Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.

Outside-in signaling through integrins and cadherins: a central mechanism to control epidermal growth and differentiation?

The process of epidermal renewal persists throughout the entire life of an organism. It begins when a keratinocyte progenitor leaves the stem cell compartment, undergoes a limited number of mitotic divisions, exits the cell cycle, and commits to terminal differentiation. At the end of this phase, the postmitotic keratinocytes detach from the basement membrane to build up the overlaying stratified epithelium. Although highly coordinated, this sequence of events is endowed with a remarkable versatility, which enables the quiescent keratinocyte to reintegrate into the cell cycle and become migratory when necessary, for example after wounding. It is this versatility that represents the Achilles heel of epithelial cells allowing for the development of severe pathologies. Over the past decade, compelling evidence has been provided that epithelial cancer cells achieve uncontrolled proliferation following hijacking of a "survival program" with PI3K/Akt and a "proliferation program" with growth factor receptor signaling at its core. Recent insights into adhesion receptor signaling now propose that integrins, but also cadherins, can centrally control these programs. It is suggested that the two types of adhesion receptors act as sensors to transmit extracellular stimuli in an outside-in mode, to inversely modulate epidermal growth factor receptor signaling and ensure cell survival. Hence, cell-matrix and cell-cell adhesion receptors likely play a more powerful and wide-ranging role than initially anticipated. This Perspective article discusses the relevance of this emerging field for epidermal growth and differentiation, which can be of importance for severe pathologies such as tumorigenesis and invasive metastasis, as well as psoriasis and Pemphigus vulgaris.

Direct adipotropic actions of atorvastatin: differentiation state-dependent induction of apoptosis, modulation of endocrine function, and inhibition of glucose uptake

Statins exert anti-inflammatory, anti-atherogenic actions. The mechanisms responsible for these effects remain only partially elucidated. Diabetes and obesity are characterized by low-grade inflammation. Metabolic and endocrine adipocyte dysfunction is known to play a crucial role in the development of these disorders and the related cardiovascular complications. Thus, direct modulation of adipocyte function may represent a mechanism of pleiotropic statin actions. We investigated effects of atorvastatin on apoptosis, differentiation, endocrine, and metabolic functions in murine white and brown adipocyte lines. Direct exposure of differentiating preadipocytes to atorvastatin strongly reduced lipid accumulation and diminished protein expression of the differentiation marker CCAAT/enhancer binding protein-beta (CEBP-beta). In fully differentiated adipocytes, however, lipid accumulation remained unchanged after chronic atorvastatin treatment. Furthermore, cell viability was reduced in response to atorvastatin treatment in proliferating and differentiating preadipocytes, but not in differentiated cells. Moreover, atorvastatin induced apoptosis and inhibited protein kinase B (AKT) phosphorylation in proliferating and differentiating preadipocytes, but not in differentiated adipocytes. On the endocrine level, direct atorvastatin treatment of differentiated white adipocytes enhanced expression of the pro-inflammatory adipokine interleukin-6 (IL-6), and downregulated expression of the insulin-mimetic and anti-inflammatory adipokines visfatin and adiponectin. Finally, these direct adipotropic endocrine effects of atorvastatin were paralleled by the acute inhibition of insulin-induced glucose uptake in differentiated white adipocytes, while protein expression of the thermogenic uncoupling protein-1 (UCP-1) in brown adipocytes remained unchanged. Taken together, our data for the first time demonstrate direct differentiation state-dependent effects of atorvastatin including apoptosis, modulation of pro-inflammatory and glucostatic adipokine expression, and insulin resistance in adipose cells. These differential interactions may explain variable clinical observations.

Generic detection of coronaviruses and differentiation at the prototype strain level by reverse transcription-PCR and nonfluorescent low-density microarray

A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.

Tissue-dependent subcellular localization of Drosophila arginine methyl-transferase 4 (DART4), a coactivator whose overexpression affects neither viability nor differentiation

Drosophila arginine methyl-transferase 4 (DART4) belongs to the type I class of arginine methyltransferases. It catalyzes the methylation of arginine residues to monomethylarginines and asymmetrical dimethylarginines. The DART4 sequence is highly similar to mammalian PRMT4/CARM1, and DART4 substrate specificity has been conserved, too. Recently it was suggested that DART4/Carmer functions in ecdysone receptor mediated apoptosis of the polytene larval salivary glands and an apparent up-regulation of DART4/Carmer mRNA levels before tissue histolysis was reported. Here we show that in Drosophila larvae, DART4 is mainly expressed in the imaginal disks and in larval brains, and to a much lesser degree in the polytene larval tissue such as salivary glands. In glands, DART4 protein is present in the cytoplasm and the nucleus. The nuclear signal emanates from the extrachromosomal domain and gets progressively restricted to the region of the nuclear lamina upon pupariation. Surprisingly, DART4 levels do not increase in salivary glands during pupariation, and overexpression of DART4 does not cause precautious cell death in the glands. Furthermore, over- and misexpression of DART4 under the control of the alpha tubulin promoter do not lead to any major problem in the life of a fly. This suggests that DART4 activity is regulated at the posttranslational level and/or that it acts as a true cofactor in vivo. We present evidence that nuclear localization of DART4 may contribute to its function because DART4 accumulation changes from a distribution with a strong cytoplasmic component during the transcriptional quiescence of the young embryo to a predominantly nuclear one at the onset of zygotic transcription.

Sex determination and temperature-induced sex differentiation in three natural populations of Nile tilapia (Oreochromis niloticus) adapted to extreme temperature conditions

As a species of major interest for aquaculture, the sex determination system (SDS) of Nile tilapia, Oreochromis niloticus, has been widely investigated. In this species, sex determination is considered to be governed by the interactions between a complex system of genetic sex determination factors (GSD) and the influence of temperature (TSD) during a critical period. Previous studies were exclusively carried out on domestic stocks with the genetic and maintenance limitations associated. Given the wide distribution and adaptation potential of the Nile tilapia, we investigated under controlled conditions the sex determination system of natural populations adapted to three extreme thermal regimes: stable extreme environments in Ethiopia, either cold temperatures in a highland lake (Lake Koka), or warm temperatures in hydrothermal springs (Lake Metahara), and an environment with large seasonal variations in Ghana (Kpandu, Lake Volta). The sex ratio analysis was conducted on progenies reared under constant basal (27 degrees C) or high (36 degrees C) temperatures during the 30 days following yolk-sac resorption. Sex ratios of the progenies reared at standard temperature suggest that the three populations share a similar complex GSD system based on a predominant male heterogametic factor with additional influences of polymorphism at this locus and/or action of minor factors. The three populations presented a clear thermosensitivity of sex differentiation, with large variations in the intensity of response depending on the parents. This confirms the presence of genotype-environment interactions in TSD of Nile tilapia. Furthermore the existence of naturally sex-reversed individuals is strongly suggested in two populations (Kpandu and Koka). However, it was not possible here to infer if the sex-inversion resulted from minor genetic factors and/or environmental influences. The present study demonstrated for the first time the conservation of a complex SDS combining polymorphic GSD and TSD components in natural populations of Nile tilapia. We discuss the evolutionary implications of our findings and highlight the importance of field investigations of sex determination. (c) 2007 Elsevier B.V. All rights reserved.

HIC1 tumour suppressor gene is suppressed in acute myeloid leukaemia and induced during granulocytic differentiation

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 (HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)-positive acute promyelocytic leukaemia receiving all-trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL-60 and U-937 cells upon ATRA-induced differentiation and in CD34+ progenitor cells after granulocyte colony-stimulating factor-induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL-60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC-rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL-60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis.

Chondrogenic differentiation of bovine synovium: bone morphogenetic proteins 2 and 7 and transforming growth factor beta1 induce the formation of different types of cartilaginous tissue

OBJECTIVE: To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor beta1 (TGFbeta1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. METHODS: Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFbeta1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. RESULTS: BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFbeta1 differed in terms of both cell phenotype and gene expression profiles. CONCLUSION: The 3 tested members of the TGFbeta superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded alpha1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.