iFATS

Adipocyte-derived factors suppress heart contraction.

iFats — Sat, 04 Sep 2010 08:16:01 +0000

Background:Obesity is strongly associated with cardiovascular diseases including systemic hypertension, coronary artery disease and heart failure. Despite several investigations the pathophysiological mechanisms involved remain unclear. We have previously shown that adipose tissue exerts a highly potent activity with an acute depressant effect on cardiomyocytes, thus suggesting direct involvement of adipose tissue in the development of heart dysfunction.Objective and Design:This study investigates the effects of adipocyte factors obtained from subcutaneous adipose tissue on the whole cardiac function by using isolated perfused rat hearts in a Langendorff mode. We recorded changes in coronary flow, developed isovolumetric left ventricular pressure, contraction rate and relaxation rate.Results:We observed a significant decrease in heart contractility parameters as well as in coronary flow within a few seconds of incubation with adipocyte factors. The cardiodepressant effects could not be blocked by the nonselective cyclooxygenase-inhibitor indomethacin. Human adipocytes release tumor necrosis factor-alpha, interleukin-6 (IL-6) and IL-1beta into extracellular medium. These cytokines were tested for their potential effect but were, however, not responsible for the cardiodepressant effect observed.Conclusion:These data indicate that human adipocytes secrete factors with a strong acute depressant effect on cardiac force generation and coronary flow due to contraction of the coronary vessels, thus suggesting a direct role of adipose tissue in the pathogenesis of cardiac dysfunction.International Journal of Obesity advance online publication, 22 June 2010; doi:10.1038/ijo.2010.121.

Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters.

iFats — Sat, 04 Sep 2010 07:59:35 +0000

BACKGROUND: We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells. RESULTS: Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro. CONCLUSIONS: This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

Geometric control of human stem cell morphology and differentiation.

iFats — Sat, 04 Sep 2010 07:58:07 +0000

During tissue morphogenesis, stem cells and progenitor cells migrate, proliferate, and differentiate, with striking changes in cell shape, size, and acting mechanical stresses. The local cellular function depends on the spatial distribution of cytokines as well as local mechanical microenvironments in which the cells reside. In this study, we controlled the organization of human adipose derived stem cells using micro-patterning technologies, to investigate the influence of multi-cellular form on spatial distribution of cellular function at an early stage of cell differentiation. The underlying role of cytoskeletal tension was probed through drug treatment. Our results show that the cultivation of stem cells on geometric patterns resulted in pattern- and position-specific cell morphology, proliferation and differentiation. The highest cell proliferation occurred in the regions with large, spreading cells (such as the outer edge of a ring and the short edges of rectangles). In contrast, stem cell differentiation co-localized with the regions containing small, elongated cells (such as the inner edge of a ring and the regions next to the short edges of rectangles). The application of drugs that inhibit the formation of actomyosin resulted in the lack of geometrically specific differentiation patterns. This study confirms the role of substrate geometry on stem cell differentiation, through associated physical forces, and provides a simple and controllable system for studying biophysical regulation of cell function.

Simple Modular Bioreactors for Tissue Engineering: A System for Characterization of Oxygen Gradients, Human Mesenchymal Stem Cell Differentiation, and Prevascularization.

iFats — Fri, 03 Sep 2010 07:56:06 +0000

Large-scale tissue engineering is limited by nutrient perfusion and mass transport limitations, especially oxygen diffusion, which restrict construct development to smaller than clinically relevant dimensions and limit the ability for in vivo integration. The goal of this work was to develop a modular approach to tissue engineering, where scaffold and tissue size, transport issues, and surgical implantation in vivo are considered from the outset. Human mesenchymal stem cells (hMSCs) were used as the model cell type, as their differentiation has been studied for several different cell lineages and often with conflicting results. Changes in the expression profiles of hMSCs differentiated under varied oxygen tensions are presented, demonstrating tissue-specific oxygen requirements for both adipogenic (20% O(2)) and chondrogenic (5% O(2)) differentiation. Oxygen and nutrient transport were enhanced by developing a bioreactor system for perfusing hMSC-seeded collagen gels using porous silk tubes, resulting in enhanced oxygen transport and cell viability within the gels. These systems are simple to use and scaled for versatility, to allow for the systematic study of relationships between cell content, oxygen, and cell function. The data may be combined with oxygen transport modeling to derive minimally sized modular units for construction of clinically relevant tissue-engineered constructs, a generic strategy that may be employed for vascularized target tissues.

Growth and Differentiation Properties of Mesenchymal Stromal Cell Populations Derived from Whole Human Umbilical Cord.

iFats — Fri, 03 Sep 2010 07:52:29 +0000

Up to 2.8 x 10(7) fibroblast-like cells displaying an abundant presence of mesenchymal stem cell (MSC) markers CD73, CD90, CD105 and a low level of HLA-I expression can be isolated from one whole human umbilical cord (UC) using a simple and highly reproducible explant culture approach. Cells derived from whole UC, similar to cells collected from separate compartments of UC, display a distinct chondrogenic and adipogenic potential. Therefore they are potential candidates for cartilage and adipose tissue engineering. Cell differentiation along the osteogenic pathway is, however, less efficient, even after the addition of 1.25-dihydroxyvitamin D3, a potent osteoinductive substance. Isolated cells are highly proliferative, tolerate cryopreservation with an average survival rate of about 75% and after thawing can be propagated further, at least over 20 population doublings before their proliferative activity begins to decline. More importantly, they synthesize numerous trophic factors including neurotrophins and factors which facilitate angiogenesis and hematopoiesis. In conclusion, cells isolated from whole UC satisfies all requirements essential for the generation of stem cell banks containing permanently available cell material for applications in the field of regenerative medicine. Nevertheless, further studies are needed to improve and adjust the methods which are already employed for adult MSC expansion and differentiation to specific properties and requirements of the primitive stem cells collected from UC. So, our data verify that the choice of individual parameters for cell propagation, such as duration of cell expansion and cell seeding density, has a substantial impact on the quality of UC-derived cell populations.

A Subset of Osteoblasts Expressing High Endogenous Levels of PPARgamma Switches Fate to Adipocytes in the Rat Calvaria Cell Culture Model.

iFats — Fri, 03 Sep 2010 07:46:46 +0000

BACKGROUND: Understanding fate choice and fate switching between the osteoblast lineage (ObL) and adipocyte lineage (AdL) is important to understand both the developmental inter-relationships between osteoblasts and adipocytes and the impact of changes in fate allocation between the two lineages in normal aging and certain diseases. The goal of this study was to determine when during lineage progression ObL cells are susceptible to an AdL fate switch by activation of endogenous peroxisome proliferator-activated receptor (PPAR)gamma. METHODOLOGY/PRINCIPAL FINDINGS: Multiple rat calvaria cells within the ObL developmental hierarchy were isolated by either fractionation on the basis of expression of alkaline phosphatase or retrospective identification of single cell-derived colonies, and treated with BRL-49653 (BRL), a synthetic ligand for PPARgamma. About 30% of the total single cell-derived colonies expressed adipogenic potential (defined cytochemically) when BRL was present. Profiling of ObL and AdL markers by qRT-PCR on amplified cRNA from over 160 colonies revealed that BRL-dependent adipogenic potential correlated with endogenous PPARgamma mRNA levels. Unexpectedly, a significant subset of relatively mature ObL cells exhibited osteo-adipogenic bipotentiality. Western blotting and immunocytochemistry confirmed that ObL cells co-expressed multiple mesenchymal lineage determinants (runt-related transcription factor 2 (Runx2), PPARgamma, Sox9 and MyoD which localized in the cytoplasm initially, and only Runx2 translocated to the nucleus during ObL progression. Notably, however, some cells exhibited both PPARgamma and Runx2 nuclear labeling with concomitant upregulation of expression of their target genes with BRL treatment. CONCLUSIONS/SIGNIFICANCE: We conclude that not only immature but a subset of relatively mature ObL cells characterized by relatively high levels of endogenous PPARgamma expression can be switched to the AdL. The fact that some ObL cells maintain capacity for adipogenic fate selection even at relatively mature developmental stages implies an unexpected plasticity with important implications in normal and pathological bone development.

Id4, a new candidate gene for senile osteoporosis, acts as a molecular switch promoting osteoblast differentiation.

iFats — Thu, 02 Sep 2010 07:44:25 +0000

Excessive accumulation of bone marrow adipocytes observed in senile osteoporosis or age-related osteopenia is caused by the unbalanced differentiation of MSCs into bone marrow adipocytes or osteoblasts. Several transcription factors are known to regulate the balance between adipocyte and osteoblast differentiation. However, the molecular mechanisms that regulate the balance between adipocyte and osteoblast differentiation in the bone marrow have yet to be elucidated. To identify candidate genes associated with senile osteoporosis, we performed genome-wide expression analyses of differentiating osteoblasts and adipocytes. Among transcription factors that were enriched in the early phase of differentiation, Id4 was identified as a key molecule affecting the differentiation of both cell types. Experiments using bone marrow-derived stromal cell line ST2 and Id4-deficient mice showed that lack of Id4 drastically reduces osteoblast differentiation and drives differentiation toward adipocytes. On the other hand knockdown of Id4 in adipogenic-induced ST2 cells increased the expression of Ppargamma2, a master regulator of adipocyte differentiation. Similar results were observed in bone marrow cells of femur and tibia of Id4-deficient mice. However the effect of Id4 on Ppargamma2 and adipocyte differentiation is unlikely to be of direct nature. The mechanism of Id4 promoting osteoblast differentiation is associated with the Id4-mediated release of Hes1 from Hes1-Hey2 complexes. Hes1 increases the stability and transcriptional activity of Runx2, a key molecule of osteoblast differentiation, which results in an enhanced osteoblast-specific gene expression. The new role of Id4 in promoting osteoblast differentiation renders it a target for preventing the onset of senile osteoporosis.

Tissue engineering and the use of stem/progenitor cells for airway epithelium repair.

iFats — Thu, 02 Sep 2010 07:42:29 +0000

Stem/progenitor cells can be used to repair defects in the airway wall, resulting from e.g., tumors, trauma, tissue reactions following long-time intubations, or diseases that are associated with epithelial damage. Several potential sources of cells for airway epithelium have been identified. These can be divided into two groups. The first group consists of endogenous progenitor cells present in the respiratory tract. This group can be subdivided according to location into (a) a ductal cell type in the submucosal glands of the proximal trachea, (b) basal cells in the intercartilaginous zones of the lower trachea and bronchi, (c) variant Clara cells (Clara v-cells) in the bronchioles and (d) at the junctions between the bronchioles and the alveolar ducts, and (e) alveolar type II cells. This classification of progenitor cell niches is, however, controversial. The second group consists of exogenous stem cells derived from other tissues in the body. This second group can be subdivided into: (a) embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, or amniotic fluid stem cells, (b) side-population cells from bone marrow or epithelial stem cells present in bone marrow or circulation and (c) fat-derived mesenchymal cells. Airway epithelial cells can be co-cultured in a system that includes a basal lamina equivalent, extracellular factors from mesenchymal fibroblasts, and in an air-liquid interface system. Recently, spheroid-based culture systems have been developed. Several clinical applications have been suggested: cystic fibrosis, acute respiratory distress syndrome, chronic obstructive lung disease, pulmonary fibrosis, pulmonary edema, and pulmonary hypertension. Clinical applications so far are few, but include subglottic stenosis, tracheomalacia, bronchiomalacia, and emphysema.

Ochratoxin A inhibits adipogenesis through the ERK-PPAR-gamma pathway in human adipose tissue-derived mesenchymal stem cells.

iFats — Thu, 02 Sep 2010 07:41:07 +0000

chratoxin A (OTA) is a ubiquitous fungal metabolite with nephrotoxic, carcinogenic and apoptotic potential. Although the toxic effects of OTA in various cell types are well characterized, it is not known whether OTA has an effect on stem cell differentiation. In this study, we demonstrate that OTA inhibits adipogenesis in human adipose tissue-derived mesenchymal stem cells (hAMSC), as indicated by decreased accumulation of intracellular lipid droplets. Furthermore, OTA significantly reduces the expression of adipocyte-specific markers, including peroxisome proliferator-activated receptor-gamma (PPAR-gamma), CCAAT enhancer binding protein-alpha (C/EBP-alpha), lipoprotein lipase (LPL) and adipocyte fatty acid-binding protein (aP2). At the molecular level, OTA phosphorylates PPAR-gamma2 through ERK activation and inhibits PPAR-gamma activity. We also found that treatment with the MEK inhibitor, PD98059, significantly blocked the OTA-induced inhibition of adipogenesis. These results indicate that OTA suppresses adipogenesis in an ERK-dependent manner. Taken together, our results suggest a novel effect of OTA on adipocyte differentiation in hAMSC and the possibility that OTA might affect the differentiation of other types of stem cells.

Isolating adipose-derived mesenchymal stem cells from lipoaspirate blood and saline fraction.

iFats — Wed, 01 Sep 2010 07:39:44 +0000

Isolation of adipose-derived stem cells (ASCs) typically involves 8+ hours of intense effort, requiring specialized equipment and reagents. Here, we present an improved technique for isolating viable populations of mesenchymal stem cells from lipoaspirate saline fractions within 30 minutes. Importantly, the cells exhibit remarkable similarities to those obtained using the traditional isolation protocols, in terms of their multipotent differentiation potential and immunophenotype. Reducing the acquisition time of ASCs is critical for advancing regenerative medicine therapeutics, and our approach provides rapid and simple techniques for enhanced isolation and expansion of patient-derived mesenchymal stem cells.