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Background: Human autologous serum (AS) and umbilical cord serum (UCS) both contain growth and neurotrophic factors that promote corneal healing. Aim: Our objectives were to compare equine AS and UCS cytokine and growth factor profiles and to assess the safety and clinical feasibility of the therapeutic use of UCS eye drops in cases of spontaneous complex ulcers. Study Design: Prospective clinical trial. Methods: Vitamin A insulin growth factor, platelet-derived growth factor-BB, transforming growth factor (TGF)-ß1 (enzyme-linked immunosorbent assay), interleukin (IL)-1ß, IL-6, interferon-γ, and monocyte chemoattractant protein 1 concentrations were determined in 10 AS collected from different horses and 10 UCS sampled at delivery. Six client-owned horses presenting with complex non-healing corneal defects of >5 mm2 were included in a clinical trial and treated with conventional therapy and conditioned UCS drops for 8-15 days. Ulcer surface and time to complete epithelialization were recorded. Results: Median concentrations of vitamin A, insulin growth factor, and platelet-derived growth factor-BB were not significantly different in AS compared with UCS (respectively, 14.5 vs. 12.05 µg/ml; 107.8 vs. 107.3 pg/ml; and 369.1 vs. 924.2 pg/ml). TGF-ß1 median concentration in UCS was significantly higher than in AS (3,245 vs. 2571pg/ml) (p = 0.04). IL-1ß, IL-6, interferon-γ, and monocyte chemoattractant protein 1 concentrations were variable in AS and undetectable in UCS. The corneal median ulcerative area was 37.2 mm2 (6.28-57.14 mm2) and had a duration of 4-186 days (median 19 days). All lesions healed within 13-42 days (median 17 days). No adverse effects nor recurrences within 1 month were noticed. Limitations: The sample size was small. Spontaneous corneal epithelial defects presented with variable clinical characteristics. There were no age-matched control horses to assess corneal healing time and rate. Conclusion and Clinical Significance: Equine UCS may be beneficial, as it contains no pro-inflammatory cytokines and a greater concentration of TGF-ß1 compared with AS. Topical UCS appears safe and may potentially be used as adjunctive therapy for equine complex non-healing ulcers.

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OBJECTIVE: To determine the feasibility of umbilical cord-derived mesenchymal stem cell (UC-MSC) transplantation into the cervical spinal cord of horses by using fluoroscopy with or without endoscopic guidance and to evaluate the neurological signs and tissue reaction after injection. STUDY DESIGN: Experimental study. ANIMALS: Eight healthy adult horses with no clinical signs of neurological disease. METHODS: After cervical ventral interbody fusion (CVIF), ten million fluorescently labeled allogeneic UC-MSC were injected into the spinal cord under endoscopic and fluoroscopic guidance (n = 5) or fluoroscopic guidance only (n = 3). Postoperative neurological examinations were performed, and horses were humanely killed 48 hours (n = 4) or 14 days (n = 4) postoperatively. Spinal tissues were examined after gross dissection and with bright field and fluorescent microscopy. RESULTS: Needle endoscopy of the cervical canal by ventral approach was associated with intraoperative spinal cord puncture (2/5) and postoperative ataxia (3/5). No intraoperative complications occurred, and one (1/3) horse developed ataxia with cell transplantation under fluoroscopy alone. Umbilical cord-derived MSC were associated with small vessels and detected up to 14 days in the spinal cord. Demyelination was observed in six of eight cases. CONCLUSION: Fluoroscopically guided intramedullary UC-MSC transplantation during CVIF avoids spinal cord trauma and decreases risk of ataxia from endoscopy. Umbilical cord-derived MSC persist in the spinal cord for up to 14 days. Cell injection promotes angiogenesis and induces demyelination of the spinal tissue. CLINICAL SIGNIFICANCE: Umbilical cord-derived MSC transplantation into the spinal cord during CVIF without endoscopy is recommended for future evaluation of cell therapy in horses affected by cervical vertebral compressive myelopathy.

Asunto(s)

Vértebras Cervicales/cirugía , Enfermedades de los Caballos/cirugía , Trasplante de Células Madre Mesenquimatosas/veterinaria , Compresión de la Médula Espinal/veterinaria , Fusión Vertebral/veterinaria , Animales , Ataxia/prevención & control , Ataxia/veterinaria , Endoscopía/efectos adversos , Endoscopía/veterinaria , Estudios de Factibilidad , Femenino , Fluoroscopía , Caballos , Masculino , Complicaciones Posoperatorias/veterinaria , Compresión de la Médula Espinal/cirugía , Fusión Vertebral/métodos

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An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Background: The growing interest in mesenchymal stromal cells (MSCs) in equine medicine, together with the development of MSC biobanking for allogeneic use, raises concerns about biosafety of such products. MSCs derived from umbilical cord (UC) carry an inherent risk of contamination by environmental conditions and vertical transmission of pathogens from broodmares. There is yet no report in the scientific literature about horses being contaminated by infected MSC products, and no consensus about systematic infectious screening of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) to ensure microbiological safety of therapeutic products. Objectives: To develop a standard protocol to ensure UC-MSC microbiological safety and to assess the risk of vertical transmission of common intracellular pathogens from broodmares to paired UC-MSCs. Study Design and Methods: Eighty-four UC and paired peripheral maternal blood (PMB) samples were collected between 2014 and 2016. Sterility was monitored by microbiological control tests. Maternal contamination was tested by systematical PMB PCR screening for 14 pathogens and a Coggins test. In case of a PCR-positive result regarding one or several pathogen(s) in PMB, a PCR analysis for the detected pathogen(s) was then conducted on the associated UC-MSCs. Results: Ten out of 84 UC samples were contaminated upon extraction and 6/84 remained positive in primo culture. The remaining 78/84 paired PMB & UC-MSC samples were evaluated for vertical transmission; 37/78 PMB samples were PCR positive for Equid herpesvirus (EHV)-1, EHV-2, EHV-5, Theileria equi, Babesia caballi, and/or Mycoplasma spp. Hepacivirus was detected in 2/27 cases and Theiler Diseases Associated Virus in 0/27 cases (not performed on all samples due to late addition). All paired UC-MSC samples tested for the specific pathogen(s) detected in PMB were negative (37/37). Main Limitations: More data are needed regarding MSC susceptibility to most pathogens detected in PMB. Conclusions: In-process microbiological controls combined with PMB PCR screening provide a comprehensive assessment of UC-MSC exposure to infectious risk, vertical transmission risk appearing inherently low.

Asunto(s)

Bacterias/aislamiento & purificación , Células Madre Mesenquimatosas/citología , Piroplasmida/aislamiento & purificación , Cordón Umbilical/citología , Virus/aislamiento & purificación , Animales , Bancos de Muestras Biológicas , Contención de Riesgos Biológicos , Femenino , Caballos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/microbiología , Leucocitos Mononucleares/parasitología , Células Madre Mesenquimatosas/microbiología , Células Madre Mesenquimatosas/parasitología

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The purpose of this prospective study was to evaluate the effects of single and repeated intra-articular administration of allogeneic, umbilical cord-derived, neonatal mesenchymal stem cells (MSC) in horses with lameness due to osteoarthritis (OA) of a metacarpophalangeal joint (MPJ). Twenty-eight horses were included. Horses were divided into two groups. Horses in group MSC1 received an MSC injection at M0 and a placebo injection at M1 (1 month after M0). Horses in group MSC2 received MSC injections at M0 and at M1. Joint injections were performed with a blinded syringe. Clinical assessment was performed by the treating veterinarian at M1, M2 and M6 (2 and 6 months after M0), including lameness evaluation, palpation and flexion of the joint. Radiographic examination of the treated joints was performed at inclusion and repeated at M6. Radiographs were anonymized and assessed by 2 ECVDI LA associate members. Short term safety assessment was performed by owner survey. A 2-month rehabilitation program was recommended to veterinarians. There was a significant improvement of the total clinical score for horses in both groups. There was no significant difference in the total clinical score between groups MSC1 and MSC2 at any time point in the study. There was no significant difference in the total radiographic OA score, osteophyte score, joint space width score and subchondral bone score between inclusion and M6. Owner-detected adverse effects to MSC injection were recorded in 18% of the horses. Lameness caused by OA improved significantly over the 6-month duration of the study after treatment with allogeneic neonatal umbilical cord-derived MSCs combined with 8 weeks rest and rehabilitation. There is no apparent clinical benefit of repeated intra-articular administration of MSCs at a 1-month interval in horses with MPJ OA when compared to the effect of a single injection.

Asunto(s)

Enfermedades de los Caballos/terapia , Caballos , Trasplante de Células Madre Mesenquimatosas , Articulación Metacarpofalángica , Articulación Metatarsofalángica , Osteoartritis/terapia , Aloinjertos , Animales , Femenino , Enfermedades de los Caballos/patología , Enfermedades de los Caballos/fisiopatología , Masculino , Osteoartritis/patología , Osteoartritis/fisiopatología

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Objective: To explore the long-term safety and efficacy of canine allogeneic mesenchymal stromal cells (MSC) administered intra-articularly as single or repeated injections in appendicular joints of dogs affected by moderate to severe refractory osteoarthritis. Study Design: 22 pet dogs were recruited into a non-randomized, open and monocentric study initially administering one cellular injection. A second injection was offered after 6 months to owners if the first injection did not produce expected results. Materials and Methods: Anti-inflammatory treatment (if prescribed) was discontinued at last one week before the onset of treatment. Each injection consisted of at least 10 million viable neonatal allogeneic mesenchymal stromal cells obtained from fetal adnexa. Medical data was collected from veterinary clinical evaluations of joints up to 6 months post-injection and owner's assessment of their dog's mobility and well-being followed for a further 2 years when possible. Results: Mild, immediate self-limiting inflammatory joint reactions were observed in 5/22 joints after the first injection, and in almost all dogs having a subsequent injection. No other MSC-related adverse medical events were reported, neither during the 6 months follow up visits, nor during the long-term (2-years) safety follow up. Veterinary clinical evaluation showed a significant and durable clinical improvement (up to 6 months) following MSC administration. Eight dogs (11 joints) were re-injected 6 months apart, sustaining clinical benefits up to 1 year. Owner's global satisfaction reached 75% at 2 years post-treatment Conclusion: Our data suggest that a single or repeated intra-articular administration of neonatal MSC in dogs with moderate to severe OA is a safe procedure and confer clinical benefits over a 24-month period. When humoral response against MSC is investigated by flow cytometry, a positive mild and transient signal was detected in only one dog from the studied cohort, this dog having had a positive clinical outcome.

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Umbilical cord blood mesenchymal stromal/stem cells (UCB-MSCs) and umbilical cord matrix MSCs (UCM-MSCs) have chondrogenic potential and are alternative sources to standard surgically derived bone marrow or adipose tissue collection for cartilage engineering. However, the majority of comparative studies explore neonatal MSCs potential only on ISCT benchmark assays accounting for some bias in the reproducibility between in vitro and in clinical studies. Therefore, we characterized equine UCB-MSCs and UCM-MSCs and investigated with particular attention their chondrogenesis potential in 3D culture with BMP-2 + TGF-ß1 in normoxia or hypoxia. We carried out an exhaustive characterization of the extracellular matrix generated by both these two types of MSCs after the induction of chondrogenesis through evaluation of hyaline cartilage, hypertrophic and osteogenic markers (mRNA, protein and histology levels). Some differences in hypoxia sensitivity and chondrogenesis were observed. UCB-MSCs differentiated into chondrocytes express an abundant, dense and a hyaline-like cartilage matrix. By contrast, despite their expression of cartilage markers, UCM-MSCs failed to express a relevant cartilage matrix after chondrogenic induction. Both MSCs types also displayed intrinsic differences at their undifferentiated basal status, UCB-MSCs expressing higher levels of chondrogenic markers whereas UCM-MSCs synthesizing higher amounts of osteogenic markers. Our results suggest that UCB-MSCs should be preferred for ex-vivo horse cartilage engineering. How those results should be translated to in vivo direct cartilage regeneration remains to be determined through dedicated study.

Asunto(s)

Condrogénesis/fisiología , Sangre Fetal/citología , Cordón Umbilical/citología , Animales , Proteína Morfogenética Ósea 2/metabolismo , Cartílago/metabolismo , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/metabolismo , Condrogénesis/genética , Colágeno Tipo I/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Sangre Fetal/fisiología , Caballos , Cartílago Hialino/metabolismo , Células Madre Mesenquimatosas/fisiología , Osteogénesis/efectos de los fármacos , Regeneración/efectos de los fármacos , Reproducibilidad de los Resultados , Ingeniería de Tejidos/métodos , Factor de Crecimiento Transformador beta1/metabolismo , Cordón Umbilical/fisiología

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As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine.

Asunto(s)

Proteína Morfogenética Ósea 2/metabolismo , Diferenciación Celular , Condrocitos/citología , Condrocitos/metabolismo , Interferencia de ARN , Animales , Biomarcadores , Proteína Morfogenética Ósea 2/farmacología , Cartílago Articular/citología , Cartílago Articular/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Hipoxia de la Célula/genética , Condrocitos/efectos de los fármacos , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Matriz Extracelular/metabolismo , Caballos , Fenotipo , ARN Interferente Pequeño/genética , Ingeniería de Tejidos

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OBJECTIVE: Compare the clinical and pressure walkway gait evolution of dogs after a tibial plateau leveling osteotomy (TPLO) for a cranial cruciate ligament rupture (CrCLR) and treatment with either a 1-month course of non-steroidal anti-inflammatory drugs (NSAIDs) or a single postoperative intra-articular (IA) injection of allogeneic neonatal mesenchymal stromal cells (MSCs). STUDY DESIGN: Prospective, double-blinded, randomized, controlled, monocentric clinical study. ANIMALS: Sixteen client-owned dogs. MATERIALS AND METHODS: Dogs with unilateral CrCLR confirmed by arthroscopy were included. Allogeneic neonatal canine MSCs were obtained from fetal adnexa retrieved after C-section performed on healthy pregnant bitches. The dogs were randomly allocated to either the "MSCs group," receiving an IA injection of MSCs after TPLO, followed by placebo for 1 month, or the "NSAIDs group," receiving IA equivalent volume of MSCs vehicle after TPLO, followed by oral NSAID for 1 month. One of the three blinded evaluators assessed the dogs in each group before and after surgery (1, 3, and 6 months). Clinical score and gait and bone healing process were assessed. The data were statistically compared between the two groups for pre- and postoperative evaluations. RESULTS: Fourteen dogs (nine in the MSCs group, five in the NSAIDs group) completed the present study. No significant difference was observed between the groups preoperatively. No local or systemic adverse effect was observed after MSCs injection at any time point considered. At 1 month after surgery, bone healing scores were significantly higher in the MSCs group. At 1, 3, and 6 months after surgery, no significant difference was observed between the two groups for clinical scores and gait evaluation. CONCLUSION: A single IA injection of allogeneic neonatal MSCs could be a safe and valuable postoperative alternative to NSAIDs for dogs requiring TPLO surgery, particularly for dogs intolerant to this class of drugs.

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In veterinary medicine, therapeutic mesenchymal stromal cells (MSC) have been traditionally isolated from adult bone marrow or adipose tissue. Neonatal tissues, normally discarded at birth from all species have become an alternative source of cells for regenerative medicine in the human clinic. These cells have been described as being more primitive, proliferative and immunosuppressive than their adult counterparts. Our objective was to examine if this phenomena holds true in dogs. Little information exists regarding canine neonatal MSC characterisation. In this study, we were able to both isolate, phenotype and assess the differentiation and immunomodulatory properties of MSC from canine foetal adnexa allowing us to compare their characteristics to their more well-known bone marrow (BM) cousins. Neonatal tissues, including amnion (AM), placenta (PL), and umbilical cord matrix (UCM) were collected from 6 canine caesarean sections. Primary cells were expanded in vitro for 5 consecutive passages and their proliferation measured. BM-MSC were isolated from 5 control dogs euthanised from other studies and grown in vitro using an identical protocol. All MSC lines were systematically evaluated for their ability to differentiate into 3 mesodermal lineages (adipocyte, osteocyte and chondrocyte) and phenotyped by cytometry and qPCR. In addition, the enzymatic activity of the key immunomodulatory marker indoleamine 2,3-dioxygenase (IDO) was evaluated for each MSC line. MSC displaying a fibroblastic appearance were successfully grown from all neonatal tissues. PL-MSC exhibited significantly higher proliferation rates than AM- and UCM-MSC (p=0.05). Cytometric analysis showed that all MSC express CD90, CD29, and CD44, while no expression of CD45, CD34 and MHC2 was detected. Molecular profiling showed expression of CD105 and CD73 in all MSC. Low levels of SOX2 mRNA was observed in all MSC, while neither NANOG, nor OCT4 were detected. All MSC differentiate into 3 mesodermal lineages. Following inflammatory stimulation, the activity of the immunomodulatory enzyme IDO was significantly higher in neonatal MSC compared to BM-MSC (p=0.009). Our results show that canine foetal adnexa cells share very similar properties to their adult equivalents but upon stimulation show significantly higher IDO immunomodulatory activity. Further studies will be needed to confirm the potential therapeutic benefits of these cells.

Asunto(s)

Perros , Células Madre Mesenquimatosas/citología , Placenta/citología , Amnios/citología , Animales , Antígenos CD/metabolismo , Diferenciación Celular , Línea Celular , Proliferación Celular , Separación Celular , Femenino , Inmunomodulación , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Células Madre Mesenquimatosas/inmunología , Embarazo , Cordón Umbilical/citología

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The gene expression pattern of glioblastoma (GBM) is well documented but the expression profile of brain adjacent to tumor is not yet analysed. This may help to understand the oncogenic pathway of GBM development. We have established the genome-wide expression profiles of samples isolated from GBM tumor mass, white matter adjacent to tumor (apparently free of tumor cells), and white matter controls by using the Affymetrix HG-U133 arrays. Array-CGH (aCGH) was also performed to detect genomic alterations. Among genes dysregulated in peritumoral white matter, 15 were over-expressed, while 42 were down-regulated when compared to white matter controls. A similar expression profile was detected in GBM cells. Growth, proliferation and cell motility/adhesion-associated genes were up-regulated while genes involved in neurogenesis were down-regulated. Furthermore, several tumor suppressor genes along with the KLRC1 (a member of natural killer receptor) were also down-regulated in the peritumoral brain tissue. Several mosaic genomic lesions were detected by aCGH, mostly in tumor samples and several GBM-associated mosaic genomic lesions were also present in the peritumoral brain tissue, with a similar mosaicism pattern. Our data could be explained by a dilution of genes expressed from tumor cells infiltrating the peritumour tissue. Alternatively, these findings could be substained by a relevant amount of "apparently normal" cells presenting a gene profile compatible with a precancerous state or even "quiescent" cancer cells. Otherwise, the recurrent tumor may arise from both infiltrating tumor cells and from an interaction and recruitment of apparently normal cells in the peritumor tissue by infiltrating tumor cells.

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Neoplasias Encefálicas/metabolismo , Encéfalo/patología , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Transcriptoma , Encéfalo/metabolismo , Neoplasias Encefálicas/genética , Adhesión Celular , Movimiento Celular , Proliferación Celular , Análisis por Conglomerados , Hibridación Genómica Comparativa , Estudio de Asociación del Genoma Completo , Glioblastoma/genética , Humanos , Neuronas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos

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Multipotent mesenchymal stem cells with extensive self-renewal properties can be easily isolated and rapidly expanded in culture from small volumes of amniotic fluid. These cells, namely, amniotic fluid-stromal cells (AFSCs), can be regarded as an attractive source for tissue engineering purposes, being phenotypically and genetically stable, plus overcoming all the safety and ethical issues related to the use of embryonic/fetal cells. LMP3 is a novel osteoinductive molecule acting upstream to the main osteogenic pathways. This study is aimed at delineating the basic molecular events underlying LMP3-induced osteogenesis, using AFSCs as a cellular model to focus on the molecular features underlying the multipotency/differentiation switch. For this purpose, AFSCs were isolated and characterized in vitro and transfected with a defective adenoviral vector expressing the human LMP3. LMP3 induced the successful osteogenic differentiation of AFSC by inducing the expression of osteogenic markers and osteospecific transcription factors. Moreover, LMP3 induced an early repression of the Kruppel-like factor-4, implicated in MSC stemness maintenance. KLF4 repression was released upon LMP3 silencing, indicating that this event could be reasonably considered among the basic molecular events that govern the proliferation/differentiation switch during LMP3-induced osteogenic differentiation of AFSC.

Asunto(s)

Líquido Amniótico/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas con Dominio LIM/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Diferenciación Celular , Células Cultivadas , Regulación hacia Abajo , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Humanos , Factor 4 Similar a Kruppel , Osteogénesis/fisiología

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The mitogen-activated protein kinases (MAPK) ERK1 and ERK2 are among the major signal transduction molecules but little is known about their specific functions in vivo. ERK activity is provided by two isoforms, ERK1 and ERK2, which are ubiquitously expressed and share activators and substrates. However, there are not in vivo studies which have reported a role for ERK1 or ERK2 in HSCs and the bone marrow microenvironment. The present study shows that the ERK1-deficient mice present a mild osteopetrosis phenotype. The lodging and the homing abilities of the ERK1(-/-) HSC are impaired, suggesting that the ERK1(-/-)-defective environment may affect the engrafment of HSCs. Serial transplantations demonstrate that ERK1 is involved in the maintenance of an appropriate medullar microenvironment, but that the intrinsic properties of HSCs are not altered by the ERK1(-/-) defective microenvironment. Deletion of ERK1 impaired in vitro and in vivo osteoclastogenesis while osteoblasts were unaffected. As osteoclasts derive from precursors of the monocyte/macrophage lineage, investigation of the monocytic compartment was performed. In vivo analysis of the myeloid lineage progenitors revealed that the frequency of CMPs increased by approximately 1.3-fold, while the frequency of GMPs significantly decreased by almost 2-fold, compared with the respective WT compartments. The overall mononuclear-phagocyte lineage development was compromised in these mice due to a reduced expression of the M-CSF receptor on myeloid progenitors. These results show that the cellular targets of ERK1 are M-CSFR-responsive cells, upstream to osteoclasts. While ERK1 is well known to be activated by M-CSF, the present results are the first to point out an ERK1-dependent M-CSFR regulation on hematopoietic progenitors. This study reinforces the hypothesis of an active cross-talk between HSCs, their progeny and bone cells in the maintenance of the homeostasis of these compartments.

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Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nicho de Células Madre , Animales , Densidad Ósea , Médula Ósea/patología , Huesos/enzimología , Huesos/patología , Compartimento Celular , Diferenciación Celular , Linaje de la Célula , Movimiento Celular , Proliferación Celular , Microambiente Celular , Eliminación de Gen , Macrófagos/patología , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 3 Activada por Mitógenos/deficiencia , Monocitos , Osteoblastos/enzimología , Osteoblastos/patología , Osteoclastos/enzimología , Osteoclastos/patología , Osteogénesis

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Several in vitro assays have been proposed to identify cancer stem cells (CSCs), including immunophenotyping, sphere assay and side population (SP) assay. CD133 antigen has been proposed as a CSC marker in colon cancer (CC). However, no functional data are available to date and conflicting results have been reported regarding its role as true CSC marker. Here we set out to identify a molecular signature associated with potential CSC. CD133(+) cells isolated from the CaCo-2 CC cell line were analysed by microarray molecular profiling compared to CD133(-) counterparts. Various differentially expressed genes were identified and the most relevant transcripts found to be over-expressed in CD133(+) cells were evaluated by quantitative RT-PCR in the CD133(+) fractions isolated from several CC cell lines. In the attempt to find a correlation between putative CSCs, isolated by means of CD133 immunophenotyping and the SP approach, we demonstrated a significant enrichment of CD133(+) cells within the SP fraction of CC cells, and comparison of the gene expression profiles revealed that Endothelin-1 (END-1) and nuclear receptor subfamily 4, group A, member 2 (NR4A2) transcripts are highly expressed in both CD133(+) and SP fractions of CC cells. Moreover, depletion of CD133 by siRNA induced a significant attenuation of END-1 and NR4A2 expression levels in CaCo-2 cells, while expression of all three molecules decreased during sodium butyrate-induced differentiation. In conclusion, we have identified a molecular signature associated with potential CSCs and showed for the first time the existence of a functional relationship between CD133, END-1 and NR4A2 expression in colon cancer cells.

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Antígenos CD/genética , Neoplasias del Colon/genética , Endotelina-1/genética , Regulación Neoplásica de la Expresión Génica/genética , Glicoproteínas/genética , Células Madre Neoplásicas/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Péptidos/genética , Antígeno AC133 , Antígenos CD/metabolismo , Western Blotting , Células CACO-2 , Separación Celular , Neoplasias del Colon/metabolismo , Endotelina-1/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Glicoproteínas/metabolismo , Humanos , Inmunofenotipificación , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Péptidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

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OBJECTIVE: To test the osteo-regenerative potential of adipose tissue-derived stromal cells (ATSCs), an attractive human source for tissue engineering, in a rat model of mandibular defect. Human dermal fibroblasts (HDFs) were used as a differentiated cellular control in the study. DESIGN: The ATSCs and HDFs were isolated from human lipoaspirate and skin biopsy specimens, respectively. Cells were characterized in vitro and then adsorbed on an osteo-conductive scaffold to be transplanted in a mandibular defect of immunosuppressed rats. Naked unseeded scaffold was used as a negative control. MAIN OUTCOME MEASURES: Bone healing was studied by computerized tomography and histologic analysis after 4, 8, and 12 weeks. RESULTS: Computed tomography showed that undifferentiated ATSCs induced successful bone healing of the mandible defect when transplanted in animals, compared with HDFs and negative controls. Histologic analysis demonstrated that the newly formed tissue in the surgical defect retained the features of compact bone. CONCLUSION: Undifferentiated human ATSCs are suitable for cell-based treatment of mandibular defects, even in the absence of previous osteogenic induction in vitro.

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Tejido Adiposo/citología , Regeneración Ósea/fisiología , Mandíbula/cirugía , Células del Estroma/trasplante , Ingeniería de Tejidos/métodos , Adulto , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Fibroblastos , Humanos , Imagenología Tridimensional , Implantes Experimentales , Masculino , Mandíbula/diagnóstico por imagen , Persona de Mediana Edad , Ratas , Ratas Wistar , Tomografía Computarizada por Rayos X , Cicatrización de Heridas

RESUMEN

BACKGROUND AIMS: Bone marrow- and adipose tissue-derived mesenchymal stromal cells (MSC) represent promising sources for regenerative medicine. However, the precise molecular mechanisms underlying MSC stemness maintenance versus differentiation are not fully understood. The aim of this study was to compare the genome-wide expression profiles of bone marrow-and adipose tissue-derived MSC, in order to identify a common molecular stemness core. METHODS: Molecular profiling was carried out using Affymetrix microarray and relevant genes were further validated by Q-PCR. RESULTS: We identified an overlapping dataset of 190 transcripts commonly regulated in both cell populations, which included several genes involved in stemness regulation (i.e. self-renewal potential and the ability to generate differentiated cells), various signaling pathways and transcription factors. In particular, we identified a central role of the Kruppel-like factor 4 (KLF4) DNA-binding protein in regulating MSC transcriptional activity. CONCLUSIONS: Our results provide new insights toward understanding the molecular basis of MSC stemness maintenance and underline the ability of KLF4 to maintain cells in an undifferentiated state.

Asunto(s)

Tejido Adiposo/citología , Células de la Médula Ósea/metabolismo , Linaje de la Célula/genética , Perfilación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adulto , Sitios de Unión , Células de la Médula Ósea/citología , Diferenciación Celular/genética , Inmunoprecipitación de Cromatina , Femenino , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Masculino , Células Madre Mesenquimatosas/citología , Persona de Mediana Edad , Modelos Biológicos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas/genética , Unión Proteica , Reproducibilidad de los Resultados , Células del Estroma/citología , Células del Estroma/metabolismo , Adulto Joven

RESUMEN

Abnormal gene promoter methylation contributes to deregulate gene expression of hematopoietic progenitors in myelodysplastic syndromes (MDS). We analyzed the gene expression profile of myelodysplastic and normal CD34+ hematopoietic stem cells (HSCs) treated in vitro with decitabine. We identified a list of candidate tumor suppressor genes, expressed at low levels in MDS HSCs and induced by hypomethylating treatment only in MDS, but not in normal HSCs. Real-time RT-PCR confirmed reduced CD9 expression in MDS CD34+ and bone marrow mononuclear cells, compared to normal controls. CD9 was specifically up-regulated by decitabine treatment in myelodysplastic CD34+ cells.

Asunto(s)

Azacitidina/análogos & derivados , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/efectos de los fármacos , Síndromes Mielodisplásicos/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD34/sangre , Antimetabolitos Antineoplásicos/farmacología , Azacitidina/farmacología , Análisis por Conglomerados , Metilación de ADN , Decitabina , Femenino , Expresión Génica/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Integrina beta1/genética , Masculino , Persona de Mediana Edad , Síndromes Mielodisplásicos/sangre , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto Joven

RESUMEN

Due to its abundance, easy retrieval, and plasticity characteristics, adipose-tissue-derived stromal cells (ATSCs) present unquestionable advantages over other adult-tissue-derived stem cells. Based on the in silico analysis of our previous data reporting the ATSC-specific expression profiles, the present study attempted to clarify and validate at the functional level the expression of the neurospecific genes expressed by ATSC both in vitro and in vivo. This allowed evidencing that ATSCs express neuro-specific trophins, metabolic genes, and neuroprotective molecules. They were in fact able to induce neurite outgrowth in vitro, along with tissue-specific commitment along the neural lineage and the expression of the TRKA neurotrophin receptor in vivo. Our observation adds useful information to recent evidence proposing these cells as a suitable tool for cell-based applications in neuroregenerative medicine.

Asunto(s)

Tejido Adiposo/citología , Moléculas de Adhesión Celular/metabolismo , Técnicas de Cocultivo/métodos , Neuritas/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Células del Estroma/metabolismo , Tejido Adiposo/metabolismo , Adulto , Animales , Diferenciación Celular , Células Cultivadas , Medios de Cultivo Condicionados , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Persona de Mediana Edad , Células PC12 , Cultivo Primario de Células , Ratas , Regulación hacia Arriba

RESUMEN

Lim mineralization protein-3 (LMP3) induces osteoblast differentiation by regulating the expression and activity of certain molecules involved in the osteogenic cascade, including those belonging to the bone morphogenetic protein (BMP) family. The complete network of molecular events involved in LMP3-mediated osteogenesis is still unknown. The aim of this study was to analyze the genome-wide gene expression profiles in human mesenchymal stem cells (hMSC) induced by exogenous LMP3 to mediate osteogenesis. For this purpose hMSC were transduced with a defective adenoviral vector expressing the human LMP3 gene and microarray analysis was performed 1 day post-adenoviral transduction. Cells transduced with the vector backbone and untransduced cells were used as independent controls in the experiments. Microarray data were independently validated by means of real-time PCR on selected transcripts. The statistical analysis of microarray data produced a list of 263 significantly (p < 0.01) differentially expressed transcripts. The biological interpretation of the results indicated, among the most noteworthy effects, the modulation of genes involved in the TGF-beta1 pathway: 88 genes coding for key regulators of the cell cycle regulatory machinery and 28 genes implicated in the regulation of cell proliferation along with the development of connective, muscular, and skeletal tissues. These results suggested that LMP3 could affect the fine balance between cell proliferation/differentiation of mesenchymal cells mostly by modulating the TGF-beta1 signaling pathway.

Asunto(s)

Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Osteogénesis/genética , Células del Estroma/citología , Transcripción Genética , Adenoviridae/genética , Matriz Ósea/metabolismo , Calcificación Fisiológica/genética , Proliferación Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas con Dominio LIM , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/metabolismo , Transducción Genética

RESUMEN

BACKGROUND: Adipose tissue-derived stromal cells (ATSCs) hold great promises in regenerative medicine. In the last decade, several studies have reported the plasticity of ATSCs toward a hepatocyte-like phenotype. Nonetheless, the molecular mechanisms underlying the conversion from a mesenchymal to an epithelial phenotype remain poorly understood. AIM: In this study, we compared the full genome expression profiles of ATSCs cultured for 4 weeks under pro-hepatogenic conditions to undifferentiated ATSCs, in order to depict the molecular events involved in ATSC hepatic transdifferentiation. METHODS: Analysis was performed using the Affymetrix human focus arrays. Sets of differentially expressed genes were functionally categorized in order to understand which pathways drive the hepatic conversion and interesting targets were validated by Q-PCR. RESULTS: ATSC-derived hepatocyte-like cells activate several genes associated with specific liver functions, including protein metabolism, innate immune response regulation, and biodegradation of toxic compounds. Furthermore, microarray analysis highlighted downregulation of transcripts associated with the mesenchymal lineage, while epithelial-related genes were overexpressed. CONCLUSION: Our data suggest that the in vitro system used in this study drove ATSCs toward a hepatic conversion through a subtle regulation of molecular pathways controlling lineage commitment that promote mesenchymal-epithelial transition.

Asunto(s)

Tejido Adiposo/citología , Hepatocitos/citología , Células del Estroma/citología , Diferenciación Celular/genética , Hepatocitos/fisiología , Humanos , Fenotipo , Células del Estroma/fisiología