Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells

Articular cartilage is an avascular connective tissue that exhibits little intrinsic capacity for repair. Articular cartilage exists in a reduced oxygen ( approximately 5%) environment in vivo; therefore, oxygen tension may be an important factor that regulates the metabolism of chondrocyte progenitors. A number of recent studies have developed tissue engineering approaches for promoting cartilage repair using undifferentiated progenitor cells seeded on biomaterial scaffolds, but little is known about how oxygen might influence these engineered tissues. Human adipose-derived adult stem (hADAS) cells isolated from the stroma of subcutaneous fat were suspended in alginate beads and cultured in control or chondrogenic media in either low oxygen (5%) or atmospheric oxygen tension (20%) for up to 14 days. Under chondrogenic conditions, low oxygen tension significantly inhibited the proliferation of hADAS cells, but induced a two-fold increase in the rate of protein synthesis and a three-fold increase in total collagen synthesis. Low oxygen tension also increased glycosaminoglycan synthesis at certain timepoints. Immunohistochemical analysis showed significant production of cartilage-associated matrix molecules, including collagen type II and chondroitin-4-sulfate. These findings suggest oxygen tension may play an important role in regulating the proliferation and metabolism of hADAS cells as they undergo chondrogenesis, and the exogenous control of oxygen tension may provide a means of increasing the overall accumulation of matrix macromolecules in tissue-engineered cartilage.     

Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen biosynthesis

It has been speculated that heart valve interstitial cells (VICs) maintain valvular tissue homeostasis through regulated extracellular matrix (primarily collagen) biosynthesis. VICs appear to be phenotypically plastic, inasmuch as they transdifferentiate into myofibroblasts during valve development, disease, and remodeling. Under normal physiological conditions, transvalvular pressures (TVPs) on the right and left side of the heart are vastly different. Hence, we hypothesize that higher left-side TVPs impose larger local tissue stress on VICs, which increases their stiffness through cytoskeletal composition, and that this relation affects collagen biosynthesis. To evaluate this hypothesis, isolated ovine VICs from the four heart valves were subjected to micropipette aspiration to assess cellular stiffness, and cytoskeletal composition and collagen biosynthesis were quantified by using the surrogates smooth muscle alpha-actin (SMA) and heat shock protein 47 (HSP47), respectively. VICs from the aortic and mitral valves were significantly stiffer (P < 0.001) than those from the pulmonary and tricuspid valves. Left-side isolated VICs contained significantly more (P < 0.001) SMA and HSP47 than right-side VICs. Mean VIC stiffness correlated well (r = 0.973) with TVP; SMA and HSP47 also correlated well (r = 0.996) with one another. Assays were repeated for VICs in situ, and, as with in vitro results, left-side VIC protein levels were significantly greater (P < 0.05). These findings suggest that VICs respond to local tissue stress by altering cellular stiffness (through SMA content) and collagen biosynthesis. This functional VIC stress-dependent biosynthetic relation may be crucial in maintaining valvular tissue homeostasis and also prove useful in understanding valvular pathologies.     

Determination of the Poisson's ratio of the cell: recovery properties of chondrocytes after release from complete micropipette aspiration

Chondrocytes in articular cartilage are regularly subjected to compression and recovery due to dynamic loading of the joint. Previous studies have investigated the elastic and viscoelastic properties of chondrocytes using micropipette aspiration techniques, but in order to calculate cell properties, these studies have generally assumed that cells are incompressible with a Poisson's ratio of 0.5. The goal of this study was to measure the Poisson's ratio and recovery properties of the chondrocyte by combining theoretical modeling with experimental measures of complete cellular aspiration and release from a micropipette. Chondrocytes isolated from non-osteoarthritic and osteoarthritic cartilage were fully aspirated into a micropipette and allowed to reach mechanical equilibrium. Cells were then extruded from the micropipette and cell volume and morphology were measured throughout the experiment. This experimental procedure was simulated with finite element analysis, modeling the chondrocyte as either a compressible two-mode viscoelastic solid, or as a biphasic viscoelastic material. By fitting the experimental data to the theoretically predicted cell response, the Poisson's ratio and the viscoelastic recovery properties of the cell were determined. The Poisson's ratio of chondrocytes was found to be 0.38 for non-osteoarthritic cartilage and 0.36 for osteoarthritic chondrocytes (no significant difference). Osteoarthritic chondrocytes showed an increased recovery time following full aspiration. In contrast to previous assumptions, these findings suggest that chondrocytes are compressible, consistent with previous studies showing cell volume changes with compression of the extracellular matrix.     

Clonal analysis of the differentiation potential of human adipose-derived adult stem cells

Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.     

Bi-directional PCR allele-specific amplification (bi-PASA) for detection of caspase-8 -652 6N ins/del promoter polymorphism (rs3834129) in breast cancer

Caspase-8 (CASP8) plays a critical role in regulating apoptosis, and its functional polymorphisms may modify cancer risk. We investigated the possible association between CASP8 -652 6N ins/del (rs3834129) and the risk of breast cancer in a sample of Iranian population. This case-control study was done on 236 breast cancer patients and 203 cancer free healthy female. We designed a rapid and simple bi-directional PCR allele-specific amplification (bi-PASA) for detection of CASP8 -652 6N ins/del polymorphism. The results showed that the CASP8 -652 6N del/dl genotype was inversely associated with breast cancer risk (OR=0.33, 95% CI=0.17-0.65, p=0.001). The frequencies of the del allele in cases and controls were 29.1% and 38.6%, respectively. An inverse association between CASP8 6N del variant and the risk of breast cancer (OR=0.66, 95% CI=0.66-0.87, p=0.002) was found. In conclusion, the result suggests that the CASP8 -652 6N del polymorphism plays a protective role in susceptibility to breast cancer in our population. Further studies in other populations with larger samples are needed to confirm these findings.     

Molecular analysis of species of Tulipa L. from Iran based on ISSR markers

Molecular characterization of Tulipa L. species can elucidate the relationships among the species and provide more information about the taxonomy of this valuable genus. In this study, the genetic relationship among 39 Tulipa accessions from Khorassan and Yazd Provinces, located in east and northeast Iran, were analyzed using inter-simple sequence repeat (ISSR) primers. Ten selected ISSR primers from 20 screened primers generated a total of 97 polymorphic DNA bands. Unweighted pair-group method of cluster analysis based on Dice similarity values separated the accessions into nine groups. Seven species were recognized within these groups, and T.?micheliana Hoog was the most frequently encountered species. The subgroups formed within both T.?micheliana and T.?lehmanniana Merckl. revealed a low level of diversity within these species. T.?biebersteiniana Schultes & Schultes fil. and T.?biflora Pallas accessions made a separate clusters. The grouping of accessions was generally consistent with principal coordinate analysis (PCA) and clearly showed the position of species in the subgenera and sections of Tulipa. These results clearly showed the usefulness of DNA fingerprinting for identification of Tulipa accessions, and it is imperative to collect and characterize more genetic variability from the other distribution areas of this genus.     

A biomechanical role for perlecan in the pericellular matrix of articular cartilage

Chondrocytes are surrounded by a narrow pericellular matrix (PCM) that is biochemically, structurally, and biomechanically distinct from the bulk extracellular matrix (ECM) of articular cartilage. While the PCM is often defined by the presence of type VI collagen, other macromolecules such as perlecan, a heparan sulfate (HS) proteoglycan, are also exclusively localized to the PCM in normal cartilage and likely contribute to PCM structural integrity and biomechanical properties. Though perlecan is essential for normal cartilage development, its exact role in the PCM is unknown. The objective of this study was to determine the biomechanical role of perlecan in the articular cartilage PCM in situ and its potential as a defining factor of the PCM. To this end, atomic force microscopy (AFM) stiffness mapping was combined with dual immunofluorescence labeling of cryosectioned porcine cartilage samples for type VI collagen and perlecan. While there was no difference in overall PCM mechanical properties between type VI collagen- and perlecan-based definitions of the PCM, within the PCM, interior regions containing both type VI collagen and perlecan exhibited lower elastic moduli than more peripheral regions rich in type VI collagen alone. Enzymatic removal of HS chains from perlecan with heparinase III increased PCM elastic moduli both overall and locally in interior regions rich in both perlecan and type VI collagen. Heparinase III digestion had no effect on ECM elastic moduli. Our findings provide new evidence for perlecan as a defining factor in both the biochemical and biomechanical properties of the PCM.     

Cytosolic,Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

Rationale

Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli.

Methods

IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro.

Results

IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner.

Conclusions

Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.     

Label-free, high-throughput measurements of dynamic changes in cell nuclei using angle-resolved low coherence interferometry

Accurate measurements of nuclear deformation, i.e., structural changes of the nucleus in response to environmental stimuli, are important for signal transduction studies. Traditionally, these measurements require labeling and imaging, and then nuclear measurement using image analysis. This approach is time-consuming, invasive, and unavoidably perturbs cellular systems. Light scattering, an emerging biophotonics technique for probing physical characteristics of living systems, offers a promising alternative. Angle-resolved low-coherence interferometry (a/LCI), a novel light scattering technique, was developed to quantify nuclear morphology for early cancer detection. In this study, a/LCI is used for the first time to noninvasively measure small changes in nuclear morphology in response to environmental stimuli. With this new application, we broaden the potential uses of a/LCI by demonstrating high-throughput measurements and by probing aspherical nuclei. To demonstrate the versatility of this approach, two distinct models relevant to current investigations in cell and tissue engineering research are used. Structural changes in cell nuclei due to subtle environmental stimuli, including substrate topography and osmotic pressure, are profiled rapidly without disrupting the cells or introducing artifacts associated with traditional measurements. Accuracy ≥ 3% is obtained for the range of nuclear geometries examined here, with the greatest deviations occurring for the more complex geometries. Given the high-throughput nature of the measurements, this deviation may be acceptable for many biological applications that seek to establish connections between morphology and function.