RESUMEN

BACKGROUND/AIMS: Although several studies have demonstrated that mesenchymal stromal cells (MSCs) exhibit beneficial immunomodulatory properties in preclinical models of allergic asthma, effects on airway remodeling have been controversial. Recent evidence has shown that MSCs modify their in vivo immunomodulatory actions depending on the specific inflammatory environment encountered. Accordingly, we assessed whether the therapeutic properties of human mesenchymal stromal cells (hMSCs) could be potentiated by conditioning these cells with serum (hMSC-serum) obtained from patients with asthma and then transplanted in an experimental model of house dust mite (HDM)-induced allergic asthma. METHODS: hMSC and hMSC-serum were administered intratracheally 24 h after the final HDM challenge. hMSC viability and inflammatory mediator production, lung mechanics and histology, bronchoalveolar lavage fluid (BALF) cellularity and biomarker levels, mitochondrial structure and function as well as macrophage polarization and phagocytic capacity were assessed. RESULTS: Serum preconditioning led to: (i) increased hMSC apoptosis and expression of transforming growth factor-ß, interleukin (IL)-10, tumor necrosis factor-α-stimulated gene 6 protein and indoleamine 2,3-dioxygenase-1; (ii) fission and reduction of the intrinsic respiratory capacity of mitochondria; and (iii) polarization of macrophages to M2 phenotype, which may be associated with a greater percentage of hMSCs phagocytosed by macrophages. Compared with mice receiving hMSCs, administration of hMSC-serum led to further reduction of collagen fiber content, eotaxin levels, total and differential cellularity and increased IL-10 levels in BALF, improving lung mechanics. hMSC-serum promoted greater M2 macrophage polarization as well as macrophage phagocytosis, mainly of apoptotic hMSCs. CONCLUSIONS: Serum from patients with asthma led to a greater percentage of hMSCs phagocytosed by macrophages and triggered immunomodulatory responses, resulting in further reductions in both inflammation and remodeling compared with non-preconditioned hMSCs.

Asunto(s)

Asma , Células Madre Mesenquimatosas , Humanos , Asma/terapia , Pulmón/patología , Macrófagos/metabolismo , Células Madre Mesenquimatosas/metabolismo , Fagocitosis

RESUMEN

Bone marrow stromal cells (BMSCs, also known as bone marrow mesenchymal stem cells) are a plastic-adherent heterogeneous cell population that contain inherent skeletal progenitors and a subset of multipotential skeletal stem cells (SSCs). Application of BMSCs in therapeutic protocols implies its isolation and expansion under good manufacturing practices (GMP). Here we describe the procedures we have found to successfully generate practical BMSCs numbers, with preserved biological potency.

Asunto(s)

Tecnología Biomédica/normas , Células de la Médula Ósea/citología , Huesos/citología , Cultivo Primario de Células/métodos , Antígenos CD34/genética , Antígenos CD34/metabolismo , Tecnología Biomédica/métodos , Células Cultivadas , Técnicas de Cocultivo/economía , Técnicas de Cocultivo/métodos , Técnicas de Cocultivo/normas , Costos y Análisis de Costo , Medio de Cultivo Libre de Suero/química , Humanos , Guías de Práctica Clínica como Asunto , Cultivo Primario de Células/economía , Cultivo Primario de Células/normas , Células del Estroma/citología , Células del Estroma/metabolismo

RESUMEN

This study evaluated physical-chemical characteristics of a vacuumed collagen-impregnated bioglass (BG) scaffolds and bone marrow stromal cells (BMSCs) behavior on those composites. scanning electron microscope and energy dispersive x-ray spectroscope demonstrated collagen (Col) was successfully introduced into BG. Vacuum impregnation system has showed efficiency for Col impregnation in BG scaffolds (approximately 20 wt %). Furthermore, mass weight decreasing and more stabilized pH were observed over time for BG/Col upon incubation in phosphate buffered saline compared to plain BG under same conditions. Calcium evaluation (Ca assay) demonstrated higher calcium uptake for BG/Col samples compared to BG. In addition, BG samples presented hydroxyapatite crystals formation on its surface after 14 days in simulated body fluid solution, and signs of initial degradation were observed for BG and BG/Col after 21 days. Fourier transform infrared spectroscopy spectra for both groups indicated peaks for hydroxyapatite formation. Finally, a significant increase of BMSCs viability for both composites was observed compared to control group, but no increase of osteogenic differentiation-related gene expressions were found. In summary, BG/Col scaffolds have improved degradation, pH equilibrium and Ca mineralization over time, accompanied by hydroxyapatite formation. Moreover, both BG and BG/Col scaffolds were biocompatible and noncytotoxic, promoting a higher cell viability compared to control. Future investigations should focus on additional molecular and in vivo studies in order to evaluate biomaterial performance for bone tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 211-222, 2019.

Asunto(s)

Células de la Médula Ósea/metabolismo , Diferenciación Celular , Proliferación Celular , Cerámica/química , Colágeno/química , Células Madre Mesenquimatosas/metabolismo , Andamios del Tejido/química , Animales , Células de la Médula Ósea/citología , Ensayo de Materiales , Células Madre Mesenquimatosas/citología , Ratas , Ratas Wistar

RESUMEN

Asthma is a chronic inflammatory disease characterized by airway inflammation and remodeling, which can lead to progressive decline of lung function. Although mesenchymal stromal cells (MSCs) have shown beneficial immunomodulatory properties in preclinical models of allergic asthma, effects on airway remodeling have been limited. Mounting evidence suggests that prior exposure of MSCs to specific inflammatory stimuli or environments can enhance their immunomodulatory properties. Therefore, we investigated whether stimulating MSCs with bronchoalveolar lavage fluid (BALF) or serum from asthmatic mice could potentiate their therapeutic properties in experimental asthma. In a house dust mite (HDM) extract asthma model in mice, unstimulated, asthmatic BALF-stimulated, or asthmatic serum-stimulated MSCs were administered intratracheally 24 hours after the final HDM challenge. Lung mechanics and histology; BALF protein, cellularity, and biomarker levels; and lymph-node and bone marrow cellularity were assessed. Compared with unstimulated or BALF-stimulated MSCs, serum-stimulated MSCs further reduced BALF levels of interleukin (IL)-4, IL-13, and eotaxin, total and differential cellularity in BALF, bone marrow and lymph nodes, and collagen fiber content, while increasing BALF IL-10 levels and improving lung function. Serum stimulation led to higher MSC apoptosis, expression of various mediators (transforming growth factor-ß, interferon-γ, IL-10, tumor necrosis factor-α-stimulated gene 6 protein, indoleamine 2,3-dioxygenase-1, and IL-1 receptor antagonist), and polarization of macrophages to M2 phenotype. In conclusion, asthmatic serum may be a novel strategy to potentiate therapeutic effects of MSCs in experimental asthma, leading to further reductions in both inflammation and remodeling than can be achieved with unstimulated MSCs. Stem Cells Translational Medicine 2019;8:301&312.

Asunto(s)

Asma/inmunología , Asma/terapia , Células Madre Mesenquimatosas/inmunología , Animales , Líquido del Lavado Bronquioalveolar/inmunología , Modelos Animales de Enfermedad , Femenino , Interleucina-10/inmunología , Interleucina-13/inmunología , Interleucina-4/inmunología , Pulmón/inmunología , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , Ratones , Ratones Endogámicos BALB C

RESUMEN

Bone tissue engineering (BTE) has the general objective of restoring and improving damaged bone. A very interesting strategy for BTE is to combine an adequate polymeric scaffold with an osteoinductive compound. Strontium is a divalent cation that can substitute calcium in hydroxyapatite and induce both anabolic and anti-catabolic effects in bone. On the other hand, systemic increases in Sr2+ levels can provoke adverse cardiovascular effects. In the present study we have developed a compatibilized blend of poly-ε-caprolactone (PCL) and polydiisopropyl fumarate (PDIPF) enriched with 1% or 5% Sr2+ and evaluated the applicability of these biomaterials for BTE, both in vitro and in vivo. In vitro, whereas Blend + 5% Sr2+ was pro-inflammatory and anti-osteogenic, Blend + 1% Sr2+ released very low quantities of the cation; was not cytotoxic for cultured macrophages; and showed improved osteocompatibility when used as a substratum for primary cultures of bone marrow stromal cells. In vivo, implants with Blend + 1% Sr2+ significantly increased bone tissue regeneration and improved fibrous bridging (vs. Blend alone), while neither inducing a local inflammatory response nor increased serum levels of Sr2+. These results indicate that our compatibilized blend of PCL-PDIPF enriched with 1% Sr2+ could be useful for BTE.

Asunto(s)

Fumaratos , Poliésteres , Polímeros , Estroncio , Ingeniería de Tejidos/métodos , Andamios del Tejido , Animales , Materiales Biocompatibles , Huesos , Supervivencia Celular , Células Cultivadas , Masculino , Ratones , Óxido Nítrico/fisiología , Células RAW 264.7 , Ratas , Células del Estroma/fisiología

RESUMEN

Diabetes mellitus is associated with a decrease in bone quality and an increase in fracture incidence. Additionally, treatment with anti-diabetic drugs can either adversely or positively affect bone metabolism. In this study we evaluated: the effect of a 3-week oral treatment with saxagliptin on femoral microarchitecture in young male non-type-2-diabetic Sprague Dawley rats; and the in vitro effect of saxagliptin and/or fetal bovine serum (FBS), insulin or insulin-like growth factor-1 (IGF1), on the proliferation, differentiation (Runx2 and PPAR-gamma expression, type-1 collagen production, osteocalcin expression, mineralization) and extracellular-regulated kinase (ERK) activation, in bone marrow stromal cells (MSC) obtained from control (untreated) rats and in MC3T3E1 osteoblast-like cells. In vivo, oral saxagliptin treatment induced a significant decrease in the femoral osteocytic and osteoblastic density of metaphyseal trabecular bone and in the average height of the proximal cartilage growth plate; and an increase in osteoclastic tartrate-resistant acid phosphatase (TRAP) activity of the primary spongiosa. In vitro, saxagliptin inhibited FBS-, insulin- and IGF1-induced ERK phosphorylation and cell proliferation, in both MSC and MC3T3E1 preosteoblasts. In the absence of growth factors, saxagliptin had no effect on ERK activation or cell proliferation. In both MSC and MC3T3E1 cells, saxagliptin in the presence of FBS inhibited Runx2 and osteocalcin expression, type-1 collagen production and mineralization, while increasing PPAR-gamma expression. In conclusion, orally administered saxagliptin induced alterations in long-bone microarchitecture that could be related to its in vitro down-regulation of the ERK signaling pathway for insulin and IGF1 in MSC, thus decreasing the osteogenic potential of these cells.

Asunto(s)

Adamantano/análogos & derivados , Células de la Médula Ósea/efectos de los fármacos , Dipéptidos/toxicidad , Inhibidores de la Dipeptidil-Peptidasa IV/toxicidad , Fémur/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Adamantano/administración & dosificación , Adamantano/toxicidad , Administración Oral , Animales , Biomarcadores/metabolismo , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Proliferación Celular/efectos de los fármacos , Dipéptidos/administración & dosificación , Inhibidores de la Dipeptidil-Peptidasa IV/administración & dosificación , Relación Dosis-Respuesta a Droga , Fémur/metabolismo , Fémur/patología , Masculino , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Células del Estroma/metabolismo , Células del Estroma/patología , Factores de Tiempo

RESUMEN

Primary human bone marrow stromal cells (hMSCs) were transfected with human telomerase reverse transcriptase (hTERT) gene with lipofection method. The hTERT transfected hMSCs of passage 100 underwent chondrogenesis induction with dexamethasone, transforming the growth factor β and vitamin C, osteogenesis induction with dexamethasone, β glycerophosphoric acid and vitamin C, and cardiomyocyte induction with 5-azacytidine. After 7, 14, 21 and 28 days of induction, immunocytochemistry was performed to detect the expressions of type I and II collagen and osteocalcin, and alizarin red staining was performed to detect the bone nodule formation in osteogenesis induction. Immunocytochemistry was carried out to detect the striated muscle actin expression in cardiomyocytes. The hMSCs undergoing successful transfection were positive for the hTERT. The hTERT transfected cells were grown in vitro successfully and passaged for 136 generations. Results showed that these cells could be induced to differentiate into chondrocytes, bone and myocardial cells. Introduction of exogenous hTERT into hMSCs could achieve immortalized hMSCs with the potential of multi-directional differentiation. Thus, these cells could be applied as seed cells in tissue engineering.

RESUMEN

In this study, in vitro cytocompatibility was investigated in the Ti-30Ta alloy after two kinds of surfaces treatments: alkaline and biomimetic treatment. Each condition was evaluated by scanning electron microscopy/energy-dispersive X-ray spectroscopy. Cellular adhesion, viability, protein expression, morphology, and differentiation were evaluated with Bone marrow stromal cells (MSCs) to investigate the short and long-term cellular response by fluorescence microscope imaging and colorimetric assays techniques. Two treatments exhibited similar results with respect to total protein content and enzyme activity as compared with alloy without treatment. However, it was observed improved of the biomineralization, bone matrix formation, enzyme activity, and MSCs functionality after biomimetic treatment. These results indicate that the biomimetic surface treatment has a high potential for enhanced osseointegration.

Asunto(s)

Aleaciones , Células Madre Mesenquimatosas/citología , Tantalio , Titanio , Animales , Microscopía Electrónica de Rastreo , Ratas , Ratas Wistar , Propiedades de Superficie

RESUMEN

Hypoxia inducible factor-1α (HIF-1α) is an important transcription factor, which plays a critical role in the formation of solid tumor and its microenviroment. The objective of the present study was to evaluate the expression and function of HIF-1α in human leukemia bone marrow stromal cells (BMSCs) and to identify the downstream targets of HIF-1α. HIF-1α expression was detected at both the RNA and protein levels using real-time PCR and immunohistochemistry, respectively. Vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) were detected in stromal cells by enzyme-linked immunosorbent assay. HIF-1α was blocked by constructing the lentiviral RNAi vector system and infecting the BMSCs. The Jurkat cell/BMSC co-cultured system was constructed by putting the two cells into the same suitable cultured media and conditions. Cell adhesion and secretion functions of stromal cells were evaluated after transfection with the lentiviral RNAi vector of HIF-1α. Increased HIF-1α mRNA and protein was detected in the nucleus of the acute myeloblastic and acute lymphoblastic leukemia compared with normal BMSCs. The lentiviral RANi vector for HIF-1α was successfully constructed and was applied to block the expression of HIF-1α. When HIF-1α of BMSCs was blocked, the expression of VEGF and SDF-1 secreted by stromal cells were decreased. When HIF-1α was blocked, the co-cultured Jurkat cell’s adhesion and migration functions were also decreased. Taken together, these results suggest that HIF-1α acts as an important transcription factor and can significantly affect the secretion and adhesion functions of leukemia BMSCs.

Asunto(s)

Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Leucemia de Células T/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adhesión Celular , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Células Jurkat , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

Las células madre estromales humanas y de roedores cultivadas pueden ser inducidas a diferenciarse en neuronas, enfatizando su utilidad potencial en la terapia celular neurorrestaurativa. Los sistemas de cultivo para la expansión de estas células describen el uso de diferentes proporciones de suero fetal, lo que motivó a estudiar qué concentración de suero fetal bovino era capaz de garantizar un adecuado rendimiento celular. Las células de la médula ósea de rata se cultivaron en medio a-MEM suplementado con 10 y 20 por cientode suero fetal bovino y se subcultivaron hasta 3 veces. La viabilidad celular de los cultivos primarios y los subcultivos estuvo por encima del 98 por ciento en ambos experimentos. Los cultivos primarios demoraron 17,4 días en confluir y los subcultivos 7,7 días. La concentración de suero fetal al 20 por ciento no aumentó significativamente la velocidad de multiplicación celular; no obstante, se obtuvo un mayor número de células estromales. El sistema de expansión in vitro podría utilizarse en estudios futuros para la expansión de las células estromales humanas, lo que sienta mejores bases para su aplicación clínica(AU)

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Cultured human and rodents stromal stem cells can be induced to differentiate into neurons, emphasizing its potential use in neurorestorative cell therapy. Cropping systems for the expansion of these cells describe the use of different ratios of fetal serum, which led to study what concentration of fetal calf serum was able to ensure an adequate cell yield. Cells from rat bone marrow were cultured in medium supplemented with a-MEM 10 and 20 percent fetal bovine serum and subcultured up to 3 times. Cell viability of primary cultures and subcultures was above 98 percent in both experiments. Primary cultures converge delayed in 17.4 days and 7.7 days subcultures. The concentration of 20 percent fetal calf serum did not significantly increase the speed of cell division, however, we obtained a greater number of stromal cells. The expansion in vitro system could be used in future studies for the expansion of human stromal cells, which feels better basis for clinical application(AU)

Asunto(s)

Células del Estroma/trasplante , Dispositivos de Expansión Tisular/normas , Ratas/genética , Técnicas de Cultivo/métodos , Suero/inmunología

RESUMEN

Las células madre estromales humanas y de roedores cultivadas pueden ser inducidas a diferenciarse en neuronas, enfatizando su utilidad potencial en la terapia celular neurorrestaurativa. Los sistemas de cultivo para la expansión de estas células describen el uso de diferentes proporciones de suero fetal, lo que motivó a estudiar qué concentración de suero fetal bovino era capaz de garantizar un adecuado rendimiento celular. Las células de la médula ósea de rata se cultivaron en medio a-MEM suplementado con 10 y 20 por cientode suero fetal bovino y se subcultivaron hasta 3 veces. La viabilidad celular de los cultivos primarios y los subcultivos estuvo por encima del 98 por ciento en ambos experimentos. Los cultivos primarios demoraron 17,4 días en confluir y los subcultivos 7,7 días. La concentración de suero fetal al 20 por ciento no aumentó significativamente la velocidad de multiplicación celular; no obstante, se obtuvo un mayor número de células estromales. El sistema de expansión in vitro podría utilizarse en estudios futuros para la expansión de las células estromales humanas, lo que sienta mejores bases para su aplicación clínica

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Cultured human and rodents stromal stem cells can be induced to differentiate into neurons, emphasizing its potential use in neurorestorative cell therapy. Cropping systems for the expansion of these cells describe the use of different ratios of fetal serum, which led to study what concentration of fetal calf serum was able to ensure an adequate cell yield. Cells from rat bone marrow were cultured in medium supplemented with a-MEM 10 and 20 percent fetal bovine serum and subcultured up to 3 times. Cell viability of primary cultures and subcultures was above 98 percent in both experiments. Primary cultures converge delayed in 17.4 days and 7.7 days subcultures. The concentration of 20 percent fetal calf serum did not significantly increase the speed of cell division, however, we obtained a greater number of stromal cells. The expansion in vitro system could be used in future studies for the expansion of human stromal cells, which feels better basis for clinical application

Asunto(s)

Células del Estroma/trasplante , Dispositivos de Expansión Tisular/normas , Ratas/genética , Suero/inmunología , Técnicas de Cultivo/métodos