphery of cells and expanded in a web-like manner along junctions in cell clusters, and Oct3/4 was restricted to the nucleus within few hours of dissociation. However, R(i) (-) cells exhibited deformation and blebbing and appeared to die over time. E-cadherin exhibited a punctate pattern along the periphery, after which it accumulated on one or both sides of the cytoplasm. Actin filaments were concentrated at the bleb bases. Oct3/4 was detected in the cytoplasm, not in the nucleus the recovery of integrated E-cadherin distribution. Quantitative real-time RT-PCR revealed RhoA upregulation and E-cadherin downregulation at 12h after dissociation. Oct3/4 gene expression was unaffected by ROCK inhibition. These results revealed that the cooperative nature of hES cells is maintained by the E-cadherin-actin cytoskeleton system along with the restricted distribution of Oct3/4 in the nucleus. RhoA activation followed by dissociation disorders this system and accelerates cell death, which is partially suppressed by ROCK inhibition.
Asunto(s)
Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/ultraestructura , Proteínas de Transporte de Catión Orgánico/metabolismo , Actinas/metabolismo , Amidas/farmacología , Animales , Cadherinas/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Citoplasma/metabolismo , Citoplasma/ultraestructura , Inhibidores Enzimáticos/farmacología , Humanos , Inmunohistoquímica , Cinética , Ratones , Piridinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Quinasas Asociadas a rho/antagonistas & inhibidoresRESUMEN
Although we previously reported the development of cell-dense thickened cardiac tissue by repeated transplantation-based vascularization of neonatal rat cardiac cell sheets, the cell sources for human cardiac cells sheets and their functions have not been fully elucidated. In this study, we developed a bioreactor to expand and induce cardiac differentiation of human induced pluripotent stem cells (hiPSCs). Bioreactor culture for 14 days produced around 8×10(7) cells/100 ml vessel and about 80% of cells were positive for cardiac troponin T. After cardiac differentiation, cardiomyocytes were cultured on temperature-responsive culture dishes and showed spontaneous and synchronous beating, even after cell sheets were detached from culture dishes. Furthermore, extracellular action potential propagation was observed between cell sheets when two cardiac cell sheets were partially overlaid. These findings suggest that cardiac cell sheets formed by hiPSC-derived cardiomyocytes might have sufficient properties for the creation of thickened cardiac tissue.
Asunto(s)
Reactores Biológicos , Diferenciación Celular , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/citología , Ingeniería de Tejidos/métodos , HumanosRESUMEN
There is a great deal of interest in differentiation of human embryonic stem cells (hESCs) into hepatocyte-like cells for application in pharmaceutical screening. Cytochrome P450 (CYP) 1A is involved in the metabolic activation of procarcinogenic compounds as well as in detoxification of drugs. We differentiated hESCs into hepatocyte-like cells (hESC-derived hepatocyte-like cells) and examined whether CYP1A was induced in these cells by typical inducers of CYP1A. hESC-derived hepatocyte-like cells expressed albumin, α-fetoprotein, CYP3A4, CYP3A7, CYP1A1, CYP1A2, and UDP-glucuronyl transferase (UGT) 1A1 mRNA. The levels of CYP1A1, CYP1A2, and UGT1A1 mRNA expression were increased by omeprazole and 3-methylcholanthrene. Furthermore, the enzyme activity of CYP1A was also increased by these compounds. In conclusion, hESC-derived hepatocyte-like cells are available for the detection of CYP1A inducers.
Asunto(s)
Citocromo P-450 CYP1A1/biosíntesis , Células Madre Embrionarias/química , Células Madre Embrionarias/citología , Hepatocitos/química , Hepatocitos/citología , Adulto , Albúminas/metabolismo , Animales , Biotransformación/efectos de los fármacos , Biotransformación/genética , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Línea Celular , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Citocromo P-450 CYP1A2/genética , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Descubrimiento de Drogas , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/enzimología , Inducción Enzimática/efectos de los fármacos , Glucuronosiltransferasa/genética , Glucuronosiltransferasa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Humanos , Inactivación Metabólica , Masculino , Metilcolantreno/farmacología , Ratones , Persona de Mediana Edad , Omeprazol/farmacología , ARN Mensajero/genética , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismoRESUMEN
Cryopreservation is an essential technique in basic research and clinical applications of human embryonic stem (hES) cells. Cryopreserved hES cells are fragile and undergo post-thaw apoptosis. We performed gene pathway analysis on cryopreserved and thawed hES cells to examine the effect of Y-27632, a Rho-associated kinase (ROCK) inhibitor, on apoptosis and associated molecular events. Y-27632 was added to the cryopreservation solution and/or the post-thaw medium of two hES cell lines (KhES-1, KhES-3). Post-thaw apoptosis was recorded as a function of time using Giemsa staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Apoptosis plateaued 12h after the untreated hES cells were thawed. Gene pathway analysis showed the activation of IL-1ß, TGF-ß, and their respective receptors (IL-1R, ACVR1C) in the mitogen-activated protein kinase (MAPK) pathway, which resulted in the upregulation of caspase-8 and -10. Quantitative RT-PCR confirmed the upregulation of IL-1ß, TGF-ß, their respective receptors, and caspase-10 and -3. As these molecules were suppressed by Y-27632, gene pathways involving these molecules probably depend on ROCK activation. The TGF-ß receptor antagonist, SB-431542, and an inhibitor of p38MAPK, SB-203580, did not affect apoptosis. Combining Y-27632 with SB-203580, however, resulted in an increase in the survival rate compared with the control. This suggests that the initiation of apoptosis depends on cytokine interactions and multiple ways exist to reduce post-thaw apoptosis in hES cells. Y-27632 can suppress cytokine interactions and the MAPK pathway, thereby reducing the occurrence of apoptosis, and is an effective cryoprotectant for hES cells.
Asunto(s)
Amidas/farmacología , Apoptosis/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/fisiología , Piridinas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Benzamidas/farmacología , Caspasa 10/biosíntesis , Caspasa 3/biosíntesis , Caspasa 8/biosíntesis , Línea Celular , Supervivencia Celular/efectos de los fármacos , Criopreservación , Crioprotectores/metabolismo , Dioxoles/farmacología , Células Madre Embrionarias/citología , Humanos , Imidazoles/farmacología , Interleucina-1beta/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
OBJECTIVE: The objective of the study was to induce transdifferentiation of human hepatoma HepG2 cells into pancreatic-like cells without direct genetic intervention. METHODS: HepG2 cells were transfected with plasmids for the hepatocyte marker protein green fluorescent protein (albumin-GFP) and the pancreatic cell marker Discosoma spp red fluorescent protein (elastase-DsRed) to create FAE-HepG2 cells. Fluorescent marker expression was used to monitor in vitro transdifferentiation stimulated 100 mM CCl4, 2 mM D-galactosamine, or 200 µM ZnCl2. Concentrations were selected for optimal cell survival rate. Transdifferentiation was also characterized by immunohistochemical detection of amylase, glucagon, and insulin and by polymerase change reaction analysis of amylase and insulin mRNA production. RESULTS: Control cells expressed albumin-GFP but no elastase-DsRed. By 30 days of culture, all 3 agents induced expression of pancreatic-like cell marker elastase-DsRed. ZnCl2 was the most effective as most cells expressed elastase-DsRed in the absence of simultaneous expression of albumin-GFP. For CCl4 and D-galactosamine, elastase-DsRed was expressed in the same cells as albumin-GFP. Cells treated by each agent also expressed amylase, insulin, and glucagon proteins and mRNAs. CONCLUSIONS: Without direct genetic intervention, select low small molecules can induce in vitro transformation of hepatoma cells into pancreatic-like cells.
Asunto(s)
Tetracloruro de Carbono/farmacología , Transdiferenciación Celular/efectos de los fármacos , Cloruros/farmacología , Galactosamina/farmacología , Páncreas/citología , Compuestos de Zinc/farmacología , Albúminas/genética , Albúminas/metabolismo , Amilasas/genética , Amilasas/metabolismo , Expresión Génica/efectos de los fármacos , Glucagón/genética , Glucagón/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células Hep G2 , Humanos , Inmunohistoquímica , Insulina/genética , Insulina/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Fluorescente , Páncreas/metabolismo , Elastasa Pancreática/genética , Elastasa Pancreática/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , TransfecciónRESUMEN
Mouse embryonic stem cells (ESCs) can be induced to form pancreatic exocrine enzyme-producing cells in vitro in a stepwise fashion that recapitulates the development in vivo. However, there is no protocol for the differentiation of pancreatic-like cells from human ESCs (hESCs). Based upon the mouse ESC model, we have induced the in vitro formation of pancreatic exocrine enzyme-producing cells from hESCs. The protocol took place in four stages. In Stage 1, embryoid bodies (EBs) were formed from dissociated hESCs and then treated with the growth factor activin A, which promoted the expression of Foxa2 and Sox17 mRNAs, markers of definitive endoderm. In Stage 2, the cells were treated with all-trans retinoic acid which promoted the transition to cells that expressed gut tube endoderm mRNA marker HNF1b. In Stage 3, the cells were treated with fibroblast growth factor 7 (FGF7), which induced expression of Pdx1 typical of pancreatic progenitor cells. In Stage 4, treatment with FGF7, glucagon-like peptide 1, and nicotinamide induced the expression amylase (AMY) mRNA, a marker for mature pancreatic exocrine cells. Immunohistochemical staining showed the expression of AMY protein at the edges of cell clusters. These cells also expressed other exocrine secretory proteins including elastase, carboxypeptidase A, chymotrypsin, and pancreatic lipase in culture. Production of these hESC-derived pancreatic enzyme-producing cells represents a critical step in the study of pancreatic organogenesis and in the development of a renewable source of human pancreatic-like exocrine cells.
Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular , Cuerpos Embrioides/citología , Páncreas Exocrino/citología , Activinas/farmacología , Amilasas/biosíntesis , Carboxipeptidasas A/biosíntesis , Quimotripsina/biosíntesis , Cuerpos Embrioides/efectos de los fármacos , Cuerpos Embrioides/enzimología , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/enzimología , Factor 7 de Crecimiento de Fibroblastos/farmacología , Péptido 1 Similar al Glucagón/farmacología , Factor Nuclear 3-beta del Hepatocito/biosíntesis , Humanos , Lipasa/biosíntesis , Niacinamida/farmacología , Páncreas Exocrino/enzimología , Elastasa Pancreática/biosíntesis , Factores de Transcripción SOXF/biosíntesis , Tretinoina/farmacologíaRESUMEN
Poor adhesion of single human embryonic stem (hES) cells after freeze-thawing causes death. To investigate mechanisms responsible for this, Rho-dependent protein kinase (ROCK) inhibitor Y-27632-treated and untreated single hES cells were analyzed for E-cadherin and F-actin distribution by immunostaining and phalloidin staining respectively and for G13 signaling pathway components by DNA microarray and quantitative polymerase chain reaction (PCR). Y-27632-treated cells clustered rapidly and maintained E-cadherin and F-actin distribution without losing Oct-3/4. Immediately after thawing, E-cadherin in untreated hES cells dotted along the membrane and then displayed eccentric cytoplasmic localization. Bleb formation and early Oct-3/4 loss occurred after F-actin network condensation in the cytoplasm. Microarray analyses and quantitative PCR indicated upregulation of two actin reorganization-associated components of the G13 signaling pathway, Arhgdib and Cdc42, in untreated cells. Considering these findings and that cell death was partly interrupted by Y-27632, E-cadherin and actin cytoskeleton network disruption through the G13 signaling pathway may cause hES cell death after freeze-thawing.
Asunto(s)
Actinas/metabolismo , Cadherinas/biosíntesis , Células Madre Embrionarias/citología , Congelación , Subunidades alfa de la Proteína de Unión al GTP G12-G13/metabolismo , Transducción de Señal , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Humanos , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Embryonic stem cells (ESCs) are potentially powerful tools for regenerative medicine and establishment of disease models. The recent progress in ESC technologies is noteworthy, but ESC differentiation into renal lineages is relatively less established. The present study aims to differentiate mouse ESCs (mESCs) into a renal progenitor pool, the intermediate mesoderm (IM), without addition of exogenous cytokines and embryoid formation. First, we treated mESCs with a combination of small molecules (Janus-associated tyrosine kinase inhibitor 1, LY294002, and CCG1423) and differentiated them into BMP7-positive cells, BMP7 being the presumed inducing factor for IM. When these cells were cultured with adding retinoic acid, expression of odd-skipped related 1 (Osr1), which is essential to IM differentiation, was enhanced. To simplify the differentiation protocol, the abovementioned four small molecules (including retinoic acid) were combined and added to the culture. Under this condition, more than one-half of the cells were positive for Osr1, and at the same time, Pax2 (another IM marker) was detected by real-time PCR. Expressions of ectodermal marker and endodermal marker were not enhanced, while mesodermal marker changed. Moreover, expression of genes indispensable to kidney development, i.e., Lim1 and WT1, was detected by RT-PCR. These results indicate the establishment of a specific, effective method for differentiation of the ESC monolayer into IM using a combination of small molecules, resulting in an attractive cell source that could be experimentally differentiated to understand nephrogenic mechanisms and cell-to-cell interactions in embryogenesis.
Asunto(s)
Proteína Morfogenética Ósea 7/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Riñón/fisiología , Mesodermo/citología , Regeneración , Anilidas/farmacología , Animales , Benzamidas/farmacología , Comunicación Celular , Linaje de la Célula , Cromonas/farmacología , Técnicas de Cocultivo , Células Madre Embrionarias/clasificación , Expresión Génica , Marcadores Genéticos , Quinasas Janus/antagonistas & inhibidores , Riñón/citología , Ratones , Morfolinas/farmacología , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
To induce hepatocytes from human embryonic stem (hES) cells easily and effectively, a simple suspension culture method that separates ES colonies with a scraper and transfers them into newly developed, nonadherent MPC (2-methacryloyloxyethyl phosphorylcholine) plates, and the staged-additional cocktail method, including growth factors, cytokines, and Lanford serum-free medium, were developed and evaluated mainly by morphological analysis. The formed embryoid bodies (EBs) showed compact cellular agglomeration until day 4 and later formed coeloms in their interior. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis showed that they are gene markers of the three germ layers. Mesenchymal cells with rough endoplasmic reticulum (rER) and extracellular matrix (ECM), and without junctions, were recognized in the interior of the EBs by transmission electron microscopy (TEM) in addition to epithelial cells. When they were stimulated by the staged-additional cocktail, they expressed albumin-positive immunoreactivity, indocyanine green (ICG) uptake, and typical ultrastructures of the hepatocytes, including bile canaliculi. These results indicate that these combined methods promote EB formation and hepatocyte differentiation from hES cells.
Asunto(s)
Células Madre Embrionarias/citología , Hepatocitos/citología , Secuencia de Bases , Línea Celular , Cartilla de ADN , Células Madre Embrionarias/ultraestructura , Humanos , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Cardiomyocytes derived from human embryonic stem (ES) cells are a potential source for cell-based therapy for heart diseases. We studied the effect of bone morphogenetic protein (BMP)-4 in the presence of fetal bovine serum (FBS) on cardiac induction from human H1 ES cells during embryoid body (EB) development. Suspension culture for 4 days with 20% FBS produced the best results for the differentiation of early mesoderm and cardiomyocytes. The addition of Noggin reduced the incidence of beating EBs from 23.6% to 5.3%, which indicated the involvement of BMP signaling in the spontaneous cardiac differentiation. In this condition, treatment with 12.5-25 ng/ml BMP-4 during the 4-day suspension optimally promoted the cardiomyocyte differentiation. The incidence of beating EBs at 25 ng/ml BMP-4 reached 95.8% on day 6 of expansion and then plateaued until day 20. In real-time PCR analysis, the cardiac development-related genes MESP1 and Nkx2.5 were upregulated in the EB outgrowths by 25 ng/ml BMP-4. The activation of BMP signaling in EBs was confirmed by the increase in the phosphorylation of Smad1/5/8 and by the nuclear localization of phospho-Smad1/5/8 and Smad4. The addition of 150 ng/ml Noggin considerably decreased the incidence of beating EBs and Nkx2.5 expression, and Noggin alone increased Nestin expression and neural differentiation in EB outgrowths. The cardiomyocytes induced by 25 ng/ml BMP-4 showed proper cell biological characteristics and a course of differentiation as judged from isoproterenol administration, gene expression, protein assay, immunoreactivity, and subcellular structures. No remarkable change in the extent of apoptosis and proliferation in the cardiomyocytes was observed by BMP-4 treatment. These findings showed that BMP-4 in combination with FBS at the appropriate time and concentrations significantly promotes cardiomyocyte induction from human ES cells.
Asunto(s)
Proteína Morfogenética Ósea 4/farmacología , Técnicas de Cultivo de Célula/métodos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Miocitos Cardíacos/citología , Agonistas Adrenérgicos beta/farmacología , Animales , Proteínas Portadoras/farmacología , Bovinos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Sangre Fetal , Expresión Génica/efectos de los fármacos , Humanos , Isoproterenol/farmacología , Mesodermo/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , SueroRESUMEN
The objective of this study was to establish Liv2, a surface marker of mouse immature hepatocytes (hepatoblasts), as a selection tool for embryonic stem (ES) cell-derived immature hepatocytes by acquiring basic data on Liv2 in normal mouse embryos and by confirming Liv2 expression in mouse ES-derived cells. The estimated molecular weight of Liv2 was 40-45 kDa, and immunoreactivity was definitively detected in the cell membrane of fetal hepatocytes on embryonic day (E) 9.5, declined gradually until E12.5,and subsequently became undetectable. Liv2 was localized on and close to the cell membrane. Embryoid bodies (EB) were formed from mouse ES cells whose undifferentiated state was confirmed with immunostaining of Nanog by the hanging drop method. A few Liv2-positive cells occurred as a cluster in EB outgrowth on day 7, but only some of these were albumin (ALB)-positive on day 13. These cells had the same pattern of immunoreactivity, i.e., localization on the cell membrane, as immature hepatocytes in the developing liver, although there were other types of cells with a different pattern of immunoreactivity that were seen only as a granular pattern in the cytoplasm and without ALB or the neuronal marker nestin. These results suggest thatLiv2 may be useful as a surface marker for immature hepatocytes derived from ES cells.This application would allow for the sole selection of immature hepatocytes and provide a useful tool for regenerative medicine.
Asunto(s)
Biomarcadores/metabolismo , Diferenciación Celular , Hepatocitos/citología , Hepatocitos/metabolismo , Hígado/embriología , Hígado/metabolismo , Animales , Biomarcadores/análisis , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Hígado/citología , Ratones , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Especificidad por SustratoRESUMEN
The influence of cell density of cells cryopreserved inside a collagen matrix at various cooling rates was investigated. Human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (20 mm in diameter and 1 mm in thickness) as an extracellular matrix to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 10(5) to 10(7) cells/cm(3). Four artificial tissues were first stacked in a test chamber, frozen at a cooling rate of 0.3 to 50 degrees C/min in a solution of Dulbecco's Modified Eagle Medium, 20% fetal bovine serum and 10% dimethylsulfoxide, kept frozen below -185 degrees C for 2 hours, and then finally thawed. Membrane integrity of fibroblasts using a trypan blue exclusion assay was evaluated as an index for post-thaw cellular viability. Results show that with increasing cell density, the post-thaw membrane integrity decreased. Therefore, in the cryopreservation of biological tissue, it seems high cell density is one factor which causes a decline in viability.
Asunto(s)
Criopreservación/métodos , Fibroblastos/citología , Conservación de Tejido/métodos , Órganos Artificiales , Recuento de Células , Supervivencia Celular , Colágeno , HumanosRESUMEN
Cryopreservation of embryonic stem (ES) cells is essential to establish them as a resource for regenerative therapy. We evaluated survival adhesion rate, cell structure, gene expression, and multipotency of frozen and thawed embryoid bodies (EBs) derived from mouse ES cells. EBs were cryopreserved using the BICELL and the Program Freezer. After one week the EBs were thawed and cultured. EBs prepared in the Program Freezer had the highest survival adhesion (Program Freezer; 55-69%, BICELL; 30-38%). Though many cells in the thawed EBs were damaged, some were not, especially those prepared in the Program Freezer. RT-PCR analysis showed that genes characteristic of the three embryonic germ layers were expressed in thawed EBs cultured for one week. EBs transplanted into mice formed teratomas consisting of cells derived from the three germ layers. In conclusion, EBs frozen in the Program Freezer had higher survival adhesion rates compared to the BICELL and formed differentiated cells characteristic of the three embryonic germ layers.