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1.
Exp Cell Res ; 374(1): 122-127, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30496759

RESUMEN

BACKGROUND: Mesenchymal stromal cells (MSCs) can be used in several clinical applications. While MSCs are frequently cultured in fetal bovine serum for in vitro experimentation, human serum supplements are required for cells to be used in patients. Here we show how different human serum supplements and in vitro manipulations used during the cell culture impact on MSC proliferation rate and expression of inflammatory molecules. METHODS: MSCs were cultured in medium supplemented with human plasma or serum combined with human platelet lysate (PL) and/or basic fibroblast growth factor (FGF2). Real time RT-PCR and western blot were used to assess expression of inflammatory cytokines. RESULTS: Serum with addition of FGF2 gave the fastest proliferation rate. However, serum with FGF2 also increased expression of genes encoding inflammatory cytokines. The most favorable expansion condition for chondrogenic differentiation and inhibition of cartilage matrix degrading enzymes was plasma supplemented with PL and FGF2. Detachment of cells using trypsin gave considerable upregulation of inflammatory cytokine mRNAs which lasted for up to 24 h, with concomitant increase in protein levels. Even the gentle act of changing medium led to upregulation of cytokine mRNA, caused by addition of fresh serum. DISCUSSION: Different culture conditions and simple cell manipulation influence proliferation rate and expression of inflammatory genes. Supplementing culture medium with allogeneic AB serum and FGF2 during monolayer expansion supported cell expansion better than other supplements, but also induced the highest levels of inflammatory cytokines and gave inferior results for chondrogenic differentiation. The importance of the composition of the culture medium and even gentle in vitro manipulation of the cells should be taken into account in the planning of procedures using in vitro expanded MSCs.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Citocinas/metabolismo , Mediadores de Inflamación/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proliferación Celular , Células Cultivadas , Condrogénesis , Citocinas/genética , Humanos , Inflamación/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo
2.
Part Fibre Toxicol ; 15(1): 21, 2018 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-29751765

RESUMEN

BACKGROUND: Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis. RESULTS: Exposure-relevant concentrations of DEP (0.12 µg/cm2) applied on the epithelial side of an alveolar 3D tri-culture, rapidly induced pro-inflammatory and aryl hydrocarbon receptor (AhR)-regulated genes in the basolateral endothelial cells. These effects seem to be due to soluble lipophilic constituents rather than particle translocation. Extractable organic material of DEP (DEP-EOM) was next fractionated with increasing polarity, chemically characterized, and examined for direct effects on pro-inflammatory and AhR-regulated genes in human microvascular endothelial (HMEC-1) cells and primary human endothelial cells (PHEC) from four healthy donors. Exposure-relevant concentrations of lipophilic DEP-EOM (0.15 µg/cm2) induced low to moderate increases in IL-1α, IL-1ß, COX2 and MMP-1 gene expression, and the MMP-1 secretion was increased. By contrast, the more polar EOM had negligible effects, even at higher concentrations. Use of pharmacological inhibitors indicated that AhR and protease-activated receptor-2 (PAR-2) were central in regulation of EOM-induced gene expression. Some effects also seemed to be attributed to redox-responses, at least at the highest exposure concentrations tested. Although the most lipophilic EOM, that contained the majority of PAHs and aliphatics, had the clearest low-concentration effects, there was no straight-forward link between chemical composition and biological effects. CONCLUSION: Lipophilic and semi-lipophilic chemicals seemed to detach from DEP, translocate through alveolar epithelial cells and trigger pro-inflammatory reactions in endothelial cells at exposure-relevant concentrations. These effects appeared to be triggered by AhR agonists, and involve PAR-2 signaling.


Asunto(s)
Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Nanopartículas/toxicidad , Hidrocarburos Policíclicos Aromáticos/toxicidad , Receptores de Hidrocarburo de Aril/metabolismo , Emisiones de Vehículos/toxicidad , Ciclooxigenasa 2/genética , Citocinas/genética , Células Endoteliales/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Inflamación , Metaloproteinasa 1 de la Matriz/genética , Microvasos/efectos de los fármacos , Microvasos/inmunología , Microvasos/metabolismo , Nanopartículas/química , Hidrocarburos Policíclicos Aromáticos/química , Transducción de Señal
3.
Eur Heart J ; 38(39): 2936-2943, 2017 Oct 14.
Artículo en Inglés | MEDLINE | ID: mdl-28431003

RESUMEN

AIMS: Intracoronary infusion of autologous nucleated bone marrow cells (BMCs) enhanced the recovery of left ventricular ejection fraction (LVEF) after ST-segment elevation myocardial infarction (STEMI) in the randomised-controlled, open-label BOOST trial. We reassessed the therapeutic potential of nucleated BMCs in the randomised placebo-controlled, double-blind BOOST-2 trial conducted in 10 centres in Germany and Norway. METHODS AND RESULTS: Using a multiple arm design, we investigated the dose-response relationship and explored whether γ-irradiation which eliminates the clonogenic potential of stem and progenitor cells has an impact on BMC efficacy. Between 9 March 2006 and 16 July 2013, 153 patients with large STEMI were randomly assigned to receive a single intracoronary infusion of placebo (control group), high-dose (hi)BMCs, low-dose (lo)BMCs, irradiated hiBMCs, or irradiated loBMCs 8.1 ± 2.6 days after percutaneous coronary intervention (PCI) in addition to guideline-recommended medical treatment. Change in LVEF from baseline (before cell infusion) to 6 months as determined by MRI was the primary endpoint. The trial is registered at Current Controlled Trials (ISRCTN17457407). Baseline LVEF was 45.0 ± 8.5% in the overall population. At 6 months, LVEF had increased by 3.3 percentage points in the control group and 4.3 percentage points in the hiBMC group. The estimated treatment effect was 1.0 percentage points (95% confidence interval, -2.6 to 4.7; P = 0.57). The treatment effect of loBMCs was 0.5 percentage points (-3.0 to 4.1; P = 0.76). Likewise, irradiated BMCs did not have significant treatment effects. BMC transfer was safe and not associated with adverse clinical events. CONCLUSION: The BOOST-2 trial does not support the use of nucleated BMCs in patients with STEMI and moderately reduced LVEF treated according to current standards of early PCI and drug therapy.


Asunto(s)
Trasplante de Médula Ósea/métodos , Infarto del Miocardio con Elevación del ST/terapia , Células de la Médula Ósea/efectos de la radiación , Método Doble Ciego , Femenino , Rayos gamma , Humanos , Infusiones Intralesiones , Angiografía por Resonancia Magnética , Masculino , Persona de Mediana Edad , Intervención Coronaria Percutánea , Trasplante de Células Madre/métodos , Células Madre/efectos de la radiación , Trasplante Autólogo , Resultado del Tratamiento , Función Ventricular Izquierda/fisiología
4.
Cytotherapy ; 18(2): 242-52, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26669908

RESUMEN

BACKGROUND AIMS: Autologous endothelial cells are promising alternative angiogenic cell sources in trials of therapeutic vasculogenesis, in the treatment of vascular diseases and in the field of tissue engineering. A population of endothelial cells (ECs) with long-term proliferative capability, endothelial colony-forming cells (ECFCs), can be isolated from human peripheral blood. ECFCs are considered an endothelial precursor population. They can be expanded in cell factories in sufficient numbers for clinical applications, but because the number of isolated primary ECs is low, the culture period required may be long. Another EC population that is easily available in the autologous setting and may be expanded in vitro through several population doublings are ECs from adipose tissue (AT-ECs). METHODS: Through extensive comparisons using whole-genome microarray analysis, morphology, phenotype and functional assays, we wanted to evaluate the potential of these EC populations for use in clinical neovascularization. RESULTS: Global gene expression profiling of ECFCs, AT-ECs and the classical EC population, human umbilical vein ECs, showed that the EC populations clustered as unique populations, but very close to each other. By cell surface phenotype and vasculogenic potential in vitro and in vivo, we also found the ECFCs to be extremely similar to AT-ECs. CONCLUSIONS: These properties, together with easy access in the autologous setting, suggest that both AT-ECs and ECFCs may be useful in trials of therapeutic neovascularization. However, AT-ECs may be a more practical alternative for obtaining large quantities of autologous ECs.


Asunto(s)
Tejido Adiposo/citología , Células Endoteliales de la Vena Umbilical Humana/citología , Neovascularización Fisiológica/fisiología , Células Madre/citología , Ingeniería de Tejidos/métodos , Diferenciación Celular , Células Cultivadas , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
5.
Acta Orthop ; 87(sup363): 1-5, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28271925

RESUMEN

The combination of modern interventional and preventive medicine has led to an epidemic of ageing. While this phenomenon is a positive consequence of an improved lifestyle and achievements in a society, the longer life expectancy is often accompanied by decline in quality of life due to musculoskeletal pain and disability. The Aarhus Regenerative Orthopaedics Symposium (AROS) 2015 was motivated by the need to address regenerative challenges in an ageing population by engaging clinicians, basic scientists, and engineers. In this position paper, we review our contemporary understanding of societal, patient-related, and basic science-related challenges in order to provide a reasoned roadmap for the future to deal with this compelling and urgent healthcare problem.


Asunto(s)
Envejecimiento/fisiología , Sistema Musculoesquelético/fisiopatología , Medicina Regenerativa/métodos , Animales , Comorbilidad , Modelos Animales de Enfermedad , Humanos , Regeneración/fisiología
6.
Stem Cells ; 32(6): 1629-35, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24449344

RESUMEN

The transcription factor SOX9 is believed to be the master regulator of chondrogenesis. SOX8 is another SOX group E transcription factor with a high degree of homology to SOX9. Here, we demonstrate that SOX8 mRNA levels decrease during in vitro dedifferentiation of human articular chondrocytes and increase during chondrogenic differentiation of mesenchymal stromal cells. Knockdown of SOX9 reduced the expression of SOX8, COL2A1, and a range of other chondrogenic molecules. SOX8 knockdown reduced the expression of a large number of overlapping chondrogenic molecules, but not SOX9. Neither siSOX9 nor siSOX8 altered expression of the hypertrophic marker gene COL10A1. siSOX9, but not siSOX8 led to upregulation of hypertrophy associated genes MMP13 and ALPL. Transfection of synthetic SOX5, 6, and 9 mRNA trio upregulated SOX8, COL2A1, and ACAN, but not COL10A1 mRNA. Replacement of synthetic SOX9 by SOX8 in the SOX trio showed similar but lower chondrogenic effect. We conclude that SOX8 expression is regulated by SOX9, and that both together with SOX5 and SOX6 are required as a SOX quartet for transcription of COL2A1 and a large number of other chondrogenic molecules. Neither SOX8 nor SOX9 affect COL10A1 expression, but SOX9 inhibits chondrocyte hypertrophy through inhibition of MMP13 and ALPL expression.


Asunto(s)
Diferenciación Celular , Condrogénesis , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Factores de Transcripción SOXE/metabolismo , Desdiferenciación Celular/genética , Diferenciación Celular/genética , Condrocitos/metabolismo , Condrogénesis/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reproducibilidad de los Resultados , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXE/genética , Transfección
7.
Biochem Biophys Res Commun ; 447(1): 139-44, 2014 Apr 25.
Artículo en Inglés | MEDLINE | ID: mdl-24704451

RESUMEN

Bone tissue engineering requires an osteoconductive scaffold, multipotent cells with regenerative capacity and bioactive molecules. In this study we investigated the osteogenic differentiation of human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) on titanium dioxide (TiO2) scaffold coated with alginate hydrogel containing various concentrations of simvastatin (SIM). The mRNA expression of osteoblast-related genes such as collagen type I alpha 1 (COL1A1), alkaline phosphatase (ALPL), osteopontin (SPP1), osteocalcin (BGLAP) and vascular endothelial growth factor A (VEGFA) was enhanced in hAD-MSCs cultured on scaffolds with SIM in comparison to scaffolds without SIM. Furthermore, the secretion of osteoprotegerin (OPG), vascular endothelial growth factor A (VEGFA), osteopontin (OPN) and osteocalcin (OC) to the cell culture medium was higher from hAD-MSCs cultured on scaffolds with SIM compared to scaffolds without SIM. The TiO2 scaffold coated with alginate hydrogel containing SIM promote osteogenic differentiation of hAD-MSCs in vitro, and demonstrate feasibility as scaffold for hAD-MSC based bone tissue engineering.


Asunto(s)
Tejido Adiposo/citología , Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Osteogénesis , Simvastatina/farmacología , Andamios del Tejido , Adulto , Supervivencia Celular , Cadena alfa 1 del Colágeno Tipo I , Femenino , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Persona de Mediana Edad , Osteocalcina/metabolismo , Osteopontina/metabolismo , Osteoprotegerina/metabolismo , ARN Mensajero/metabolismo , Ingeniería de Tejidos/métodos , Titanio , Factor A de Crecimiento Endotelial Vascular/metabolismo
8.
Mol Ther ; 21(6): 1169-81, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23568258

RESUMEN

Synthetic microRNAs regulate gene expression when transfected into cells, and may be used in strategies for molecular therapy both in vitro and in vivo. Liposomal transfection reagents are frequently used as delivery vehicles in both settings. Here, we report on the immunological off-target effects observed following liposome transfection of synthetic microRNA-145 into human mesenchymal stem cells and human articular chondrocytes (hAC). The immune response was independent on endosome delivery and toll-like receptors (TLRs) but was mediated by retinoic acid inducible-gene 1 (RIG-I). Upregulation of immune genes required liposomal delivery, as no immune response was observed after electroporation of smiR-145 directly in to the cytosol, suggesting a new role of RIG-I. Immune response was seen both with blunt ended and 2-nucleotide 3' overhang versions of synthetic miR-145, and occurred in the absence of a 5'ppp cap. Mutations in a centrally placed poly (UUUU) sequence reduced, but did not abolish the immune response. Interestingly, exposure to liposomes alone led to upregulation of several immune genes, including RIG-I mRNA. However, this process was not mediated by RIG-I. This insight is important for researchers to avoid unexpected results from gene transfer experiments in vitro and unwanted immune responses following the use of lipid-based transfection reagents in vivo.


Asunto(s)
ARN Helicasas DEAD-box/genética , Liposomas/química , Células Madre Mesenquimatosas/efectos de los fármacos , MicroARNs/genética , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Biología Computacional , Citocinas/metabolismo , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Células HEK293 , Humanos , Células Madre Mesenquimatosas/patología , MicroARNs/metabolismo , Análisis por Micromatrices , Microscopía Confocal , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Inmunológicos , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Transfección , Regulación hacia Arriba
9.
Arthrosc Sports Med Rehabil ; 6(2): 100909, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38495635

RESUMEN

Purpose: To compare the functional and patient-reported outcome measures after autologous chondrocyte implantation (ACI) and arthroscopic debridement (AD) in symptomatic, isolated cartilage injuries larger than 2 cm2 in patients aged 18 to 50 years. Methods: Twenty-eight patients were included and randomized to ACI (n = 15) or AD (n = 13) and followed for 2 years. The primary outcome was the change in the Knee injury and Osteoarthritis Outcome Score (KOOS) Quality of Life (QoL) subscale. Results: The mean age at inclusion was 34.1 (standard deviation [SD] 8.5) years. There were 19 (68%) male patients. The mean size of the lesion was 4.2 (SD 1.7) cm2. There was a statistically significant and clinically meaningful improvement in patient-reported outcome measures from baseline to 2 years in both groups. The improvement from baseline to final follow-up for the primary endpoint (the KOOS QoL subscale) was larger for the AD group (39.8, SD 9.4) compared with the ACI group (23.8, SD 6.7), but this difference was not statistically significant (P = .17). However, according to a mixed linear model there were statistically significantly greater scores in the AD group for several KOOS subscales at several time points, including KOOS QoL, KOOS pain, and KOOS sport and recreation at 2 years. Conclusions: This study indicates that AD followed by supervised physiotherapy is equal to or better than ACI followed by supervised physiotherapy in patients with isolated cartilage lesions of the knee larger than 2 cm2. The improvement in KOOS QoL score from baseline to 2 years was clinically meaningful for both groups (23.8 points for ACI and 39.8 points AD), and larger for the AD group by 16 points. Level of Evidence: Level I, prospective randomized controlled trial.

10.
BMC Genomics ; 14: 105, 2013 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-23414147

RESUMEN

BACKGROUND: For safe clinical application of engineered cartilage made from mesenchymal stem cells (MSCs), molecular mechanisms for chondrogenic differentiation must be known in detail. Changes in gene expression and extracellular matrix synthesis have been extensively studied, but the epigenomic modifications underlying these changes have not been described. To this end we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations. RESULTS: We see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5' end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set. CONCLUSIONS: Histone modifications, rather than DNA methylation, provide the primary epigenetic control of early differentiation of MSCs towards the chondrogenic lineage.


Asunto(s)
Epigénesis Genética , Genoma Humano , Células Madre Mesenquimatosas/citología , Diferenciación Celular/genética , Linaje de la Célula , Células Cultivadas , Condrogénesis/genética , Metilación de ADN , Regulación de la Expresión Génica , Histonas/genética , Humanos , Regiones Promotoras Genéticas , Elementos Reguladores de la Transcripción , Sitio de Iniciación de la Transcripción
11.
Cancer Immunol Immunother ; 62(9): 1499-509, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23817721

RESUMEN

BACKGROUND: The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting. METHODS: We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9-18 vaccines containing 10(7) cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456. RESULTS: Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test). CONCLUSION: These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.


Asunto(s)
Neoplasias Encefálicas/terapia , Vacunas contra el Cáncer/administración & dosificación , Células Dendríticas/inmunología , Glioblastoma/terapia , Inmunoterapia Adoptiva/métodos , Células Madre Neoplásicas/inmunología , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/patología , Vacunas contra el Cáncer/inmunología , Terapia Combinada , Células Dendríticas/patología , Supervivencia sin Enfermedad , Femenino , Glioblastoma/inmunología , Glioblastoma/patología , Humanos , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/patología , ARN Mensajero/genética , Telomerasa/genética , Telomerasa/inmunología , Transfección
12.
Front Bioeng Biotechnol ; 11: 1116513, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36896010

RESUMEN

Focal lesions of articular cartilage give rise to pain and reduced joint function and may, if left untreated, lead to osteoarthritis. Implantation of in vitro generated, scaffold-free autologous cartilage discs may represent the best treatment option. Here we compare articular chondrocytes (ACs) and bone marrow-derived mesenchymal stromal cells (MSCs) for their ability to make scaffold-free cartilage discs. Articular chondrocytes produced more extracellular matrix per seeded cell than mesenchymal stromal cells. Quantitative proteomics analysis showed that articular chondrocyte discs contained more articular cartilage proteins, while mesenchymal stromal cell discs had more proteins associated with cartilage hypertrophy and bone formation. Sequencing analysis revealed more microRNAs associated with normal cartilage in articular chondrocyte discs, and large-scale target predictions, performed for the first time for in vitro chondrogenesis, suggested that differential expression of microRNAs in the two disc types were important mechanisms behind differential synthesis of proteins. We conclude that articular chondrocytes should be preferred over mesenchymal stromal cells for tissue engineering of articular cartilage.

13.
Basic Clin Pharmacol Toxicol ; 132(1): 83-97, 2023 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-36214226

RESUMEN

Exposure to fine particulate matter (PM2.5 ) from incomplete fossil fuel combustion (coal, oil, gas and diesel) has been linked to increased morbidity and mortality due to metabolic diseases. PM2.5 exaggerate adipose inflammation and insulin resistance in mice with diet-induced obesity. Here, we elucidate the hypothesis that such systemic effects may be triggered by adhered particle components affecting adipose tissue directly. Studying adipocytes differentiated from primary human mesenchymal stem cells, we found that lipophilic organic chemicals (OC) from diesel exhaust particles induced inflammation-associated genes and increased secretion of the chemokine CXLC8/interleukin-8 as well as matrix metalloprotease 1. The oxidative stress response gene haem oxygenase-1 and tumour necrosis factor alpha were seemingly not affected, while aryl hydrocarbon receptor-regulated genes, cytochrome P450 1A1 (CYP1A1) and CYP1B1 and plasminogen activator inhibitor-2, were clearly up-regulated. Finally, expression of ß-adrenergic receptor, known to regulate adipocyte homoeostasis, was down-regulated by exposure to these lipophilic OC. Our results indicate that low concentrations of OC from combustion particles have the potential to modify expression of genes in adipocytes that may be linked to metabolic disease. Further studies on mechanisms linking PM exposure and metabolic diseases are warranted.


Asunto(s)
Contaminantes Atmosféricos , Células Madre Mesenquimatosas , Humanos , Ratones , Animales , Emisiones de Vehículos/toxicidad , Material Particulado/toxicidad , Compuestos Orgánicos , Adipocitos/química , Inflamación , Contaminantes Atmosféricos/toxicidad
14.
J Cell Physiol ; 226(11): 2908-15, 2011 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-21302288

RESUMEN

Human mesenchymal stem cells (hMSCs) may be used for therapeutic applications. Culture conditions such as the serum source may impact on cell quality and the onset of replicative senescence. We have examined the effect of culturing hMSCs in autologous serum (AS) versus fetal bovine serum (FBS) on factors involved in in vitro replicative senescence. hMSCs from four donors were cultured in 10% FBS or 10% AS until they reached senescence. Cells were harvested at early passage and near senescence to study factors known to be involved in cellular senescence. The number of population doublings till senescence was similar for cells cultured in FBS, but varied greatly for hMSCs cultured in AS. FBS cells accumulated in S phase of cell cycle. This could not be explained by increased expression of cell cycle inhibitor proteins. Heat shock proteins were upregulated in AS compared to FBS cells. Reactive oxygen species and nitric oxide were upregulated in senescent FBS cells. Telomeres were shorter in senescent cells, more significantly in FBS cells. The source of serum was a determinant for the time till senescence in cultured hMSC. Serum source affected aspects of cell cycle regulation and the levels of heat shock proteins. Several mechanisms are likely to be responsible for replicative senescence in hMSC. Insight into the molecular details of how serum factors impacts on these mechanisms is important for the safe use of hMSCs in clinical applications.


Asunto(s)
Células de la Médula Ósea/fisiología , Senescencia Celular , Sangre Fetal/metabolismo , Células Madre Mesenquimatosas/fisiología , Animales , Células de la Médula Ósea/metabolismo , Bovinos , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Choque Térmico/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Regulación hacia Arriba
15.
J Cell Biochem ; 112(2): 684-93, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21268090

RESUMEN

To obtain sufficient numbers of cells for tissue engineering applications, human bone marrow-derived mesenchymal stem cells (hBM-MSC) are commonly cultured as monolayers in incubators containing room air. In this study, we investigated whether three-dimensional (3D) culture conditions and incubator gas concentrations more similar to those observed in vivo impacted on cell expansion, differentiation capability, or phenotype of hBM-MSC. We found that 3D culture alone increased the expression of some molecules involved in osteogenic and adipogenic differentiation. In contrast, 3D culture did not induce chondrogenic differentiation, but enhanced the response to the chondrogenic differentiation medium. Changing the oxygen concentration to 6% and the carbon dioxide concentration to 7.5% did not impact on the results of any of our assays, showing that the hyperoxia of room air is not detrimental to hBM-MSC proliferation, differentiation, or phenotype.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/fisiología , Células Madre Mesenquimatosas/citología , Diferenciación Celular/genética , Células Cultivadas , Citometría de Flujo , Humanos , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
16.
Osteoarthr Cartil Open ; 3(3): 100189, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-36474808

RESUMEN

Objective: MicroRNA-140-3p is the most prevalent form of canonical miR-140 in native chondrocytes. IsomiRs are sequence variants of microRNAs with potentially distinct functionalities. Here we present functional studies of canonical microRNA-140-3p and two of its most prevalent isomiRs, a 5' isomiR and a 3' isomiR, in an inflammation-induced model of osteoarthritis (OA). Method: Canonical miR-140-3p, the 5' isomiR and the 3' isomiR were overexpressed separately in chondrocytes from three donors and subsequently subjected to an inflammatory milieu mediated by interleukin 1 beta and tumor necrosis factor alpha. RNA sequencing was performed on the cells to investigate the altered transcriptomes, RT-qPCR was performed to validate important observations, and western blot analysis was carried out to further study key inflammatory molecules. Results: The three microRNAs downregulated many of the same genes. However, the 5' isomiR showed a much greater target spectrum compared to the other two miRNAs, and downregulated cascades of genes downstream of interferon beta, interferon gamma and interleukin 1 beta as well as genes involved in several other inflammatory and antiviral pathways. In addition the 5' isomiR downregulated practically all HLA class II and class I genes. Conclusion: Introduction of the 5' isomiR led to downregulation of genes essential for some of the most important inflammation cascades and virtual silencing of genes responsible for antigen presentation. These observations may indicate a very promising therapeutic potential for the 5' isomiR for OA and several inflammatory conditions, particularly HLA associated immune conditions including many arthritic diseases.

17.
Cartilage ; 13(2_suppl): 774S-784S, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-31072202

RESUMEN

OBJECTIVE: To investigate the heterogeneity of in vitro expanded chondrocytes used for autologous chondrocyte implantation. METHODS: Human articular chondrocytes were expanded in vitro for 14 days, sorted into 86 single cells using fluorescence-activated cell sorting and subjected to single-cell RNA sequencing. Principal component, Cross R2 hierarchical clustering, and differential gene expression analyses were used for data evaluation. Flow cytometry and single-cell RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) was used to validate the results of the RNA sequencing data Polyclonal chondrocyte populations from the same donor were differentiated in vitro toward the osteogenic and adipogenic lineages. RESULTS: There was considerable variation in gene expression between individual cells, but we found no evidence for separate cell subpopulations based on principal component, hierarchical clustering, and differential gene expression analysis. Most of the cells expressed all the markers defining mesenchymal stem cells, and as polyclonal chondrocyte populations from the same donor were shown to differentiate into osteocytes and adipocytes in vitro, these cells formally qualify as mesenchymal stem cells. CONCLUSIONS: In vitro expanded chondrocytes consist of one single population of cells with heterogeneity in gene expression between the cells. Dedifferentiated chondrocytes qualify as mesenchymal stem cells as they fulfill all the criteria suggested by the International Society for Cellular Therapy.


Asunto(s)
Condrocitos , Células Madre Mesenquimatosas , Adipocitos , Diferenciación Celular , Condrocitos/metabolismo , Humanos , Análisis de Secuencia de ARN
18.
Cartilage ; 13(1_suppl): 1237S-1249S, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-33858229

RESUMEN

OBJECTIVE: Despite new strategies in tissue engineering, cartilage repair remains a major challenge. Our aim is to treat patients with focal lesions of articular cartilage with autologous hyaline cartilage implants using a scaffold-free approach. In this article, we describe experiments to optimize production of scaffold-free cartilage discs. DESIGN: Articular chondrocytes were expanded in vitro, seeded in transwell inserts and redifferentiated using established chondrogenic components. Experimental variables included testing 2 different expansion media, adding bone morphogenetic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), growth/differentiation factor 5 (GDF5), or fibroblast growth factor 18 (FGF18) to the differentiation medium and allowing the disc to float freely in large wells. Cartilage discs were analyzed by weight and thickness, real-time RT-qPCR (reverse transcriptase qualitative polymerase chain reaction), fluorescence immunostaining, transmission electron microscopy, second harmonic generation imaging, and measurement of Young's modulus. RESULTS: Addition of BMP2 to the chondrogenic differentiation medium (CDM) was essential for stable disc formation, while IGF1, GDF5, and FGF18 were redundant. Allowing discs to float freely in CDM on a moving platform increased disc thickness compared with discs kept continuously in transwell inserts. Discs cultured for 6 weeks reached a thickness of almost 2 mm and Young's modulus of >200 kPa. There was abundant type II collagen. Collagen fibrils were 25 nm thick, with a tendency to be organized perpendicular to the disc surface. CONCLUSION: Scaffold-free engineering using BMP2 and providing free movement in CDM produced firm, elastic cartilage discs with abundant type II collagen. This approach may potentially be used in clinical trials.


Asunto(s)
Cartílago Articular/cirugía , Condrocitos , Ingeniería de Tejidos , Células Cultivadas , Condrogénesis , Colágeno Tipo II , Humanos
19.
Exp Cell Res ; 315(15): 2558-67, 2009 Sep 10.
Artículo en Inglés | MEDLINE | ID: mdl-19477173

RESUMEN

Adipose-tissue derived mesenchymal stem cells (AT-MSCs) are a promising tool for use in cell-based therapies. However, in vitro expansion is required to obtain clinically relevant cell numbers, and this might increase the chance of genomic instability. DNA repair is crucial for maintaining DNA integrity. Here we have compared the initial step of base excision repair in uncultured and cultured AT-MSCs by analysis of base removal activities and expression levels of relevant DNA glycosylases. Uracil, 5-hydroxyuracil and ethenoadenine removal activities were upregulated in cultured cells compared to uncultured cells. In contrast, both the 8-oxo-7,8-dihydroguanine (8-oxoG) removal activity and the concentration of 8-oxoG bases in the DNA were reduced in the cultured cells. Gene expression analysis showed no substantial changes in mRNA expression. The glycosylase activities remained stable through at least 12 passages, suggesting that DNA repair is proficient through the period required for in vitro expansion of AT-MSCs to clinically relevant numbers.


Asunto(s)
ADN Glicosilasas/metabolismo , Reparación del ADN , Células Madre Mesenquimatosas/fisiología , Tejido Adiposo/citología , Animales , Células Cultivadas , Daño del ADN , ADN Glicosilasas/genética , Perfilación de la Expresión Génica , Guanina/análogos & derivados , Guanina/metabolismo , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Uracilo/análogos & derivados , Uracilo/metabolismo
20.
Knee Surg Sports Traumatol Arthrosc ; 18(10): 1407-16, 2010 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-20020100

RESUMEN

Treatment of focal lesions of the articular cartilage of the knee using chondrocytes in a hyaluronic acid (HA) scaffold is already being investigated in clinical trials. An alternative may be to use mesenchymal stem cells (MSC). We have compared articular chondrocytes with MSC from human bone marrow (BM) and adipose tissue (AT), all cultured in HA scaffolds, for their ability to express genes and synthesize proteins associated with chondrogenesis. The cells were expanded in monolayer cultures. After seeding into the scaffold, the chondrocytes were maintained in medium, while the two MSC populations were given a chondrogenic differentiation medium. Chondrogenesis was assessed by real-time RT-PCR for chondrocyte-associated genes, by immunohistochemistry and by ELISA for collagens in the supernatant. Redifferentiation of the dedifferentiated chondrocytes in the HA scaffold was shown by a modest increase in type II collagen mRNA (COL2A1) and reduction in COL1A1. BM-MSC expressed 600-fold higher levels of COL2A1 than chondrocytes after 3 weeks in the scaffold. The levels of aggrecan (AGC1) and COL1A1 were similar for chondrocyte and BM-MSC scaffold cultures, while COL10A1 was higher in the BM-MSC. AT-MSC expressed levels of COL2A1 and COL1A1 similar to chondrocytes, but less AGC1 and COL10A1. Surprisingly, little collagen II protein was observed in the scaffold. Instead, collagen II was found in the culture medium. Chondrogenesis in HA scaffolds was more efficient using BM-MSC than AT-MSC or chondrocytes. Some of the secreted collagen II escaped entrapment in the extracellular space and was detected in the culture medium.


Asunto(s)
Células de la Médula Ósea/metabolismo , Condrogénesis/fisiología , Células Madre Mesenquimatosas/metabolismo , Ingeniería de Tejidos/métodos , Andamios del Tejido , Adulto , Análisis de Varianza , Células de la Médula Ósea/citología , Cartílago Articular/cirugía , Diferenciación Celular/fisiología , Células Cultivadas , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ácido Hialurónico/metabolismo , Inmunohistoquímica , Masculino , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto Joven
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