RESUMEN
Insulin resistance (IR) is one of the characteristic features of equine metabolic syndrome (EMS). Presently, the only therapies of choice are caloric restrictions combined with mineral supplementation, which might improve insulin sensitivity. In this study we investigated the effect of Haematococcus pluvialis algae water extract enriched in bioaccumulation process in magnesium ions (Hp_Mg(II)) on equine adipose derived mesenchymal stromal stem cells, in which insulin resistance was induced by palmitic acid (IR-EqASCs). For this purpose, chemical characterization of H. pluvialis was performed with special emphasis on the analysis of minerals composition, total phenolic and carotenoids contents, as well as scavenging activity. To examine the influence of H. pluvialis extract on IR-EqASCs, various methods of molecular biology and microscopic observations (i.e., immunofluorescence staining, SEM, gene expression by RT-qPCR, proliferative and metabolic cells activity analysis) were applied to investigate in vitro viability, oxidative stress markers and apoptosis-related factor accumulation, along with insulin resistance-related genes expression. Obtained results show, that Hp_Mg(II) significantly improves proliferative and metabolic activity of IR-EqASCs, shortens their population doubling time, improves their clonogenic potential and reduces expression of apoptosis related genes. Moreover, anti-oxidative effect of extract was presented.
Asunto(s)
Tejido Adiposo/citología , Resistencia a la Insulina , Magnesio/farmacología , Microalgas/química , Animales , Antioxidantes/farmacología , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica/efectos de los fármacos , Caballos , Resistencia a la Insulina/genética , Iones , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Oxidación-Reducción , Ácido Palmítico , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , AguaRESUMEN
Heterotopic ossification (HO) is a pathological condition characterized by the deposition of mineralized tissue in ectopic locations such as the skeletal muscle. The precise cellular origin and molecular mechanisms underlying HO are still debated. In our study we focus on the differentiation of mesoangioblasts (MABs), a population of multipotent skeletal muscle precursors. High-content screening for small molecules that perturb MAB differentiation decisions identified Idoxuridine (IdU), an antiviral and radiotherapy adjuvant, as a molecule that promotes MAB osteogenic differentiation while inhibiting myogenesis. IdU-dependent osteogenesis does not rely on the canonical BMP-2/SMADs osteogenic pathway. At pro-osteogenic conditions IdU induces a mild DNA Damage Response (DDR) that activates ATM and p38 eventually promoting the phosphorylation of the osteogenesis master regulator RUNX2. By interfering with this pathway IdU-induced osteogenesis is severely impaired. Overall, our study suggests that induction of the DDR promotes osteogenesis in muscle resident MABs thereby offering a new mechanism that may be involved in the ectopic deposition of mineralized tissue in the muscle.
Asunto(s)
Diferenciación Celular , Daño del ADN , Células Madre Multipotentes/patología , Músculo Esquelético/patología , Osteogénesis , Animales , Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular/efectos de los fármacos , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Idoxuridina/farmacología , Células Madre Multipotentes/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Osificación Heterotópica , Proteínas Smad/metabolismoRESUMEN
BACKGROUND: Mesenchymal stem cell-derived conditioned medium (MSC-CM) has emerged as a promising cell-free tool for restoring degenerative diseases and treating traumatic injuries. The present study describes the effect of selenium as a reactive oxygen species (ROS) scavenger and its additive effect with basic fibroblast growth factor (bFGF) on in vitro expansion of amniotic fluid (AF)-MSCs and the paracrine actions of AF-MSC-CM as well as the associated cellular and molecular mechanisms. METHODS: In this study, we obtained CM from human AF-MSCs cultured with selenium. The stemness of selenium-treated AF-MSCs was evaluated by cell growth and differentiation potential. Human fibroblasts were treated with AF-MSC-CM and analyzed for cell signaling changes. For in vivo wound healing assay, ICR mice with a full-thickness skin wound were used. RESULTS: Selenium played a critical role in in vitro expansion of AF-MSCs through activation of the AKT-ERK1/2, Smad2, and Stat3 signaling pathways along with inactivation of GSK3ß. When administered together with bFGF, it showed remarkable effect in inhibiting ROS accumulation and preserving their multipotency. Proliferation and migration of human dermal fibroblasts and in vivo wound healing were improved in the CMs derived from AF-MSCs exposed to selenium and bFGF, which was caused by the Smad2, AKT-MEK1/2-ERK, and NFκB signaling triggered by the paracrine factors of AF-MSCs, such as TGF-ß, VEGF, and IL-6. Our results suggest the following: (a) supplementation of selenium in AF-MSC culture contributes to in vitro expansion and preservation of multipotency, (b) ROS accumulation causes progressive losses in proliferative and differentiation potential, (c) the separate activities of bFGF and selenium in MSCs exert an additive effect when used together, and (d) the additive combination improves the therapeutic effects of AF-MSC-derived CMs on tissue repair and regeneration. CONCLUSION: Antioxidants, such as selenium, should be considered as an essential supplement for eliciting the paracrine effects of MSC-CMs.
Asunto(s)
Líquido Amniótico/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Células Madre Mesenquimatosas/citología , Comunicación Paracrina , Selenio/farmacología , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo Condicionados/farmacología , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Espacio Intracelular/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos ICR , Modelos Biológicos , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Comunicación Paracrina/efectos de los fármacos , Fenotipo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Heterotopic ossification (HO) develops in the extremities of wounded service members and is common in the setting of high-energy penetrating injuries and blast-related amputations. No safe and effective prophylaxis modality has been identified for this patient population. Palovarotene has been shown to reduce bone formation in traumatic and genetic models of HO. The purpose of this study was to determine the effects of Palovarotene on inflammation, progenitor cell proliferation, and gene expression following a blast-related amputation in a rodent model (n = 72 animals), as well as the ability of Raman spectroscopy to detect early HO before radiographic changes are present. Treatment with Palovarotene was found to dampen the systemic inflammatory response including the cytokines IL-6 (p = 0.01), TNF-α (p = 0.001), and IFN-γ (p = 0.03) as well as the local inflammatory response via a 76% reduction in the cellular infiltration at post-operative day (POD)-7 (p = 0.03). Palovarotene decreased osteogenic connective tissue progenitor (CTP-O) colonies by as much as 98% both in vitro (p = 0.04) and in vivo (p = 0.01). Palovarotene treated animals exhibited significantly decreased expression of osteo- and chondrogenic genes by POD-7, including BMP4 (p = 0.02). Finally, Raman spectroscopy was able to detect differences between the two groups by POD-1 (p < 0.001). These results indicate that Palovarotene inhibits traumatic HO formation through multiple inter-related mechanisms including anti-inflammatory, anti-proliferative, and gene expression modulation. Further, that Raman spectroscopy is able to detect markers of early HO formation before it becomes radiographically evident, which could facilitate earlier diagnosis and treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1135-1144, 2018.
Asunto(s)
Células Madre Multipotentes/efectos de los fármacos , Osificación Heterotópica/prevención & control , Osteogénesis/efectos de los fármacos , Pirazoles/uso terapéutico , Estilbenos/uso terapéutico , Animales , Traumatismos por Explosión/complicaciones , Proliferación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Expresión Génica/efectos de los fármacos , Masculino , Osificación Heterotópica/etiología , Pirazoles/farmacología , Ratas Sprague-Dawley , Espectrometría Raman , Estilbenos/farmacología , Síndrome de Respuesta Inflamatoria Sistémica/prevención & control , Heridas Relacionadas con la Guerra/complicacionesRESUMEN
The extensive use of mesenchymal stem cells (MCS) in tissue engineering and cell therapy increases the necessity to improve their expansion. Among these, porcine MCS are valuable models for tissue engineering and are classically expanded in static T-flasks. In this work, different processes of stirred cultures were evaluated and compared. First, the effect of glucose, glutamine, antioxidant, and growth factors concentrations on porcine MSC expansion were analyzed in a suitable medium by performing kinetic studies. Results showed that a lower glucose concentration (5.5 mM) enabled to increase maximal cell concentration by 40 % compared with a higher one (25 mM), while addition of 2 to 6 mM of glutamine increased maximal cell concentration by more than 25 % compared with no glutamine supplementation. Moreover, supplementation with 1 µM thioctic acid increased maximal cell concentration by 40 % compared with no supplementation. Using this adapted medium, microcarriers cultures were performed and compared with T-flasks expansion. Porcine MSC were shown to be able to proliferate on the five types of microcarriers tested. Moreover, cultures on Cytodex 1, Cytopore 2, and Cultispher G exhibited a MSC growth rate more than 40 % higher compared with expansion in T-flasks, while MSC metabolism was similar.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Mesenquimatosas/citología , Microesferas , Amoníaco/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Glucosa/farmacología , Glutamina/farmacología , Cinética , Ácido Láctico/biosíntesis , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Sus scrofaRESUMEN
The design of bioactive three-dimensional (3D) scaffolds is a major focus in bone tissue engineering. Incorporation of growth factors into bioprinted scaffolds offers many new possibilities regarding both biological and architectural properties of the scaffolds. This study investigates whether the sustained release of bone morphogenetic protein 2 (BMP-2) influences osteogenicity of tissue engineered bioprinted constructs. BMP-2 loaded on gelatin microparticles (GMPs) was used as a sustained release system, which was dispersed in hydrogel-based constructs and compared to direct inclusion of BMP-2 in alginate or control GMPs. The constructs were supplemented with goat multipotent stromal cells (gMSCs) and biphasic calcium phosphate to study osteogenic differentiation and bone formation respectively. BMP-2 release kinetics and bioactivity showed continuous release for three weeks coinciding with osteogenicity. Osteogenic differentiation and bone formation of bioprinted GMP containing constructs were investigated after subcutaneous implantation in mice or rats. BMP-2 significantly increased bone formation, which was not influenced by the release timing. We showed that 3D printing of controlled release particles is feasible and that the released BMP-2 directs osteogenic differentiation in vitro and in vivo.
Asunto(s)
Alginatos/farmacología , Bioimpresión/métodos , Proteína Morfogenética Ósea 2/metabolismo , Osteogénesis , Animales , Diferenciación Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Femenino , Gelatina/farmacología , Ácido Glucurónico/farmacología , Cabras , Ácidos Hexurónicos/farmacología , Humanos , Hidroxiapatitas/farmacología , Ratones Desnudos , Microesferas , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Osteocalcina/metabolismo , Osteogénesis/efectos de los fármacos , Ratas Wistar , Células del Estroma/citología , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Tejido Subcutáneo/metabolismo , Andamios del Tejido/químicaRESUMEN
Development of in vitro systems, such as those based on embryonic stem cell differentiation, depends on the selection of adequate test and training compounds. We recommend the use of two classes of positive controls, the "gold standard compounds" for which developmental neurotoxicity (DNT) has been proven in man, and the "pathway compounds" that are known to disrupt signalling pathways and key processes relevant for neuronal differentiation. We introduce the concept of toxicity endophenotypes (TEP) as changes in neuronal connectivity resulting from exposure to developmental toxicants. Thus, TEPs provide the scientific rationale for modeling DNT with simple in vitro models of key neurodevelopmental events. In this context, we discuss scientific and technical aspects of the test compound selection process. We suggest to include compounds with unspecific toxicity, besides negative control compounds, and we recommend tandem approaches to determine relative toxicities instead of absolute measures. Finally, we discuss how to avoid pitfalls by distinguishing between unspecific forms of cytotoxicity and specific developmental neurotoxicity. A compilation of compound lists corresponding to the above-discussed principles supplement this review.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Modelos Neurológicos , Neurogénesis/efectos de los fármacos , Neurotoxinas/toxicidad , Animales , Muerte Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Humanos , Técnicas In Vitro , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Sistema Nervioso/efectos de los fármacos , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Neuritas/efectos de los fármacos , FenotipoRESUMEN
Osteoporosis is a reduction in skeletal mass due to an imbalance between bone formation and bone resorption. Therefore, the identification of specific stimulators of bone formation is of therapeutic significance in the treatment of osteoporosis. Salicylideneamino-2-thiophenol (Sal-2) consists of two benzene rings, has been reported to possess antioxidant activity, and is an effective remedy for fever and rheumatic diseases. However, until now the effects of osteoblastic bone formation by Sal-2 were unknown. In this study, we investigated the effects of Sal-2 on osteogenic differentiation of multipotent bone marrow stromal stem cells by alizarin red S staining for osteogenic differentiation, RT-PCR and western blot for alkaline phosphatase (ALP) activity and signaling pathways, FACS analysis and immunofluorescence staining for CD44 and CD51 expression, calcium assays, and immunofluorescence staining for signaling pathways. We found that Sal-2 enhanced the osteogenic differentiation of multipotent bone marrow stromal stem cells. Sal-2 treatment induced the expression and activity of ALP, and enhanced the levels of CD44 and CD51 expression as well as Ca2+ content, in multipotent bone marrow stromal stem cells. Moreover, we found that Sal-2-induced osteogenic differentiation and expression of osteogenesis-related molecules involve the activation of the MAPK and nuclear factor-κB pathways. Our findings provide insight into both the mechanism and effects of Sal-2 on osteogenic differentiation and demonstrate that Sal-2 may be a beneficial adjuvant in stimulating bone formation in osteoporotic diseases.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Multipotentes/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Salicilatos/farmacología , Compuestos de Sulfhidrilo/farmacología , Fosfatasa Alcalina/biosíntesis , Fosfatasa Alcalina/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Calcio/análisis , Diferenciación Celular/fisiología , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Receptores de Hialuranos/biosíntesis , Integrina alfaV/biosíntesis , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Madre Multipotentes/metabolismo , FN-kappa B/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
1,25-dihydroxyvitamin D3 (1,25D3) was reported to induce premature organismal aging in fibroblast growth factor-23 (Fgf23) and klotho deficient mice, which is of main interest as 1,25D3 supplementation of its precursor cholecalciferol is used in basic osteoporosis treatment. We wanted to know if 1,25D3 is able to modulate aging processes on a cellular level in human mesenchymal stem cells (hMSC). Effects of 100 nM 1,25D3 on hMSC were analyzed by cell proliferation and apoptosis assay, ß-galactosidase staining, VDR and surface marker immunocytochemistry, RT-PCR of 1,25D3-responsive, quiescence- and replicative senescence-associated genes. 1,25D3 treatment significantly inhibited hMSC proliferation and apoptosis after 72 h and delayed the development of replicative senescence in long-term cultures according to ß-galactosidase staining and P16 expression. Cell morphology changed from a fibroblast like appearance to broad and rounded shapes. Long term treatment did not induce lineage commitment in terms of osteogenic pathways but maintained their clonogenic capacity, their surface marker characteristics (expression of CD73, CD90, CD105) and their multipotency to develop towards the chondrogenic, adipogenic and osteogenic pathways. In conclusion, 1,25D3 delays replicative senescence in primary hMSC while the pro-aging effects seen in mouse models might mainly be due to elevated systemic phosphate levels, which propagate organismal aging.
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Senescencia Celular/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Multipotentes/citología , Vitamina D/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Transformada , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Factor-23 de Crecimiento de Fibroblastos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Inmunofenotipificación , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Ratones , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de Calcitriol/metabolismo , Factores de Tiempo , Vitamina D/farmacología , beta-Galactosidasa/metabolismoRESUMEN
We investigate the effect of bladder-derived acellular matrix (ACM) on bone marrow mesenchymal stem cells (BM-MSC) growth, survival, and differentiation, and evaluate the effect of collagen I and IV on BM-MSC differentiation potential to SMC. BM-MSCs isolated from CD1(_) mice were characterized by surface markers and differentiation into different lineages. BM-MSC SMC potential was further evaluated in stem cell medium alone or supplemented with TGF-ß1 and recombinant human platelet-derived growth factor (PDGF-BB) on plastic, collagen I and IV using western blot. Furthermore, BM-MSCs were seeded on porcine derived ACM-fortified with hyaluronic acid and cultured in Mesencult+-growth factors, bone, or fat induction media for 3 weeks. Seeded constructs were evaluated by H&E, Ki67 assay, Oil red O, and Alizarin red stain. SMC differentiation was semiquantified via immunohistochemistry. BM-MSCs differentiated into fat and bone when induced. In Mesencult, BM-MSCs differentiated into SMC, expressing α-SMA, calponin, and MHC. BM-MSCs cultured on collagen I and IV reduced expression of SMC and MHC compared to plastic. On ACM-HA, BM-MSCs maintained multipotent state by differentiating to bone and fat when induced. In Mesencult, BM-MSC-seeded ACM-HA expressed α-SMA, calponin, and MHC. TGF-ß1 and PDGF-BB enhanced SMC differentiation on collagens and ACM-HA. SMC proteins expression by BM-MSC varies depending on culture substrate. SMC markers are expressed higher on plastic and lower on collagen I, IV, and ACM-HA, suggesting these substrates preferentially maintain undifferentiated state of BM-MSC, which could be advantageous for incorporation of cell-seeded grafts to permit host modulation of tissue regeneration.
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Células de la Médula Ósea/citología , Matriz Extracelular/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Multipotentes/citología , Proteínas Proto-Oncogénicas c-sis/farmacología , Factor de Crecimiento Transformador beta1/farmacología , Vejiga Urinaria/metabolismo , Actinas/metabolismo , Animales , Becaplermina , Biomarcadores/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Proteínas de Unión al Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/farmacología , Colágeno Tipo IV/farmacología , Medios de Cultivo/farmacología , Matriz Extracelular/efectos de los fármacos , Humanos , Ácido Hialurónico/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Proteínas de Microfilamentos/metabolismo , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Plásticos , Sus scrofa , CalponinasRESUMEN
OBJECTIVE: To study the proliferation effect of neural stem cells (NSCs) of ginsenoside Rg1 in vitro. METHOD: NSCs from the embryonic rat (E14) were isolated, 10 mg x L(-1) BrdU were added in the medium. The NSCs were incubated together with 1, 10 micromol x L(-1) ginsenoside Rg1 for 48 hours, control group were setup. Then BrdU positive cell number were detected and counted by fluorescence microscop. The Hes1 and Mash1 mRNA expression were detected by real-time RT-PCR. RESULT: The number of BrdU positive cells in 10 micromol x L(-1) ginsenoside Rg1 group was increased significantly compared with the control group (40.5 +/- 10.9 vs 26.2 +/- 6.0, P < 0.01), and the Hes1 mRNA expression were increased significantly (1.00 +/- 0.11) vs (1.28 +/- 0.14), P < 0.05. CONCLUSION: ginsenoside Rg1 can promote the proliferation of neural stem cells in vitro, and this effect may be induced by the up-regulation Hes1 expression.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Ginsenósidos/farmacología , Neuronas/efectos de los fármacos , Animales , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/fisiología , Neuronas/fisiología , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The extracellular matrix (ECM) is a component of neural cell niches and regulates multiple functions of diverse cell types. To date, limited information is available concerning its biological effects on the growth properties of oligodendrocyte progenitor cells (OPCs). In the present study, we examined effects of several ECM components, i.e., fibronectin, laminin, and Matrigel, on the survival, proliferation, migration, process extension, and purity of OPCs isolated from embryonic day 15 rat spinal cords. All three ECM components enhanced these biological properties of the OPCs compared with a non-ECM substrate, poly-D-lysine. However, the extents of their effects were somewhat different. Among these ECMs, fibronectin showed the strongest effect on almost all aspects of the growth properties of OPCs, implying that this molecule is a better substrate for the growth of OPCs in vitro. Because of its survival- and growth-promoting effects on OPCs, fibronectin may be considered as a candidate substrate for enhancing OPC-mediated repair under conditions when exogenous delivery or endogenous stimulation of OPCs is applied.
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Proteínas de la Matriz Extracelular/farmacología , Células Madre Multipotentes/efectos de los fármacos , Oligodendroglía/citología , Animales , Apoptosis/efectos de los fármacos , Técnicas de Cultivo de Célula/instrumentación , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Extensiones de la Superficie Celular/efectos de los fármacos , Extensiones de la Superficie Celular/ultraestructura , Células Cultivadas/efectos de los fármacos , Colágeno/farmacología , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Fibronectinas/farmacología , Laminina/farmacología , Células Madre Multipotentes/citología , Proteoglicanos/farmacología , Ratas , Ratas Wistar , Médula Espinal/citología , Médula Espinal/embriologíaRESUMEN
Genetic modification of hematopoietic stem cells holds great promise in the treatment of hematopoietic disorders. However, clinical application of gene delivery has been limited, in part, by low gene transfer efficiency. To overcome this problem, we investigated the effect of retronectin (RN) on lentiviral-mediated gene delivery into hematopoietic progenitor cells (HPCs) derived from bone marrow both in vitro and in vivo. RN has been shown to enhance transduction by promoting colocalization of lentivirus and target cells. We found that RN enhanced lentiviral transfer of the VENUS transgene into cultured c-Kit(+) Lin(-) HPCs. As a complementary approach, in vivo gene delivery was performed by subjecting mice to intra-bone marrow injection of lentivirus or a mixture of RN and lentivirus. We found that co-injection with RN increased the number of VENUS-expressing c-Kit(+) Lin(-) HPCs in bone marrow by 2-fold. Further analysis of VENUS expression in colony-forming cells from the bone marrow of these animals revealed that RN increased gene delivery among these cells by 4-fold. In conclusion, RN is effective in enhancing lentivirus-mediated gene delivery into HPCs.
Asunto(s)
Fibronectinas/farmacología , Técnicas de Transferencia de Gen , Células Madre Hematopoyéticas/efectos de los fármacos , Lentivirus/genética , Proteínas Recombinantes/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células Cultivadas , Evaluación Preclínica de Medicamentos , Fibronectinas/química , Células Madre Hematopoyéticas/metabolismo , Humanos , Lentivirus/fisiología , Ratones , Ratones Endogámicos C57BL , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismo , Fragmentos de Péptidos/farmacología , Estructura Terciaria de Proteína , Regulación hacia ArribaRESUMEN
OBJECTIVE: Cytomegalovirus (CMV) infection was greatly common in the world. CMV infection produces usually mild or asymptomatic infections in individuals with normal immune responses, whereas it may cause serious disease in immunosuppressive patients. Clinical manifestations include suppression of myelopoiesis, a mononucleosis like syndrome, hepatosplenomegaly, lymphadenopathy, thrombocytopenia, and hemolytic anemia. In patients undergoing bone marrow transplantation CMV remains the most common infectious causes of morbidity and mortality. But the treatment drugs with specific effect for CMV was fewer at the present. This study was to investigate the effect of CMV on proliferation of colony forming unit granulocyte-macrophage (CFU-GM), CFU-erythroid (CFU-E), brust forming unit-erythroid (BFU-E), CFU-multipotential (CFU-Mix) and CFU-megakaryocyte (CFU-Mk) progenitor cells of cord blood (CB) with the presence of ganciclovir (GCV) and astragalus membranaceus in vitro. METHODS: Twenty CB samples were collected from fetal umbilical vein of normal term spontaneous delivery neonates. The colony forming unit-assay was applied to observe the suppression effect of CMV-AD169 strain on CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk of CB with the presence of GCV and astragalus membranaceus in vitro. The technique of PCR was used to demonstrate the existence of CMV-AD169 DNA in the colony cells of cultured CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk. RESULTS: (1) The numbers of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk colonies in CMV infection groups were significantly less than those in blank and mock group, respectively. The last time of colonies in groups with CMV infection was significantly shorten compared with the blank and mock group. (2) CMV-DNA was positively detected in the colony cells of CMV infection groups by PCR, while negative in the control groups. (3) The lasting time of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk colonies infected with CMV extended significantly with the presence of astragalus membranaceus and GCV, and the numbers of those increased significantly compared with the CMV infection group, respectively. The increasing rate of colonies was 27.2%, 45.2%, 49.1%, 39.0% and 11.9% with astragalus membranaceus group, 37.4%, 74.2%, 71.7%, 67.4% and 38.9% with GCV group, 53.6%, 83.8%, 88.7%, 87.8% and 61.5% with astragalus membranaceus and GCV group, respectively. CONCLUSIONS: The differentiation and proliferation of CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk were significantly inhibited after infected with CMV-AD169 strain. The suppression effect of CMV-AD169 on CFU-GM, CFU-E, BFU-E, CFU-Mix and CFU-Mk was inhibited with the presence of GCV and astragalus membranaceus in vitro. This suggested that CMV-AD169 may be inhibited or killed by GCV and Astragalus Membranaceus in vitro.
Asunto(s)
Astragalus propinquus/química , Citomegalovirus/efectos de los fármacos , Medicamentos Herbarios Chinos/farmacología , Ganciclovir/farmacología , Células Madre Hematopoyéticas/efectos de los fármacos , Antivirales/farmacología , División Celular/efectos de los fármacos , Infecciones por Citomegalovirus/tratamiento farmacológico , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/efectos de los fármacos , Células Precursoras Eritroides/metabolismo , Sangre Fetal/citología , Sangre Fetal/efectos de los fármacos , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Multipotentes/citología , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/metabolismoRESUMEN
Granulocyte-colony stimulating factor (G-CSF) has been reported to mobilize bone marrow multi-potent stem cells, which differentiate into cardiac myocytes after myocardial infarction (MI). However, there have not been any reports regarding the effect of G-CSF on stem cell infiltration in the MI site. Hearts of mice that had undergone coronary occlusion were isolated and digested with collagenase. Infiltrating cells in the heart were collected using Percoll density gradients. The infiltrating cells were sorted for side population (SP) cells using Hoechst 33342 dye. Hundreds of infiltrating SP cells were found in the heart from 1 to 14 d after MI. There were only a few SP cells in hearts without infarction. Infiltrating SP cells were increased in the 4-d G-CSF treated group compared with the vehicle group (1106 +/- 106 vs. 323 +/- 26/heart, P < 0.05). The infiltration of inflammatory cells was not influenced by the G-CSF treatment. In a separate series of experiments, we confirmed that the infiltrating SP cells were derived from bone marrow. That is, SP cells in the infarcted hearts of mice, which had been transplanted with bone marrow from ROSA 26 (beta-galactosidase transgenic) mice, were positive for beta-galactosidase. In the immunohistochemical examination, Sca-1(+)/CD45(-) cells were existed in the infarcted site after MI. Therefore, SP cells may infiltrate into infarcted heart. G-CSF augmented this kind of stem cell infiltration without increasing inflammatory cells. These results suggest that G-CSF may enhance myocardial regeneration without aggravated inflammation in the infarcted heart.