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1.  Cardiac fibroblast-derived 3D extracellular matrix seeded with mesenchymal stem cells as a novel device to transfer cells to the ischemic myocardium 
Demonstrate a novel manufacturing method to generate extracellular matrix scaffolds from cardiac fibroblasts (CF-ECM) as a therapeutic mesenchymal stem cell-transfer device.
Materials and Methods
Rat CF were cultured at high-density (~1.6×105/cm2) for 10–14 days. Cell sheets were removed from the culture dish by incubation with EDTA and decellularized with water and peracetic acid. CF-ECM was characterized by mass spectrometry, immunofluorescence and scanning electron microscopy. CF-ECM seeded with human embryonic stem cell derived mesenchymal stromal cells (hEMSCs) were transferred into a mouse myocardial infarction model. 48 hours later, mouse hearts were excised and examined for CF-ECM scaffold retention and cell transfer.
CF-ECM scaffolds are composed of fibronectin (82%), collagens type I (13%), type III (3.4%), type V (0.2%), type II (0.1%) elastin (1.3%) and 18 non-structural bioactive molecules. Scaffolds remained intact on the mouse heart for 48 hours without the use of sutures or glue. Identified hEMSCs were distributed from the epicardium to the endocardium.
High density cardiac fibroblast culture can be used to generate CF-ECM scaffolds. CF-ECM scaffolds seeded with hEMSCs can be maintained on the heart without suture or glue. hEMSC are successfully delivered throughout the myocardium.
PMCID: PMC3964807  PMID: 24683428
Cardiac Fibroblast; Extracellular Matrix; Stem Cell; Cardiac; Regeneration; Heart Failure; Myocardial Infarction
2.  A high-fat diet decreases AMPK activity in multiple tissues in the absence of hyperglycemia or systemic inflammation in rats 
Consumption of a high-fat diet (HFD) in experimental animal models initiates a series of molecular events and outcomes, including insulin resistance and obesity, that mimic the metabolic syndrome in humans. The relationship among, and order of, the molecular events linking a diet high in fat to pathologies is often unclear. In the present study, we provide several novel insights into the relationship between a HFD and AMP-activated protein kinase (AMPK), a key regulator of cellular metabolism and whole-body energy balance. HFD substantially decreased the activities of both iso-forms of AMPK in white adipose tissue, heart, and liver. These decreases in AMPK activity occurred in the absence of decreased AMPK transcription, systemic inflammation, hyperglycemia, or elevated levels of free fatty acids. The HFD-induced decrease in AMPK activity was associated with systemic insulin resistance and hyperleptinemia. In blood, >98 % of AMPK activity was localized in agranulocytes as the α1 isoform. In contrast to the solid tissues studied, AMPK activities were not altered by HFD in granulocytes or agranulocytes. We conclude that HFD-induced obesity causes a broad, non-tissue, or isoform-specific lowering of AMPK activity. Given the central position AMPK plays in whole-body energy balance, this decreased AMPK activity may play a previously unrecognized role in obesity and its associated pathologies.
PMCID: PMC3644018  PMID: 22941749
Obesity; Diabetes; Adipose; Blood
3.  High-content adhesion assay to address limited cell samples† 
Cell adhesion is a broad topic in cell biology that involves physical interactions between cells and other cells or the surrounding extracellular matrix, and is implicated in major research areas including cancer, development, tissue engineering, and regenerative medicine. While current methods have contributed significantly to our understanding of cell adhesion, these methods are unsuitable for tackling many biological questions requiring intermediate numbers of cells (102–105), including small animal biopsies, clinical samples, and rare cell isolates. To overcome this fundamental limitation, we developed a new assay to quantify the adhesion of ~102–103 cells at a time on engineered substrates, and examined the adhesion strength and population heterogeneity via distribution-based modeling. We validated the platform by testing adhesion strength of cancer cells from three different cancer types (breast, prostate, and multiple myeloma) on both IL-1β activated and non-activated endothelial monolayers, and observed significantly increased adhesion for each cancer cell type upon endothelial activation, while identifying and quantifying distinct subpopulations of cell-substrate interactions. We then applied the assay to characterize adhesion of primary bone marrow stromal cells to different cardiac fibroblast-derived matrix substrates to demonstrate the ability to study limited cell populations in the context of cardiac cell-based therapies. Overall, these results demonstrate the sensitivity and robustness of the assay as well as its ability to enable extraction of high content, functional data from limited and potentially rare primary samples. We anticipate this method will enable a new class of biological studies with potential impact in basic and translational research.
PMCID: PMC3832292  PMID: 23426645
4.  Caloric restriction does not alter effects of aging in cardiac side population cells 
Age  2010;33(3):351-361.
The aged heart displays a loss of cardiomyocyte number and function, possibly due to the senescence and decreased regenerative potential that has been observed in some cardiac progenitor cells. An important cardiac progenitor that has not been studied in the context of aging is the cardiac side population (CSP) cell. To address this, flow cytometry-assisted cell sorting was used to isolate CSP cells from adult (6–10 months old) and aged (24–32 months old) C57Bl/6 mice that were fed either a control diet or an anti-aging diet (caloric restriction, CR). Aging caused a 2.3-fold increase in the total number of CSP cells and a 3.2-fold increase in the cardiomyogenic sca1+/CD31− subpopulation. Aging did not affect markers of proliferation or senescence, including telomerase activity and expression of cell cycle genes, in sca1+/CD31− CSP cells. In contrast, the aged cells had reduced expression of genes associated with differentiation, including smooth muscle actin and cardiac muscle actin (5.1- and 3.2-fold, respectively). None of these age effects were altered by CR diet. Therefore, it appears that the manner in which CSP cells age is distinct from the aging of post-mitotic tissue (and perhaps other progenitor cells) that can often be attenuated by CR.
PMCID: PMC3168602  PMID: 20922487
Adult stem cell; Cardiac regeneration; CR; CSP; Sca1; CD31
5.  Caloric restriction attenuates the age-associated increase of adipose-derived stem cells but further reduces their proliferative capacity 
Age  2010;33(2):107-118.
White adipose tissue is a promising source of mesenchymal stem cells. Currently, little is known about the effect of age and caloric restriction (CR) on adipose-derived stem cells (ASC). This is important for three reasons: firstly, age and CR cause extensive remodeling of WAT; it is currently unknown how this remodeling affects the resident stem cell population. Secondly, stem cell senescence has been theorized as one of the causes of aging and could reduce the utility of a stem cell as a reagent. Thirdly, the mechanism by which CR extends lifespan is currently not known, one theory postulates that CR maintains the resident stem cell population in youthful “fit” state. For the purpose of this study, we define ASC as lineage negative (lin−)/CD34+(low)/CD31−. We show that aging increases the abundance of ASC and the expression of Cdkn2a 9.8-fold and Isl1 60.6-fold. This would suggest that aging causes an accumulation of non-replicative ASC. CR reduced the percentage of ASC in the lin− SVF while also reducing colony forming ability. Therefore, CR appears to have anti-proliferative effects on ASC that may be advantageous from the perspective of cancer, but our data raises the possibility that it may be disadvantageous for regenerative medicine applications.
PMCID: PMC3127466  PMID: 20628827
Stem cells; Aging; Caloric restriction; Adipose tissue; Regeneration; Cancer

Results 1-5 (5)