RESUMEN
The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.
Asunto(s)
Criopreservación , Hepatocitos , Hígado Artificial , Esferoides Celulares , Hepatocitos/metabolismo , Hepatocitos/citología , Criopreservación/métodos , Esferoides Celulares/metabolismo , Esferoides Celulares/citología , Animales , Supervivencia Celular , Masculino , Temperatura , Ratas , Urea/metabolismo , Humanos , Amoníaco/metabolismo , Fallo Hepático Agudo/terapia , Fallo Hepático Agudo/metabolismo , Hígado/metabolismo , Hígado/citologíaRESUMEN
To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients.
RESUMEN
Bioartificial livers (BAL) may offer acute liver failure (ALF) patients an opportunity for cure without liver transplantation. We evaluated the efficacy of a spheroid-based BAL system, containing aggregates of porcine hepatocytes, in a porcine model of ALF. ALF pigs were divided into three groups. The control group consisted of treatment naïve pigs (n = 5), blank group consisted of pigs that were attached to the BAL system not containing hepatocytes for 12 hours (n = 5) and BAL group consisted of pigs that were attached to the BAL containing hepatocytes for 12 hours (n = 5). Increase in serum ammonia levels were significantly greater in the blank group (P < 0.01) and control group (P < 0.01), compared to the BAL group during the treatment period. Increase in ICP was significantly greater in the control group compared to the BAL group (P = 0.01). Survival was significantly prolonged in the BAL group compared to the blank group (P = 0.03). A BAL system with a bioreactor containing hepatocyte spheroids showed effective clearance of serum ammonia, preservation of renal function and delayed ICP increase in a porcine model of ALF.
Asunto(s)
Células Inmovilizadas , Hepatocitos , Fallo Hepático Agudo , Hígado Artificial , Esferoides Celulares , Animales , Células Inmovilizadas/metabolismo , Células Inmovilizadas/patología , Modelos Animales de Enfermedad , Hepatocitos/metabolismo , Hepatocitos/patología , Fallo Hepático Agudo/metabolismo , Fallo Hepático Agudo/patología , Fallo Hepático Agudo/terapia , Masculino , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , PorcinosRESUMEN
Biophysical wave stimulus has been used as an effective tool to promote cellular maturation and differentiation in the construction of engineered tissue. Pulsed electromagnetic fields (PEMFs) and sound waves have been selected as effective stimuli that can promote neural differentiation. The aim of this study was to investigate the synergistic effect of PEMFs and sound waves on the neural differentiation potential in vitro and in vivo using human bone marrow mesenchymal stem cells (hBM-MSCs). In vitro, neural-related genes in hBM-MSCs were accelerated by the combined exposure to both waves more than by individual exposure to PEMFs or sound waves. The combined wave also up-regulated the expression of neural and synaptic-related proteins in a three-dimensional (3-D) culture system through the phosphorylation of extracellular signal-related kinase. In a mouse model of photochemically induced ischemia, exposure to the combined wave reduced the infarction volume and improved post-injury behavioral activity. These results indicate that a combined stimulus of biophysical waves, PEMFs and sound can enhance and possibly affect the differentiation of MSCs into neural cells. Our study is meaningful for highlighting the potential of combined wave for neurogenic effects and providing new therapeutic approaches for neural cell therapy. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:201-211, 2017.
Asunto(s)
Diferenciación Celular/efectos de la radiación , Células Madre Mesenquimatosas/efectos de la radiación , Células-Madre Neurales/efectos de la radiación , Osteogénesis/efectos de la radiación , Células de la Médula Ósea/citología , Proliferación Celular/efectos de la radiación , Campos Electromagnéticos , Regulación del Desarrollo de la Expresión Génica/efectos de la radiación , Humanos , Neuronas/citología , Neuronas/efectos de la radiación , SonidoRESUMEN
Various animal models of stroke have been developed to simulate the human stroke with the development of the ischemic method facilitates preclinical stroke research. The photothrombotic ischemia model, based on the intravascular photochemical reaction, is widely used for in vivo studies. However, this study has limitations, which generated a relatively small-sized infarction model on superficial cortex compared to that of the MCAO stroke model. In this study, the photothorombosis mouse model is adapted and the optimum conditions for generation of cell death and deficits with high reproducibility is determined. The extent of damage within the cortex was assessed by infarct volume and cellular/behavioral analyses. In this model, the neural cell death and inflammatory responses is detected; moreover, the degree of behavioral impairment is correlated with the brain infarct volume. Further, to enhance the understanding of neural repair, the effect of neural differentiation by transplantation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) is analyzed. The authors demonstrated that transplantation of BM-MSCs promoted the neural differentiation and behavioral performance in their photothrombosis model. Therefore, this research was meaningful to provide a stable animal model of stroke with low variability. Moreover, this model will facilitate development of novel MSC-based therapeutics for stroke.
Asunto(s)
Isquemia Encefálica/terapia , Trombosis Intracraneal/terapia , Trasplante de Células Madre Mesenquimatosas , Accidente Cerebrovascular/terapia , Animales , Células de la Médula Ósea/citología , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Humanos , Trombosis Intracraneal/genética , Trombosis Intracraneal/patología , Células Madre Mesenquimatosas , Ratones , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/fisiopatología , Volumen SistólicoRESUMEN
Effects of mechanical vibration on cell activity and behavior remain controversial: There has been evidence on both positive and negative effects. Furthermore, research on the anterior cruciate ligament (ACL) has as yet been limited and the frequency-related effects remain unknown, even though ACL injury is common and an injured ACL hardly spontaneously recovers. The object of this work was to address the influence of mechanical vibration on ACL fibroblasts, to determine the effects of frequencies, and to further study this effect at the cellular level. We found that sonic vibration affected ACL fibroblasts' proliferation and metabolism in a frequency-dependent manner, and 20 Hz gave rise to the most ACL cell activity and comprehensively increased extracellular matrix (ECM) contents, including collagen type I, collagen type III, fibronectin, elastin, tenascin, glycosaminoglycan (GAG), and the cytoskeleton protein vimentin. Thus, our results indicate that sonic vibration possesses frequency-dependent effects on proliferation and productivity of ACL fibroblast with an optimal frequency of 20 Hz under the present stimulation conditions, providing further information for future research in how vibrational stimulation manipulates ACL cellular behavior.
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Ligamento Cruzado Anterior/citología , Matriz Extracelular/metabolismo , Fibroblastos/citología , Adulto , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Elastina/metabolismo , Femenino , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Sonicación , Tenascina/metabolismo , Vibración , Vimentina/metabolismoRESUMEN
Even though the inducing effect of electromagnetic fields (EMF) on the neural differentiation of human bone marrow mesenchymal stem cells (hBM-MSCs) is a distinctive, the underlying mechanism of differentiation remains unclear. To find out the signaling pathways involved in the neural differentiation of BM-MSCs by EMF, we examined the CREB phosphorylation and Akt or ERK activation as an upstream of CREB. In hBM-MSCs treated with ELF-EMF (50 Hz, 1 mT), the expression of neural markers such as NF-L, MAP2, and NeuroD1 increased at 6 days and phosphorylation of Akt and CREB but not ERK increased at 90 min in BM-MSCs. Moreover, EMF increased phosphorylation of epidermal growth factor receptor (EGFR) as an upstream receptor tyrosine kinase of PI3K/Akt at 90 min. It has been well documented that ELF-MF exposure may alter cellular processes by increasing intracellular reactive oxygen species (ROS) concentrations. Thus, we examined EMF-induced ROS production in BM-MSCs. Moreover, pretreatment with a ROS scavenger, N-acetylcystein, and an EGFR inhibitor, AG-1478, prevented the phosphorylation of EGFR and downstream molecules. These results suggest that EMF induce neural differentiation through activation of EGFR signaling and mild generation of ROS.
Asunto(s)
Células de la Médula Ósea/citología , Diferenciación Celular , Campos Electromagnéticos , Receptores ErbB/metabolismo , Células Madre Mesenquimatosas/citología , Especies Reactivas de Oxígeno/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Humanos , FosforilaciónRESUMEN
Adult stem cells are considered multipotent. Especially, human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have the potential to differentiate into nerve type cells. Electromagnetic fields (EMFs) are widely distributed in the environment, and recently there have been many reports on the biological effects of EMFs. hBM-MSCs are weak and sensitive pluripotent stem cells, therefore extremely low frequency-electromagnetic fields (ELF-EMFs) could be affect the changes of biological functions within the cells. In our experiments, ELF-EMFs inhibited the growth of hBM-MSCs in 12 days exposure. Their gene level was changed and expression of the neural stem cell marker like nestin was decreased but the neural cell markers like MAP2, NEUROD1, NF-L, and Tau were induced. In immunofluorescence study, we confirmed the expression of each protein of neural cells. And also both oligodendrocyte and astrocyte related proteins like O4 and GFAP were expressed by ELF-EMFs. We suggest that EMFs can induce neural differentiation in BM-MSCs without any chemicals or differentiation factors.
Asunto(s)
Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Células Madre Mesenquimatosas/química , Células Madre Mesenquimatosas/citología , Células-Madre Neurales/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular , Campos Electromagnéticos , Expresión Génica , Humanos , Células Madre Mesenquimatosas/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/química , Células-Madre Neurales/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMEN
AIMS: Adipose tissue-derived stem cells (AT-MSCs) have been proposed as a new source for nervous tissue damage due to their capacity of neural differentiation potential including neurons, oligodendrocytes and astrocytes. Recently, many studies have demonstrated that sub-sonic vibration (SSV) is an effective cell differentiation method but there have been no studies on the effect of SSV about AT-MSC differentiation into neural-like cells in vitro. Therefore, we examined the effect of SSV on AT-MSCs to investigate the differentiation potential of neural-like cells. MAIN METHODS: We assessed the changes in AT-MSCs by SSV during 4 days at 10, 20, 30 and 40 Hz (1.0 V). After stimulation, they were analyzed by RT-PCR, Western blot and immunohistological analysis using neural cell type-specific genes and antibodies. Further, to confirm the neural differentiation, we investigated adipogenic genes for RT-PCR analysis. For a mechanism study, we analyzed activation levels in time course of ERK phosphorylation after SSV. KEY FINDINGS: After 4-day SSV exposure, we observed morphological changes of AT-MSCs. Further, SSV induced gene/protein levels of neural markers while inhibiting adipogenesis and they were mainly upregulated at 30 Hz. In addition, phosphorylated ERK level was increased in a time-dependent manner upon 30 Hz SSV for 6h. SIGNIFICANCE: These results demonstrated that SSV affects AT-MSCs differentiation potential and 30 Hz SSV affected neural differentiation on AT-MSCs.
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Tejido Adiposo/metabolismo , Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/metabolismo , Neuronas/metabolismo , Vibración , Adipogénesis/fisiología , Tejido Adiposo/citología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Fosforilación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Regulación hacia Arriba/fisiologíaRESUMEN
AIMS: Adult stem cells, such as umbilical cord-derived mesenchymal stem cells (UC-MSCs), have the potential to differentiate into various types of cells, including neurons. Research has shown that mechanical stimulation induces a response in MSCs, specifically, low and high intensity sub-sonic vibration (SSV) has been shown to facilitate wound healing. In this study, the effects of SSV were examined by assessing the proliferation and differentiation properties of MSCs. MAIN METHODS: hUC-MSCs were isolated from Wharton's jelly, including the smooth muscle layer of the umbilical cord. During subculture, the cells were passaged every 5-6 days using nonhematopoietic stem cell media. To measure the effect of sonic vibration, SSV was applied to these cells continuously for 5 days. KEY FINDINGS: In this study, the morphology of hUC-MSCs was altered to resemble neurons by SSV. Further, the mRNA and protein levels of neuron-specific markers, including MAP2, NF-L, and NeuroD1, increased. In addition, other neural cell markers, such as GFAP and O4, were increased. These results suggest that hUC-MSCs differentiated into neural cells upon SSV nonselectively. In a mechanism study, the ERK level increased in a time-dependent manner upon SSV for 12 h. SIGNIFICANCE: The results of this study suggest that SSV caused hUC-MSCs to differentiate into neural cells via ERK activation.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/fisiología , Neuronas/citología , Cordón Umbilical/citología , Vibración , Biomarcadores/metabolismo , Western Blotting , Cartilla de ADN/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inmunohistoquímica , Estimulación Física , Reacción en Cadena en Tiempo Real de la Polimerasa , Gelatina de Wharton/citologíaRESUMEN
In this study, we evaluated the effect of mechanical stimulation on the differentiation of umbilical cord-derived mesenchymal stem cells (UC-MSCs) in osteogenic medium using a Flexcell system that imposed cyclic uniaxial mechanical stimulation at a strain of 0%, 5%, or 10% (5 s of stretch and 15 s of relaxation) for 10 days. The expression of MSC surface antigens (CD73, CD90, and CD105) was significantly decreased as strain increased. Mechanical stimulation inhibited the growth of UC-MSCs and slightly raised lactate dehydrogenase production. Mechanically stimulated groups produced more elastin and sulfated glycosaminoglycan than unstimulated groups and these increases were in proportion to the degree of strain. Reverse transcription-polymerase chain reaction analysis revealed that mechanical stimulation induced a significant increase in the mRNA expression of osteoblast differentiation markers. The mRNA levels of osteopontin, osteonectin, and type I collagen in the 5% and 10% strained groups were significantly higher than those in the 0% strained group. From the Western blot analysis, UC-MSCs produced bone sialoprotein and vimentin in a mechanical strain-dependent manner. Thus, cyclic mechanical loading was able to enhance the differentiation of human UC-MSCs into osteoblast-like cells as determined by osteogenic gene and protein expression. Furthermore, this finding has important implications for the use of the combination of mechanical and osteogenic differentiation media for UC-MSCs in tissue engineering and regenerative medicine.
Asunto(s)
Mecanotransducción Celular/fisiología , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Estimulación Física/métodos , Cordón Umbilical/citología , Antígenos de Superficie/metabolismo , Biomarcadores/metabolismo , Diferenciación Celular , Aumento de la Célula , Proliferación Celular , Supervivencia Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Elastina/metabolismo , Expresión Génica , Glicosaminoglicanos/metabolismo , Humanos , L-Lactato Deshidrogenasa/metabolismo , Mecanorreceptores/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Madre Mesenquimatosas/enzimología , Osteoblastos/metabolismo , Osteonectina/genética , Osteonectina/metabolismo , Osteopontina/genética , Osteopontina/metabolismo , ARN Mensajero/metabolismoRESUMEN
AIMS: Although low and high intensity sub-sonic vibrations (SSV) have been shown to facilitate wound healing, very few studies have investigated the effects of SSV on 3T3-L1 preadipocytes. Therefore, the present study was undertaken to investigate the influence of SSV on the proliferation and maturation of 3T3-L1 preadipocytes. MAIN METHODS: To evaluate the effect of SSV on 3T3-L1 cell proliferation, the cells were maintained in an apparatus that administered SSV (0.5 V) for 3 days at a frequency of 10, 20, 30, or 40 Hz. In addition, to study the effect of SSV on 3T3-L1 cell maturation, the cells were stimulated with SSV for 6 days at a frequency of 10, 20, 30, or 45 Hz. KEY FINDINGS: Sub-sonic vibrations inhibited the proliferation of 3T3-L1 preadipocytes at frequencies of 20 and 30 Hz. Triglyceride levels in cells subjected to SSV at frequencies ranging from 10 to 30 Hz increased compared with those measured in control cells. The expression of adipogenic genes, such as PPAR-γ and C/EBP-α, markedly increased in response to SSV at 20 Hz and 30 Hz during maturation. SIGNIFICANCE: These results suggest that SSV affected adipogenic gene expression at 20 and 30 Hz.
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Células 3T3-L1/citología , Proliferación Celular , Regulación de la Expresión Génica/fisiología , Vibración/uso terapéutico , Células 3T3-L1/química , Animales , Bromodesoxiuridina , Cartilla de ADN/genética , L-Lactato Deshidrogenasa/análisis , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales de Tetrazolio , Tiazoles , Triglicéridos/análisisRESUMEN
BACKGROUND: The methods currently used for treating alopecia have some limitations. The drug treatment is so temporary that medication discontinuance may progress depilation immediately. The number of hair transplantation restricts because total transplantable hair number is no increase. To overcome these problems, researchers have attempted the in vitro culturing of hair follicle cells and implanting these cells in the treatment area. OBJECTIVES: In the present study, culture-expanded mesenchymal stem cells (MSCs) that do not possess aggregative activity were used to produce self-aggregated cell-aggregated spheroidal dermal papilla like tissues (DPLTs) with the aid of a special culture condition in vitro, and hair bulb structure inductive capacity pertinent to the aggregative activity was then evaluated. Then hair inducing activity of self-aggregated DPLTs employing MSCs was tested in athymic mice. METHODS: We isolated and cultivated MSCs from bone marrow and umbilical cord in vitro. After propagated MSCs underwent preconditioning in dermal papilla forming medium (DPFM), then subcultured MSCs formed self-aggregated DPLTs. We compared real human scalp dermal papilla cells (hDPCs) with DPLTs employing DPCs, DPLTs employing hBM-MSCs and DPLTs employing hUC-MSCs. RESULTS: Light microscopy and immunohistochemical staining were used to confirm that reconstructed DPLTs generated by this procedure had the size, shape, and expression of protein similar to actual DP. CONCLUSIONS: The DPLTs have the same hair bulb structure inductive ability as natural DPLTs in vitro. Transplanted DPLTs can induce new hair follicle in athymic mice. As a result, UC-MSCs and BM-MSCs may be an applicable and novel cell source for the generation of human hair cell therapy.
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Células de la Médula Ósea/citología , Folículo Piloso/citología , Folículo Piloso/crecimiento & desarrollo , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Animales , Diferenciación Celular , Proliferación Celular , Trasplante de Células , Células Cultivadas , Humanos , Ratones , Ratones DesnudosRESUMEN
The purpose of this study was to evaluate the biocompatibility of silk and collagen-hyaluronan (HA) in vitro by assessing anterior cruciate ligament (ACL) cell and T-lymphocyte cultures on scaffolds. The use of composite scaffolds as artificial ligaments in ACL reconstruction and their effects on angiogenesis were evaluated in vivo. The silk scaffold was knitted by hand and dry coated with collagen-HA, whereas the composite silk scaffold was made by covering a silk scaffold with a lyophilized collagen-HA substrate. The initial attachment and proliferation of human ACL cells on the composite silk scaffold was superior to the attachment and proliferation observed on the silk scaffold. The immune response was higher in both scaffolds after 72 h (p < 0.05) compared with the control culture condition without scaffolding, as assessed by T-lymphocyte cultures in vitro. There was no significant difference in the immune response in vitro between the silk and composite silk scaffolds. Silk and composite silk scaffolds were implanted as artificial ligaments in ACLs removed from the knees of dogs, and they were harvested 6 weeks after implantation. On gross examination, the onset of an inflammatory tissue reaction, such as synovitis, was seen in both the silk scaffold and the composite silk scaffold groups. An histological evaluation of the artificial ligament implants revealed the presence of monocytes in the silk composite scaffold and the absence of giant cells in all cases. MT staining in the composite silk scaffold-grafted group showed granulation tissue consisting of fibroblasts, lymphocytes, monocytes, and collagen fibers. In addition, CD31 staining revealed the formation of new blood vessels. On the other hand, no reparative tissues, such as blood vessels, collagen, and cells, were observed in the silk scaffold-grafted group. These results suggest that the lyophilized collagen-HA substrate is biocompatible in vitro and enhances new blood vessel and cell migration in vivo.
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Ligamento Cruzado Anterior/cirugía , Neovascularización Fisiológica , Ingeniería de Tejidos , Animales , Ligamento Cruzado Anterior/irrigación sanguínea , Ligamento Cruzado Anterior/citología , Fenómenos Biomecánicos , Movimiento Celular , Proliferación Celular , Células Cultivadas , Colágeno/administración & dosificación , Perros , Humanos , Ácido Hialurónico/administración & dosificación , Ensayo de MaterialesRESUMEN
Umbilical cord blood (UCB) is a rich source of hematopoietic stem cells that possesses practical and ethical advantages. We previously reported a novel UCB-derived adult stem cells which we termed umbilical cord blood-derived multipotent progenitor cells' (MPCs). MPCs were capable of differentiating into functional neuronal cells. Under appropriate conditions lasting several days or weeks, we now show that the MPCs differentiate into hepatocyte-like cells in vitro; their properties were verified using reverse transcription-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, periodic acid-Schiff (PAS) staining of accumulated glycogen and an enzyme-linked immunosorbent assay (ELISA). We also found that hepatic differentiated cells expressed hepatocyte specific markers, such as albumin, hepatocyte nuclear factor (HNF)-1alpha, HNF4, cytokeratin (CK)-8, CK-18, tyrosine amino transferase (TAT), and CYP2B6. Moreover, albumin was secreted, which suggests that MPCs from UCB possess multi-differentiation potential and have the capacity to differentiate into functional cells of hepatic lineage in vitro.
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Diferenciación Celular/fisiología , Sangre Fetal/citología , Hepatocitos/fisiología , Hígado/fisiología , Células Madre Multipotentes/fisiología , Adulto , Biomarcadores/metabolismo , Forma de la Célula , Células Cultivadas , Niño , Medios de Cultivo Condicionados , Factor 4 de Crecimiento de Fibroblastos/metabolismo , Hepatocitos/citología , Humanos , Hígado/citología , Células Madre Multipotentes/citologíaRESUMEN
Mesenchymal stem cells (MSCs) derived from bone marrow are an important tool in tissue engineering and cell-based therapies because of their multipotent capacity. Majority of studies on MSCs have investigated the roles of growth factors, cytokines, and hormones. Antioxidants such as ascorbic acid can be used to expand MSCs while preserving their differentiation ability. Moreover, ascorbic acid can also stimulate MSC proliferation without reciprocal loss of phenotype and differentiation potency. In this study, we evaluated the effects of ascorbic acid on the proliferation, differentiation, extracellular matrix (ECM) secretion of MSCs. The MSCs were cultured in media containing various concentrations (0-500 microM) of L-ascorbate-2-phosphate (Asc-2-P) for 2 weeks, following which they were differentiated into adipocytes and osteoblasts. Ascorbic acid stimulated ECM secretion (collagen and glycosaminoglycan) and cell proliferation. Moreover, the phenotypes of the experimental groups as well as the differentiation potential of MSCs remained unchanged. The apparent absence of decreased cell density or morphologic change is consistent with the toxicity observed with 5-250 microM concentrations of Asc-2-P. The results demonstrate that MSC proliferation or differentiation depends on ascorbic acid concentration.
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Ácido Ascórbico/administración & dosificación , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Células de la Médula Ósea/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Células Madre Mesenquimatosas/efectos de los fármacosRESUMEN
Glycogen storage disease type I (GSD-I) is a group of autosomal recessive disorders with an incidence of 1 in 100,000. The two major subtypes are GSD-Ia, caused by a deficiency of glucose-6-phosphatase (G6Pase), and GSD-Ib, caused by a deficiency of glucose-6-phosphate transporter (G6PT). We report that a substantial improvement was achieved following several infusions of hepatocytes in a patient with GSD-Ib. Hepatocytes were isolated from the unused cadaveric whole livers of two donors. At the first transplantation, approximately 2 x 10(9) cells (2% of the estimated recipient's total hepatocytes) were infused. Seven days later 1 x 10(9) (1% of liver mass) cryopreserved hepatocytes from the same donor were infused, and an additional 3 x 10(9) (3% of liver mass) cells from the second donor were infused 1 month after the second transplantation. After the hepatocyte transplantation, the patient showed no hypoglycemic symptoms despite the discontinuation of cornstarch meals. Liver biopsies on posttransplantation days 20 and 250 showed a normal level of glucose-6-phosphatase activity in presolubilization assay that was very low before transplantation. This was the first and successful clinical hepatocyte transplantation in Korea. In this study, hepatocyte transplantation allowed a normal diet in a patient with GSD-Ib, with substantial improvement in their quality of life. Hepatocyte transplantation might be an alternative to liver transplantation and dietary therapy in GSD-Ib.
Asunto(s)
Glucosa-6-Fosfatasa/metabolismo , Glucosa-6-Fosfato/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/metabolismo , Enfermedad del Almacenamiento de Glucógeno Tipo I/terapia , Hepatocitos/trasplante , Adolescente , Cadáver , Estudios de Seguimiento , Glucosa-6-Fosfato/deficiencia , Enfermedad del Almacenamiento de Glucógeno Tipo I/patología , Hepatocitos/enzimología , Humanos , Inmunosupresores/uso terapéutico , Corea (Geográfico) , Hígado/citología , Hígado/inmunología , Masculino , Calidad de Vida , Inmunología del Trasplante/efectos de los fármacos , Trasplantes , Resultado del TratamientoRESUMEN
Rabbit corneal epithelium was reconstructed using tilting dynamic culture with a self-manufactured, amniotic membrane (AM) supporter and a lyophilized amniotic membrane (LAM). Rabbit corneal epithelial (RCE) cells were cultured and cryopreserved after isolation from the limbus. The second- and third-passage RCE cells were plated onto the epithelial side of the LAM of Ahn's AM supporter. Two days later, the air-liquid interface culture was maintained with third-passage RCE cells for 6 days and second-passage corneal epithelial cells for 9 days. The average viability of thawed RCE cells, assessed using trypan blue dye exclusion, was 77.42%. The reconstructed corneal epithelium was characterized by histological (hematoxylin and eosin) and immunohistochemical staining (proliferating cell nuclear antigen) for light microscopy, and by reverse transcriptase-polymerase chain reaction, glucose assay, and transmission electron microscopy. The basal layer of the reconstructed corneal epithelium was well formed, and the epithelium was tightly constructed due to the increase in cell proliferation and differentiation caused by the tilting dynamic culture, as opposed to static culture. Tilting dynamic culture was useful for the reconstruction of the epithelium using easily damaged epithelial cells and resulted in more stratum cell layers. Moreover, cytokeratin (CK3) mRNA expression in tilting dynamic cultured third-passage RCE cells seeded onto AM was greater than in static cultured third-passage RCE cells. The morphology of the reconstructed corneal epithelium on LAM by tilting dynamic culture for 9 days resembled that of the skin epidermis. This was thought to be because the tilting dynamic culture not only accelerated the proliferation and differentiation of cells by physical or mechanical stimulation, but also ensured that the supply of medium was delivered to the basal cells more efficiently. Thus, the reconstruction of the corneal epithelium using LAM and tilting dynamic culture was considered to be a good in vitro model for autologous or allogeneic transplantation of corneal epithelium and skin epidermis in patients with damaged epithelia.