Chondrogenesis of adult stem cells from adipose tissue and bone marrow: induction by growth factors and cartilage-derived matrix.

OBJECTIVES: Adipose-derived stem cells (ASCs) and bone marrow-derived mesenchymal stem cells (MSCs) are multipotent adult stem cells with potential for use in cartilage tissue engineering. We hypothesized that these cells show distinct responses to different chondrogenic culture conditions and extracellular matrices, illustrating important differences between cell types. METHODS: Human ASCs and MSCs were chondrogenically differentiated in alginate beads or a novel scaffold of reconstituted native cartilage-derived matrix with a range of growth factors, including dexamethasone, transforming growth factor beta3, and bone morphogenetic protein 6. Constructs were analyzed for gene expression and matrix synthesis. RESULTS: Chondrogenic growth factors induced a chondrocytic phenotype in both ASCs and MSCs in alginate beads or cartilage-derived matrix. MSCs demonstrated enhanced type II collagen gene expression and matrix synthesis as well as a greater propensity for the hypertrophic chondrocyte phenotype. ASCs had higher upregulation of aggrecan gene expression in response to bone morphogenetic protein 6 (857-fold), while MSCs responded more favorably to transforming growth factor beta3 (573-fold increase). CONCLUSIONS: ASCs and MSCs are distinct cell types as illustrated by their unique responses to growth factor-based chondrogenic induction. This chondrogenic induction is affected by the composition of the scaffold and the presence of serum.

Ligament-derived matrix stimulates a ligamentous phenotype in human adipose-derived stem cells.

Human adipose stem cells (hASCs) can differentiate into a variety of phenotypes. Native extracellular matrix (e.g., demineralized bone matrix or small intestinal submucosa) can influence the growth and differentiation of stem cells. The hypothesis of this study was that a novel ligament-derived matrix (LDM) would enhance expression of a ligamentous phenotype in hASCs compared to collagen gel alone. LDM prepared using phosphate-buffered saline or 0.1% peracetic acid was mixed with collagen gel (COL) and was evaluated for its ability to induce proliferation, differentiation, and extracellular matrix synthesis in hASCs over 28 days in culture at different seeding densities (0, 0.25 x 10(6), 1 x 10(6), or 2 x 10(6) hASC/mL). Biochemical and gene expression data were analyzed using analysis of variance. Fisher's least significant difference test was used to determine differences between treatments following analysis of variance. hASCs in either LDM or COL demonstrated changes in gene expression consistent with ligament development. hASCs cultured with LDM demonstrated more dsDNA content, sulfated-glycosaminoglycan accumulation, and type I and III collagen synthesis, and released more sulfated-glycosaminoglycan and collagen into the medium compared to hASCs in COL (p

Assessment of LD matrix measures for the analysis of biological pathway association.

Complex diseases will have multiple functional sites, and it will be invaluable to understand the cross-locus interaction in terms of linkage disequilibrium (LD) between those sites (epistasis) in addition to the haplotype-LD effects. We investigated the statistical properties of a class of matrix-based statistics to assess this epistasis. These statistical methods include two LD contrast tests (Zaykin et al., 2006) and partial least squares regression (Wang et al., 2008). To estimate Type 1 error rates and power, we simulated multiple two-variant disease models using the SIMLA software package. SIMLA allows for the joint action of up to two disease genes in the simulated data with all possible multiplicative interaction effects between them. Our goal was to detect an interaction between multiple disease-causing variants by means of their linkage disequilibrium (LD) patterns with other markers. We measured the effects of marginal disease effect size, haplotype LD, disease prevalence and minor allele frequency have on cross-locus interaction (epistasis). In the setting of strong allele effects and strong interaction, the correlation between the two disease genes was weak (r=0.2). In a complex system with multiple correlations (both marginal and interaction), it was difficult to determine the source of a significant result. Despite these complications, the partial least squares and modified LD contrast methods maintained adequate power to detect the epistatic effects; however, for many of the analyses we often could not separate interaction from a strong marginal effect. While we did not exhaust the entire parameter space of possible models, we do provide guidance on the effects that population parameters have on cross-locus interaction.

Integrin-mediated interactions with extracellular matrix proteins for nucleus pulposus cells of the human intervertebral disc.

The extracellular matrix (ECM) of the human intervertebral disc is rich in molecules that interact with cells through integrin-mediated attachments. Porcine nucleus pulposus (NP) cells have been shown to interact with laminin (LM) isoforms LM-111 and LM-511 through select integrins that regulate biosynthesis and cell attachment. Since human NP cells lose many phenotypic characteristics with age, attachment and interaction with the ECM may be altered. Expression of LM-binding integrins was quantified for human NP cells using flow cytometry. The cell-ECM attachment mechanism was determined by quantifying cell attachment to LM-111, LM-511, or type II collagen after functionally blocking specific integrin subunits. Human NP cells express integrins β1, α3, and α5, with over 70% of cells positive for each subunit. Blocking subunit β1 inhibited NP cell attachment to all substrates. Blocking subunits α1, α2, α3, and α5 simultaneously, but not individually, inhibits NP cell attachment to laminins. While integrin α6β1 mediated porcine NP cell attachment to LM-111, we found integrins α3, α5, and β1 instead contributed to human NP cell attachment. These findings identify integrin subunits that may mediate interactions with the ECM for human NP cells and could be used to promote cell attachment, survival, and biosynthesis in cell-based therapeutics.

The role of extracellular matrix elasticity and composition in regulating the nucleus pulposus cell phenotype in the intervertebral disc: a narrative review.

Intervertebral disc (IVD) disorders are a major contributor to disability and societal health care costs. Nucleus pulposus (NP) cells of the IVD exhibit changes in both phenotype and morphology with aging-related IVD degeneration that may impact the onset and progression of IVD pathology. Studies have demonstrated that immature NP cell interactions with their extracellular matrix (ECM) may be key regulators of cellular phenotype, metabolism and morphology. The objective of this article is to review our recent experience with studies of NP cell-ECM interactions that reveal how ECM cues can be manipulated to promote an immature NP cell phenotype and morphology. Findings demonstrate the importance of a soft (<700 Pa), laminin-containing ECM in regulating healthy, immature NP cells. Knowledge of NP cell-ECM interactions can be used for development of tissue engineering or cell delivery strategies to treat IVD-related disorders.

Interstitial engraftment of adipose-derived stem cells into an acellular dermal matrix results in improved inward angiogenesis and tissue incorporation.

Acellular dermal matrices (ADM) are commonly used in reconstructive procedures and rely on host cell invasion to become incorporated into host tissues. We investigated different approaches to adipose-derived stem cells (ASCs) engraftment into ADM to enhance this process. Lewis rat adipose-derived stem cells were isolated and grafted (3.0 × 10(5) cells) to porcine ADM disks (1.5 mm thick × 6 mm diameter) using either passive onlay or interstitial injection seeding techniques. Following incubation, seeding efficiency and seeded cell viability were measured in vitro. In addition, Eighteen Lewis rats underwent subcutaneous placement of ADM disk either as control or seeded with PKH67 labeled ASCs. ADM disks were seeded with ASCs using either onlay or injection techniques. On day 7 and or 14, ADM disks were harvested and analyzed for host cell infiltration. Onlay and injection techniques resulted in unique seeding patterns; however cell seeding efficiency and cell viability were similar. In-vivo studies showed significantly increased host cell infiltration towards the ASCs foci following injection seeding in comparison to control group (p < 0.05). Moreover, regional endothelial cell invasion was significantly greater in ASCs injected grafts in comparison to onlay seeding (p < 0.05). ADM can successfully be engrafted with ASCs. Interstitial engraftment of ASCs into ADM via injection enhances regional infiltration of host cells and angiogenesis, whereas onlay seeding showed relatively broad and superficial cell infiltration. These findings may be applied to improve the incorporation of avascular engineered constructs.

Contribution of bacterial outer membrane vesicles to innate bacterial defense.

BACKGROUND: Outer membrane vesicles (OMVs) are constitutively produced by Gram-negative bacteria throughout growth and have proposed roles in virulence, inflammation, and the response to envelope stress. Here we investigate outer membrane vesiculation as a bacterial mechanism for immediate short-term protection against outer membrane acting stressors. Antimicrobial peptides as well as bacteriophage were used to examine the effectiveness of OMV protection. RESULTS: We found that a hyper-vesiculating mutant of Escherichia coli survived treatment by antimicrobial peptides (AMPs) polymyxin B and colistin better than the wild-type. Supplementation of E. coli cultures with purified outer membrane vesicles provided substantial protection against AMPs, and AMPs significantly induced vesiculation. Vesicle-mediated protection and induction of vesiculation were also observed for a human pathogen, enterotoxigenic E. coli (ETEC), challenged with polymyxin B. When ETEC with was incubated with low concentrations of vesicles concomitant with polymyxin B treatment, bacterial survival increased immediately, and the culture gained resistance to polymyxin B. By contrast, high levels of vesicles also provided immediate protection but prevented acquisition of resistance. Co-incubation of T4 bacteriophage and OMVs showed fast, irreversible binding. The efficiency of T4 infection was significantly reduced by the formation of complexes with the OMVs. CONCLUSIONS: These data reveal a role for OMVs in contributing to innate bacterial defense by adsorption of antimicrobial peptides and bacteriophage. Given the increase in vesiculation in response to the antimicrobial peptides, and loss in efficiency of infection with the T4-OMV complex, we conclude that OMV production may be an important factor in neutralizing environmental agents that target the outer membrane of Gram-negative bacteria.

Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response.

Conditions that impair protein folding in the Gram-negative bacterial envelope cause stress. The destabilizing effects of stress in this compartment are recognized and countered by a number of signal transduction mechanisms. Data presented here reveal another facet of the complex bacterial stress response, release of outer membrane vesicles. Native vesicles are composed of outer membrane and periplasmic material, and they are released from the bacterial surface without loss of membrane integrity. Here we demonstrate that the quantity of vesicle release correlates directly with the level of protein accumulation in the cell envelope. Accumulation of material occurs under stress, and is exacerbated upon impairment of the normal housekeeping and stress-responsive mechanisms of the cell. Mutations that cause increased vesiculation enhance bacterial survival upon challenge with stressing agents or accumulation of toxic misfolded proteins. Preferential packaging of a misfolded protein mimic into vesicles for removal indicates that the vesiculation process can act to selectively eliminate unwanted material. Our results demonstrate that production of bacterial outer membrane vesicles is a fully independent, general envelope stress response. In addition to identifying a novel mechanism for alleviating stress, this work provides physiological relevance for vesicle production as a protective mechanism.

Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells.

BACKGROUND: Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. RESULTS: Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER) marker, TRAPalpha, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939), an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. CONCLUSION: These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat.

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.