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Although the proportion of ulcer patients with medical problems among the elderly has increased with the extension of human life expectancy, treatment efficiency is drastically low, incurring substantial social costs. MSCs have independent regeneration potential, making them useful in clinical trials of difficult-to-treat diseases. In particular, ADMSCs are promising in the stem cell therapy industry as they can be obtained in vast amounts using non-invasive methods. Furthermore, studies are underway to enhance the regeneration potential of ADMSCs using cytokines, growth factors, and gene delivery to generate highly functional ADMSCs. In this study, key regulators of wound healing, SOCS-1, -3, and -5, were combined to maximize the regenerative potential of ADMSCs in pressure ulcer treatments. After transfecting SOCS-1, -3, -5, and SOCS-com into ADMSCs using a non-viral method, the expression of the inflammatory factors TNF-alpha, INF-gamma, and IL-10 was confirmed. ADMSCs transfected with SOCS-com showed decreased overall expression of inflammatory factors and increased expression of anti-inflammatory factors. Based on these results, we implanted ADMSCs transfected with SOCS-com into a pressure ulcer mouse model to observe their subsequent wound-healing effects. Notably, SOCS-com improved wound closure in ulcers, and reconstruction of the epidermis and dermis was observed. The healing mechanism of ADMSCs transfected with SOCS-com was examined by RNA sequencing. Gene analysis results confirmed that expression changes occurred in genes of key regulators of wound healing, such as chemokines, MMP-1, 9, CSF-2, and IL-33, and that such genetic changes enhanced wound healing in ulcers. Based on these results, we demonstrate the potential of ADMSCs transfected with SOCS-com as an ulcer treatment tool.
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Tejido Adiposo , Úlcera por Presión , Ratones , Animales , Humanos , Anciano , Tejido Adiposo/metabolismo , Úlcera , Úlcera por Presión/genética , Úlcera por Presión/terapia , Úlcera por Presión/metabolismo , Cicatrización de Heridas/genética , Modelos Animales de EnfermedadRESUMEN
Recent advancements in next-generation sequencing (NGS) technologies allow the simultaneous identification of targeted copy number alterations (CNAs) as well as somatic mutations using the same panel-based NGS data. We investigated whether CNAs detected by the targeted NGS data provided additional clinical implications, over somatic mutations, in myelodysplastic syndrome (MDS). Targeted deep sequencing of 28 well-known MDS-related genes was performed for 266 patients with MDS. Overall, 215 (80.8 %) patients were found to have at least one somatic mutation; 67 (25.2 %) had at least one CNA; 227 (85.3 %) had either a somatic mutation or CNA; and 12 had CNA without somatic mutations. Considering the clinical variables and somatic mutations alone, multivariate analysis demonstrated that sex, revised International Prognostic Scoring System (IPSS-R), and NRAS and TP53 mutations were independent prognostic factors for overall survival. For AML-free survival, these factors were sex, IPSS-R, and mutations in NRAS, DNMT3A, and complex karyotype/TP53 mutations. When we consider clinical variables along with somatic mutations and CNAs, genetic alterations in TET2, LAMB4, U2AF1, and CBL showed additional significant impact on the survivals. In conclusion, our study suggests that the concurrent detection of somatic mutations and targeted CNAs may provide clinically useful information for the prognosis of MDS patients.
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Variaciones en el Número de Copia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Síndromes Mielodisplásicos , Proteínas de Neoplasias/genética , Adolescente , Adulto , Anciano , Niño , Supervivencia sin Enfermedad , Femenino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/mortalidad , Tasa de SupervivenciaRESUMEN
Allogeneic stem cell transplantation is currently the only curative treatment option for myelodysplastic syndromes (MDS). Pre-transplant debulking treatment have been employed for advanced MDS and we previously reported that marrow response (blast ≤ 5%) following the bridging therapy with hypomethylating agent was an independent favorable factor for survival; however, it is still not clear which patients will respond to hypomethylating agent and which genomic features can predict the response. In this study, we performed RNAseq for 23 MDS patients among which 14 (61%) and 9 (39%) patients showed marrow complete remission and primary resistance to azacitidine, respectively. Differential expression-based analyses of treatment-naive, baseline gene expression profiles revealed that molecular functions representing mitochondria and apoptosis were up-regulated in responders. In contrast, we identified genes involved in the Wnt pathway were relatively up-regulated in non-responders. In independent validation cohorts of MDS patients, the expression of gene sets specific to non-responders and responders distinguished the patients with favorable prognosis and those responded to azacitidine highlighting the prognostic and predictive implication. In addition, a systems biology approach identified genes involved in ubiquitination, such as UBC and PFDN2, which may be key players in the regulation of differential gene expression in treatment responders and non-responders. Taken together, identifying the gene expression signature may advance our understanding of the molecular mechanisms of azacitidine and may also serve to predict patient responses to drug treatment.
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Azacitidina/farmacología , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/mortalidad , Pronóstico , Reproducibilidad de los Resultados , Transcriptoma , Resultado del TratamientoRESUMEN
BACKGROUND: Successful prevention of post-transplantation relapse after donor lymphocyte infusion (DLI) depends on its capability to mediate an effective graft-versus-leukemia (GVL) response while minimizing DLI-related toxicity, including graft-versus-host disease (GVHD). METHODS: We assessed the effects of decitabine (DEC), a hypomethylating agent, upon allogeneic immune reaction in a murine model of DLI. RESULTS: Significantly greater tumor growth retardation and survival prolongation occurred in mice administered with 1.0 mg/kg DEC for 5 days (DEC-1.0) than in control or DEC-0.1 mice. Upon prompt DEC and DLI co-administration, dendritic cells (DCs) were activated; DEC-1.0/DLI induced severe GVHD, and survival was significantly lower than with DLI alone or DEC-0.1/DLI treatments. IFN-γ and CD28 levels were higher in splenic DCs of DEC-1.0 mice than in those of control mice. Assessment of delayed DLI co-administration with DEC, when IFN-γ levels were normalized to control levels, revealed that DEC-1.0/DLI successfully facilitated tumor management without causing severe GVHD. CONCLUSIONS: Our results suggest that DEC primes allogeneic immune reactions of DLI via DC activation, and GVHD and GVL effects are separable through optimal DLI timing based on DEC-induced increase in IFN-γ expression levels.
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BACKGROUND: Cancer is characterized by uncontrolled cellular proliferation, and Polo-like kinase 1 (PLK1), a key regulator of the cell cycle, is overexpressed in many cancers, including acute leukemia and lymphoma. However, the dynamics of PLK1 transcription in myelodysplastic syndromes (MDS) are unknown. This study aimed to investigate the transcript dynamics of PLK1 and determine its role in the pathophysiology of MDS. METHODS: PLK1 mRNA obtained from the bone marrow samples of 67 patients with MDS, 16 patients with secondary acute myeloid leukemia (sAML), and 10 healthy controls were analyzed using quantitative real-time PCR and compared according to various clinical parameters. RESULTS: The median PLK1 expression levels differed slightly, but not significantly, between MDS and sAML patients [661.21 (range, 29.38-8,987.31) vs. 1,462.05 (32.22-5,734.09), respectively], but were significantly higher (P<0.001) than the levels in the healthy controls [19.0 (1.60-49.90)]. Further analyses of PLK1 levels according to the WHO classification of MDS, prognostic risk groups, karyotype risk groups, marrow blast percentage, and depth of cytopenia did not reveal any significant associations. In patients progressing to sAML, PLK1 expression levels differed significantly according to the presence or absence of resistance to hypomethylation treatment (2,470.58 vs. 415.98, P=0.03). CONCLUSION: PLK1 is upregulated in MDS patients; however, its role in the pathophysiology of MDS is unclear. Gene upregulation in cases with pharmacotherapeutic resistance warrants further investigation.
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BACKGROUND: The aim of this study was to investigate if epigenetically modified human mesenchymal stromal cells (hMSCs) can regulate the Th17-related immune responses. METHODS: We tested epigenetic drug combinations at various doses and selected the four combinations that resulted in maximal interleukin (IL)-10 and indoleamine 2,3-dioxygenase gene expression in hMSCs. We examined the effects of epigenetically modified hMSCs (epi-hMSCs) on CD4+ T-cell proliferation and inflammatory cytokine secretion under Th0- and Th17-polarizing conditions using mixed lymphocyte reactions and enzyme-linked immunosorbent assays (ELISAs). We determined Th17 cytokine levels and the percentage of Th17 cells among synovial fluid mononuclear cells (SFMCs) from rheumatoid arthritis (RA) patients by ELISA and flow cytometry. RESULTS: Epi-hMSCs inhibited the development of IL-17-producing cells in culture. The percentages of IL-17+ and interferon (IFN)-γ+ cells among peripheral blood mononuclear cells from healthy donors were lower under both the Th0 and Th17 conditions in the presence of epi-hMSCs than in the presence of no or untreated hMSCs. Epi-hMSC-treated RA patient SFMCs secreted lower levels of IL-17 and IFN-γ than RA patient SFMCs cultured without hMSCs or with untreated hMSCs. CONCLUSIONS: An optimal combination of hypomethylating agents and histone deacetylase inhibitors can enhance the immunomodulatory potential of hMSCs, which may be useful for RA treatment.
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Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Células Madre Mesenquimatosas/metabolismo , Células Th17/citología , Células Th17/metabolismo , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Epigénesis Genética/genética , Citometría de Flujo , Voluntarios Sanos , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Líquido Sinovial/citologíaRESUMEN
OBJECTIVE: In order to identify specific mesenchymal stromal cell (MSC) populations with enhanced therapeutic efficacy, we evaluated the functional changes associated with the stable expression of CD200, which is associated with immune regulatory function and osteogenic differentiation, in human bone marrow-derived MSCs (CD200/MSCs). RESULTS: We detected significantly greater osteogenesis and chondrogenesis in CD200/MSCs than in mock-transfected MSCs. In addition, the immune regulatory function of MSCs in mixed lymphocyte reactions was enhanced by CD200 gene transfection. In CD200/MSCs, the secretion of inflammatory cytokines, i.e., IL-6 and IL-8, was reduced, and levels of the anti-inflammatory factors IL-10, FOXP3, and indoleamine 2,3-dioxygenase 1 were elevated. Finally, CD200 transfection increased the stemness of MSCs, as evidenced by greater colony numbers in colony-forming unit fibroblast assays and analyses of NANOG and OCT-4 expression. CONCLUSIONS: These results suggest that CD200/MSCs have therapeutic applications, and further in-depth research should focus on the development of a clinically applicable cell-based therapeutic strategy.
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Antígenos CD/biosíntesis , Células de la Médula Ósea/fisiología , Diferenciación Celular , Expresión Génica , Células Madre Mesenquimatosas/fisiología , Células Cultivadas , Condrogénesis , Humanos , Factores Inmunológicos/metabolismo , OsteogénesisRESUMEN
BACKGROUND: Mesenchymal stromal cells (MSCs) are useful for cell therapy because of their potential for multilineage differentiation. However, MSCs that are expanded in traditional two-dimensional (2D) culture systems eventually lose their differentiation abilities. Therefore, we investigated whether azacitidine (AZA) supplementation and three-dimensional culture (3D) could improve the differentiation properties of MSCs. METHODS: 2D- or 3D-cultured MSCs which were prepared according to the conventional or hanging-drop culture method respectively, were treated with or without AZA (1 µM for 72 h), and their osteogenic and adipogenic differentiation potential were determined and compared. RESULTS: AZA treatment did not affect the cell apoptosis or viability in both 2D- and 3D-cultured MSCs. However, compared to conventionally cultured 2D-MSCs, AZA-treated 2D-MSCs showed marginally increased differentiation abilities. In contrast, 3D-MSCs showed significantly increased osteogenic and adipogenic differentiation ability. When 3D culture was performed in the presence of AZA, the osteogenic differentiation ability was further increased, whereas adipogenic differentiation was not affected. CONCLUSION: 3D culture efficiently promoted the multilineage differentiation of MSCs, and in combination with AZA, it could help MSCs to acquire greater osteogenic differentiation ability. This optimized culture method can enhance the therapeutic potential of MSCs.
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OBJECTIVE: To enhance the differentiation of mesenchymal stem cells (MSCs) and their epigenetic status by modification using hypomethylating agents (HMAs) and histone deacetylase inhibitors (HDACs). RESULTS: Treatment with 5-azacytidine or 5-azacytidine plus trichostatin A (TSA) increased expression of Runx-2, BDNF and Sox-9 compared with the control or TSA groups. Maximal increases of 4.1-, 4.5-, and 8.3-fold in Runx-2, BDNF, and Sox-9 transcript levels, respectively, were observed in the 5-azacytidine plus TSA group. Similar to the expression pattern of key regulatory molecules, differentiation to each lineage was also enhanced considerably in the 5-azacytidine or in the 5-azacytidine plus TSA groups. Quantitative analyses at the protein level showed 8.9-, 26.8-, 27.9-, and 28.5-fold upregulation of osterix, MAP-2, nestin, and type II collagen), respectively. CONCLUSION: HMAs and HDACs enhanced in vitro differentiation of MSCs, which was maximized when the two drugs were combined, with HMA having the dominant effect.
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Azacitidina/farmacología , Epigénesis Genética/efectos de los fármacos , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Condrogénesis/efectos de los fármacos , Quimioterapia Combinada , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Células Madre Mesenquimatosas/citología , Neurogénesis/efectos de los fármacos , Osteogénesis/efectos de los fármacosRESUMEN
INTRODUCTION: Despite good physical and biological properties, mineral trioxide aggregate (MTA) has a long setting time. A hydration accelerator could decrease the setting time of MTA. This study assessed the biocompatibility of MTA mixed with hydration accelerators (calcium chloride and low-dose citric acid) and investigated the effect of these materials on osteoblast differentiation. METHODS: Cell viability was evaluated by the EZ-Cytox assay kit (Daeil Lab Service, Seoul, Korea). The gene expressions of osteocalcin and bone sialoprotein were detected by reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. The mineralization behavior was evaluated with alizarin red staining. RESULTS: There was no statistically significant difference in cell viability between experimental groups. The messenger RNA level of osteogenic genes significantly increased in MTA mixed with hydration accelerators compared with the control and MTA mixed with water. MTA mixed with the hydration accelerators resulted in similar mineralization compared with MTA mixed with water. CONCLUSIONS: Hydration accelerators increase the osteogenic effect and show a similar effect on the mineralization of MTA, which may have clinical applications.
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Compuestos de Aluminio/farmacología , Materiales Biocompatibles/farmacología , Cloruro de Calcio/farmacología , Compuestos de Calcio/farmacología , Ácido Cítrico/farmacología , Osteoblastos/efectos de los fármacos , Óxidos/farmacología , Silicatos/farmacología , Células 3T3 , Compuestos de Aluminio/química , Animales , Materiales Biocompatibles/química , Cloruro de Calcio/química , Compuestos de Calcio/química , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ácido Cítrico/química , Combinación de Medicamentos , Sialoproteína de Unión a Integrina/análisis , Sialoproteína de Unión a Integrina/efectos de los fármacos , Ensayo de Materiales , Ratones , Osteocalcina/análisis , Osteocalcina/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Óxidos/química , Silicatos/química , Factores de Tiempo , Agua/químicaRESUMEN
Although the kinase receptor TrkA may play an important role in acute myeloid leukemia (AML), its involvement in other types of leukemia has not been reported. Furthermore, how it contributes to leukemogenesis is unknown. Here, we describe a molecular network that is important for TrkA function in leukemogenesis. We found that TrkA is frequently overexpressed in other types of leukemia such as acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS) including AML. In addition, TrkA was overexpressed in patients with MDS or secondary AML evolving from MDS. TrkA induced significant hematological malignancies by inducing PLK-1 and Twist-1, and enhanced survival and proliferation of leukemia, which was correlated with activation of the phosphatidylinositol 3-kinase/Akt/mTOR pathway. Moreover, endogenous TrkA associated with c-Src complexes was detected in leukemia. Suppression of c-Src activation by TrkA resulted in markedly decreased expression of PLK-1 and Twist-1 via suppressed activation of Akt/mTOR cascades. These data suggest that TrkA plays a key role in leukemogenesis and reveal an unexpected physiological role for TrkA in the pathogenesis of leukemia. These data have important implications for understanding various hematological malignancies.
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Leucemia/enzimología , Leucemia/patología , Receptor trkA/metabolismo , Familia-src Quinasas/metabolismo , Proteína Tirosina Quinasa CSK , Proteínas de Ciclo Celular/biosíntesis , Proliferación Celular , Activación Enzimática , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Redes y Vías Metabólicas , Síndromes Mielodisplásicos/enzimología , Síndromes Mielodisplásicos/patología , Proteínas Nucleares/biosíntesis , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Tumorales Cultivadas , Proteína 1 Relacionada con Twist/biosíntesis , Quinasa Tipo Polo 1RESUMEN
It has been suggested that activation of receptor PTKs is important for leukemogenesis and leukemia cell response to targeted therapy in hematological malignancies including leukemia. PTKs induce activation of the PI3K/Akt/mTOR pathway, which can result in prevention of apoptosis. Here, we describe an important role of the TrkC-associated molecular network in the process of leukemogenesis. TrkC was found to be frequently overexpressed in human leukemia cells and leukemia subtypes. In U937 human leukemia cells, blockade of TrkC using small hairpin RNA (shRNA) specific to TrkC or K562a, a specific inhibitor of TrkC, resulted in a significant decrease in growth and survival of the cells, which was closely associated with reduced mTOR level and Akt activity. In addition, TrkC enhances the survival and proliferation of leukemia, which is correlated with activation of the PI3K/Akt pathway. Moreover, TrkC significantly inhibits apoptosis via induction of the expression of PLK-1 and Twist-1 through activation of AKT/mTor pathway; therefore, it plays a key role in leukemogenesis. These findings reveal an unexpected physiological role for TrkC in the pathogenesis of leukemia and have important implications for understanding various hematological malignancies.
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Leucemia/metabolismo , Leucemia/patología , Receptor trkC/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Apoptosis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Regulación Leucémica de la Expresión Génica , Células HL-60 , Humanos , Leucemia/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor trkC/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Células Tumorales Cultivadas , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , Células U937 , Quinasa Tipo Polo 1RESUMEN
Dietary protein restriction during lactation affects lipid metabolism and food intake in rats. The goals of this study were to determine the effect of a low-protein diet on a liver damage in lactating rats, to determine whether dietary protein restriction of lactating dams affects the liver health of their offspring and to elucidate the molecular mechanisms underlying the development of hepatic damage. Lactating Sprague-Dawley rats were fed either a control 20% protein diet or an 8% low-protein diet for 11 or 23 days, respectively. After weaning, the offspring were continuously fed either the same control diet or the low-protein diet for an additional 22 days. Feeding a low-protein diet during lactation caused steatohepatitis with severe steatosis, lobular inflammation, ballooning degeneration and fibrosis. Offspring nourished by dams fed a low-protein diet showed simple hepatic steatosis. Combined effects of increased lipogenesis, decreased fatty acid oxidation and impaired very-low-density lipoprotein secretion were responsible for the development of hepatic steatosis. Hepatic up-regulation of genes linked to oxidative stress including nicotinamide adenine dinucleotide phosphate oxidase, inflammation and fibrogenesis supports the development of steatohepatitis in protein-restricted lactating rats. Furthermore, protein-restricted lactating rats showed activation of the leptin/signal transducers and activators of the transcription 3 signaling pathway. Taken together, oxidative stress induced by up-regulation of nicotinamide adenine dinucleotide phosphate oxidase with activation of leptin/signal transducers and activators of the transcription 3 signaling was responsible for development of steatohepatitis in protein-restricted lactating rats. Our findings suggest that protein malnutrition has a potential to induce steatohepatitis/hepatic steatosis in lactating mothers and infants during breast-feeding.
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Dieta con Restricción de Proteínas/efectos adversos , Proteínas en la Dieta/administración & dosificación , Hígado Graso/fisiopatología , Leptina/genética , Factor de Transcripción STAT3/metabolismo , Animales , Glucemia/análisis , Western Blotting , Femenino , Células Estrelladas Hepáticas/citología , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Lactancia/fisiología , Leptina/sangre , Lipoproteínas VLDL/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Estrés Oxidativo/efectos de los fármacos , Carbonilación Proteica , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT3/genética , Transducción de Señal , Regulación hacia ArribaRESUMEN
We developed a chondrogenic scaffold system in which plasmid DNA (pDNA) containing SOX trio (SOX-5, -6, and -9) genes was incorporated into a PLGA scaffold and slowly released to transfect adipose stem cells (ASCs) seeded in the scaffold. The purpose of this study was to test the in vitro and in vivo efficacy of the system to induce chondrogenic differentiation of ASCs. The pDNA/PEI-PEG complex-incorporated PLGA/Pluronic F127 porous scaffolds were fabricated by a precipitation/particulate leaching method. The following five kinds of pDNA were incorporated into the scaffolds: 1) pECFP-C1 vector without an interposed gene (control group); 2) SOX-5 plasmids; 3) SOX-6 plasmids; 4) SOX-9 plasmids; and 5) one-third doses of each plasmid (SOX-5, -6, and -9). ASCs were seeded on pDNA-incorporated PLGA scaffolds and cultured in chondrogenic media for 21 days. ASCs were also isolated from rabbits, seeded in pDNA-incorporated PLGA scaffolds, and then implanted in the osteochondral defect created on the patellar groove. The rabbits were sacrificed and analyzed grossly and microscopically 8 weeks after implantation. The percentage of transfected cells was highest on day 14, around 70%. After 21 days, PLGA scaffolds incorporated with each gene showed markedly increased expression of the corresponding gene and protein. Glycosaminoglycan (GAG) assay and Safranin-O staining showed an increased proteoglycan production in SOX trio pDNA-incorporated scaffolds. The COL2A1 gene and protein were notably increased in SOX trio pDNA-incorporated scaffolds than in the control, while COL10A1 protein expression decreased. Gross and histological findings from the in vivo study showed enhanced cartilage regeneration in ASCs/SOX trio pDNA-incorporated PLGA scaffolds.
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Adipocitos/fisiología , Condrogénesis/fisiología , Ácido Láctico/química , Ácido Poliglicólico/química , Factor de Transcripción SOX9/genética , Factores de Transcripción SOXD/genética , Células Madre/fisiología , Andamios del Tejido/química , Adipocitos/citología , Adulto , Animales , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Biomarcadores/metabolismo , ADN/química , ADN/genética , ADN/metabolismo , Femenino , Humanos , Ácido Láctico/metabolismo , Ensayo de Materiales , Persona de Mediana Edad , Plásmidos/genética , Ácido Poliglicólico/metabolismo , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Conejos , Células Madre/citologíaRESUMEN
The purpose of this study was to test the hypothesis that the SOX trio genes (SOX-5, SOX-6, and SOX-9) have a lower level of expression during the chondrogenic differentiation of mesenchymal stem cells (MSCs) compared with chondrocytes and that the electroporation-mediated gene transfer of SOX trio promotes chondrogenesis from human MSCs. An in vitro pellet culture was carried out using MSCs or chondrocytes at passage 3 and analyzed after 7 and 21 days. Then, MSCs were transfected with SOX trio genes and analyzed for the expression of chondrogenic markers after 21 days of in vitro culture. Without transforming growth factor-ß1, the untransfected MSCs had a lower level of SOX trio gene and protein expression than chondrocytes. However, the level of SOX-9 gene expression increased in MSCs when treated with transforming growth factor-ß1. GAG level significantly increased 7-fold in MSCs co-transfected with SOX trio, which was corroborated by Safranin-O staining. SOX trio co-transfection significantly increased COL2A1 gene and protein and decreased COL10A1 protein in MSCs. It is concluded that the SOX trio have a significantly lower expression in human MSCs than in chondrocytes and that the electroporation-mediated co-transfection of SOX trio enhances chondrogenesis and suppresses hypertrophy of human MSCs.
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Condrogénesis/genética , Electroporación/métodos , Células Madre Mesenquimatosas/metabolismo , Factores de Transcripción SOX/genética , Transfección/métodos , Anciano , Apoptosis/genética , Biomarcadores/metabolismo , Recuento de Células , Proliferación Celular , Condrocitos/citología , Condrocitos/metabolismo , Colágeno/genética , Colágeno/metabolismo , ADN/metabolismo , Regulación de la Expresión Génica , Glicosaminoglicanos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción SOX/metabolismo , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXD/genética , Factores de Transcripción SOXD/metabolismoRESUMEN
The long-term effects (~3 weeks) of two Wnt inhibitors (dickkopf [DKK]-1 and secreted frizzled-related protein [sFRP]-1), on the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was determined. Wnt inhibitors significantly increased the amount of glycosaminoglycan (GAG) in treated pellets (P<0.05). The gene expression of COL2A1 increased and COL1A1 decreased while the gene expression of SOX-9 and COL10A1 did not change significantly after three weeks of in vitro culture. The protein expression of type II collagen significantly increased (P<0.05) and that of type I collagen significantly decreased (P<0.05) while SOX-9 and type X collagen protein expression was unaffected. These findings suggest that Wnt inhibitors promote the chondrogenic differentiation of hMSCs when treated for three weeks.
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Condrogénesis , Células Madre Mesenquimatosas/fisiología , Proteínas Wnt/antagonistas & inhibidores , Técnicas de Cultivo de Célula , Diferenciación Celular , Colágeno Tipo I/biosíntesis , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo II/biosíntesis , Colágeno Tipo X/biosíntesis , Perfilación de la Expresión Génica , Glicosaminoglicanos/análisis , Humanos , Células Madre Mesenquimatosas/química , Factor de Transcripción SOX9/biosíntesisRESUMEN
This study tested the hypothesis that mitogen-activated protein kinase inhibitors suppress hypertrophy and enhance chondrogenesis during chondrogenesis of multipotent mesenchymal stromal cells (MSCs). The effects of PD98059 (an extracellular signal-regulated kinase-1/2 inhibitor) and SB203580 (a p38 inhibitor) were tested on bone marrow-derived MSCs (BMMSCs) and adipose tissue-derived MSCs (ATMSCs). In vitro pellet cultures were carried out using 2.5 x 10(5) MSCs in a chondrogenic medium containing 5 ng/mL of transforming growth factor-beta(2) (TGF-beta(2)) for BMMSCs, and 5 ng/mL of TGF-beta(2) and 100 ng/mL of bone morphogenetic protein-7 (BMP-7) for ATMSCs. From the 14th day of culture, the pellets were additionally treated with PD98059 or SB203580. After 14 more days of in vitro culture, pellets were harvested for analysis. PD98059 increased DNA content and glycosaminoglycan amount in BMMSCs and ATMSCs, whereas SB203580 had little effect. Collagen type I (COL1A1) mRNA decreased to almost a quarter in BMMSCs treated with PD98059. The mRNA levels of collagen type II (COL2A1) and SRY (sex determining region Y)-box 9 (SOX-9) increased several fold in both cells after PD98059 treatment, whereas SB203580 had only a slight effect. The gene expression of collagen type X (COL10A1) and runt-related transcription factor 2 (Runx-2) decreased by half after PD98059 treatment in BMMSCs, and decreased further in ATMSCs. SB203580 elevated COL10A1 and Runx-2 gene expression in both cell types. Safranin-O staining and immunohistochemistry generally mirrored findings from real-time PCR except for diminished expression of type I collagen in ATMSCS, and more pronounced decrease in type X collagen and Runx-2 in BMMSCs after PD98059 treatment. Our study demonstrated that PD98059 suppressed hypertrophy and promoted chondrogenesis of MSCs, and provides a ground for using them in cartilage tissue engineering.
Asunto(s)
Tejido Adiposo/citología , Células de la Médula Ósea/citología , Condrogénesis/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Células Madre Multipotentes/citología , Inhibidores de Proteínas Quinasas/farmacología , Células del Estroma/citología , Adulto , Recuento de Células , Colágeno/genética , Colágeno/metabolismo , ADN/metabolismo , Flavonoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Imidazoles/farmacología , Inmunohistoquímica , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/enzimología , Persona de Mediana Edad , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Células Madre Multipotentes/efectos de los fármacos , Células Madre Multipotentes/enzimología , Proteoglicanos/biosíntesis , Piridinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/efectos de los fármacos , Células del Estroma/enzimología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We tested the in vitro feasibility of porous PCL (poly(epsilon-caprolactone)) as a scaffold for cartilage tissue engineering from mesenchymal stem cells (MSCs) and determined the effects of various surface treatments. Three porous PCL scaffold modifications were examined: (1) PCL/Pluronic F127, (2) PCL/collagen, and (3) PCL/Pluronic F127/collagen, in addition to (4) PCL-only. MSCs (5 x 10(5)) were seeded in PCL scaffolds of pore size 100-150 microm, and after 3 weeks of in vitro culture, MSC-scaffolds were investigated for gross appearance, DNA amount, glycosaminoglycan (GAG) content, chondrogenic gene expression, and histology. Grossly, the cell-scaffold complexes became harder, and were more easily manipulated with a forceps after 3 weeks of culture. The three surface-treated scaffolds had higher DNA contents than did the PCL-only scaffold, and the GAG contents in PCL/collagen and PCL/F127/collagen scaffolds were higher than those seen in the PCL-only scaffold. Real-time PCR showed that Sox-9 and COL2A1 mRNA levels were remarkably elevated in PCL/collagen and PCL/F127/collagen scaffolds versus the PCL-only scaffold. On the other hand, Col1A1 and Col10A1 mRNA levels were lower in the three modified PCL scaffolds than in the PCL-only scaffold. Histological findings generally concurred with GAG and RT-PCR findings, and demonstrated the affinity of PCL-based scaffolds for MSCs and the potentials of these scaffold to induce chondrogenic differentiation. Cells showed more differentiated appearance and more abundant extracellular matrix formation in PCL/collagen and PCL/collagen/F127 scaffolds. Our findings suggest that PCL-based porous scaffolds may be useful carriers for MSC transplantation in the cartilage tissue engineering field, and that collagen-based surface modifications further enhance the chondrogenic differentiation of MSCs.
Asunto(s)
Materiales Biocompatibles/química , Condrogénesis/fisiología , Células Madre Mesenquimatosas/fisiología , Poliésteres/química , Andamios del Tejido/química , Anciano , Anciano de 80 o más Años , Células de la Médula Ósea/fisiología , Separación Celular , Células Cultivadas , Colágeno Tipo I/biosíntesis , Colorantes , ADN/biosíntesis , ADN/genética , Femenino , Gliceraldehído-3-Fosfato Deshidrogenasas/biosíntesis , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Glicosaminoglicanos/química , Humanos , Masculino , Persona de Mediana Edad , Fenazinas , Poloxámero , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción SOX9/biosíntesis , Factor de Transcripción SOX9/genética , TensoactivosRESUMEN
Evidence for the epigenetic regulation of hematopoietic stem cells (HSCs) is growing, but the genome-wide epigenetic signature of HSCs and its functional significance remain unclear. In this study, from a genome-wide comparison of CpG methylation in human CD34(+) and CD34(-) cells, we identified a characteristic undermethylation dip around the transcription start site of promoters and an overmethylation of flanking regions in undifferentiated CD34(+) cells. This "bivalent-like" CpG methylation pattern around the transcription start site was more prominent in genes not associated with CpG islands (CGI(-)) than CGI(+) genes. Undifferentiated hematopoietic cells also exhibited dynamic chromatin associated with active transcription and a higher turnover of histone acetylation than terminally differentiated cells. Interestingly, inhibition of chromatin condensation by chemical treatment (5-azacytidine, trichostatin A) enhanced the self-renewal of "stimulated" HSCs in reconstituting bone marrows but not "steady-state" HSCs in stationary phase bone marrows. In contrast, similar treatments on more mature cells caused partial phenotypic dedifferentiation and apoptosis at levels correlated with their hematopoietic differentiation. Taken together, our study reveals that the undifferentiated state of hematopoietic cells is characterized by a unique epigenetic signature, which includes dynamic chromatin structures and an epigenetic plasticity that correlates to level of undifferentiation.
Asunto(s)
Diferenciación Celular/genética , Metilación de ADN/genética , Células Madre Hematopoyéticas/citología , Acetilación , Animales , Antígenos CD34/biosíntesis , Western Blotting , Islas de CpG , Epigénesis Genética/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Sitio de Iniciación de la TranscripciónRESUMEN
This study was conducted to determine whether short-term administration of transforming growth factor (TGF)-beta would be as effective for inducing chondrogenesis in human mesenchymal stem cells (hMSCs) as continuous treatment. Four groups of hMSCs were cultured in a monolayer for 3 days followed by a pellet culture for 3 weeks under various conditions: group A, the control group, no growth factors treated; group B, 5 ng/ml of TGF-beta(2) was treated for 3 days in monolayer culture; group C, 5 ng/ml of TGF-beta(2) was treated for 3 days in a monolayer culture and the initial 3 days of pellet culture; group D, 5 ng/ml of TGF-beta(2) was treated for 3 days in a monolayer culture and the initial 10 days of pellet culture; group E, 5 ng/ml of TGF-beta(2) was continuously treated throughout the culture period. Glycosaminoglycan contents significantly increased in group E only. Real-time PCR indicated that expression of Sox-9, type II collagen, type II procollagen B and type X collagen increased with longer duration of TGF-beta(2) treatment. The histological findings showed that longer duration of TGF-beta(2) treatment led to significantly better quality of chondrogenesis. This study demonstrated that longer duration of TGF-beta treatment is necessary for effective chondrogenesis in hMSCs from bone marrow.