Faculty Bibliography

In our studies to develop a simple and reliable method for the generation of recombinant adenoviruses (Fotadar et al . 2003), we noted that the E. coli (BJ5183 and DH5alpha) survived a heat-shock of 45 degrees C for 5 minutes in 0.1 M calcium chloride. As a result, we investigated the growth of E. coli (DH5alpha) at elevated temperatures. We hereby demonstrate that contrary to previous observations (Cooper et al . 2001 and Bronikowski et al . 2001) E. coli can grow consistently at a temperature as high as 49 degrees C, in spite of the fact that growth beyond 40 degrees C can generally be prohibitive. Hence, it is quite likely that these E. coli (DH5alpha) may have acquired mutations which permit them to grow reproducibly at 49 degrees C. Growth of E. coli above 49 degrees C (up to 53 degrees C) was also observed, but this was sporadic and not reproducible. This result could extend the utility of these organisms for cloning and manipulations requiring high temperature.

Undifferentiated cells have been identified in the prenatal blastocyst, inner cell mass, and gonadal ridges of rodents and primates, including humans. After isolation these cells express molecular and immunological markers for embryonic cells, capabilities for extended self-renewal, and telomerase activity. When allowed to differentiate, embryonic stem cells express phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. When implanted in vivo, undifferentiated noninduced embryonic stem cells formed teratomas. In this report we describe a cell clone isolated from postnatal rat skeletal muscle and derived by repetitive single-cell clonogenic analysis. In the undifferentiated state it consists of very small cells having a high ratio of nucleus to cytoplasm. The clone expresses molecular and immunological markers for embryonic stem cells. It exhibits telomerase activity, which is consistent with its extended capability for self-renewal. When induced to differentiate, it expressed phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. The clone was designated as a postnatal pluripotent epiblastic-like stem cell (PPELSC). The undifferentiated clone was transfected with a genomic marker and assayed for alterations in stem cell characteristics. No alterations were noted. The labeled clone, when implanted into heart after injury, incorporated into myocardial tissues undergoing repair. The labeled clone was subjected to directed lineage induction in vitro, resulting in the formation of islet-like structures (ILSs) that secreted insulin in response to a glucose challenge. This study suggests that embryonic-like stem cells are retained within postnatal mammals and have the potential for use in gene therapy and tissue engineering.

We have developed a counter rotating cone extrusion device to produce the next generation of three-dimensional collagen scaffold for tissue engineering. The device can produce a continuously varying fibril angle from the lumen to the outside of a 5-mm-diameter collagen tube, similar to the pattern of heart muscle cells in the intact heart. Our scaffold is a novel, oriented, type I collagen, tubular scaffold. We selected collagen because we believe there are important signals from the collagen both geometrically and biochemically that elicit the in vivo -like phenotypic response from the cardiomyocytes. We have shown that cardiomyocytes can be cultured in these tubes and resemble an in vivo phenotype. This new model system will provide important information leading to the design and construction of a functional, biologically based assist device.

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher orders. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50-70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Several studies have shown that disruption of the normal expression patterns of platelet-derived growth factor (PDGF) ligands and receptors during development results in gross cardiac defects and embryonic or neonatal death. However, little is known about the specific role that PDGF plays in the differentiation of cardiac myocytes. In experiments complementing studies that utilized naturally-occurring Patch mice lacking the PDGFr alpha, or knockout animals lacking a PDGF ligand or receptor, we used rat and mouse whole-embryo culture (WEC) techniques to increase the exposure of embryos to the PDGF-AA or -BB ligands. Following a 48-hr culture period, we analyzed heart growth and cardiac myocyte differentiation. Exposure of rat embryos to 50 ng/ml of PDGF-AA resulted in a 42% increase in total protein levels in the heart, but did not result in a significant increase in heart growth, as determined by measurements of the atrioventricular length and the left ventricular length and width. Exposure of embryos to 50 ng/ml of PDGF-BB resulted in a 77% increase in total protein levels and a significant (P < 0.05) 8-15% increase in the measured heart parameters. Although a comparison of control and PDGF-AA-treated embryos showed no increase in the overall size of the heart, confocal microscopy showed an increase in the size and number of myofibrillar bundles in the developing myocardium. In addition, transmission electron microscopy (TEM) revealed an increase in the presence of sarcomeres, indicating that myofibrils were more highly differentiated in these areas of the treated embryos. In PDGF-BB-treated embryos, the compact zone of the myocardium was thicker and, as shown by confocal microscopy and TEM, f-actin and well-developed sarcomeres were more prevalent, indicating that the myofibrils were more differentiated in the treated embryos than in the control embryos. These studies indicate that increased exposure of embryonic hearts to PDGF-AA or -BB increases the rate of myocardial development.

The cardiac fibroblast is the principal cell type responsible for extracellular matrix (ECM) synthesis in the heart during growth and pathophysiological conditions. A dynamic interaction exists between the cardiac ECM and fibroblasts that is sensitive to the local mechanical and chemical tissue environment. We propose here that cardiac fibroblasts structurally and functionally adapt to changing local environments by altering their expression of receptor integrins. Changes in the extracellular environment are communicated in part by integrins, which link the ECM to the cell and regulate phenotype and function. In this report, we analyze integrin protein expression, migration and gel contraction by cardiac fibroblasts from rats subjected to 10 weeks of treadmill exercise (XTR), experimental hypertension (HYP) or controls (CONT). Immunoprecipitation shows that beta1 protein increases in XTR and HYP. Also, alpha1 and alpha2 integrins are lower in XTR and HYP, and alpha5 integrin is higher in XTR and lower in HYP. Functional assays show that XTR and HYP migrate slower on collagen, while XTR migrate faster and HYP slower on fibronectin. Cell isolation procedure, population expansion number or a general adaptation to culture conditions does not explain the differences observed. No significant differences in collagen gel contraction are detected. These results indicate that cardiac fibroblasts retain their in vivo patterns in vitro for a limited number of population expansions. This tissue-specific phenotype is exhibited in early passage (< or =6). However, by late passage (>8), cells begin to show adaptation to the in vitro conditions. These results show that cardiac fibroblasts respond to changing environments in pathophysiological conditions by modulating integrin expression, which is associated with changes in cell migration. They also suggest a pragmatic use for primary cardiac fibroblasts as a model to study the cardiac matrix remodeled by physiological (exercise) and pathological (hypertension) stressors.

We have examined whether cadaver dissection by first year medical students (MIs) affected their performance in two test measures: the NBME Gross Anatomy and Embryology Subject Exam (dissection-relevant questions only), and practical exams given at the end of each major section within the course. The dissections for the entire course were divided into 18 regional dissection units and each student was assigned to dissect one third of the regional units; the other two-thirds of the material was learned from the partner-prosected cadavers. Performance for each student on the exams was then assessed as a function of the regions those students actually dissected. While the results indicated a small performance advantage for MIs answering questions on material they had dissected on the NBME Subject Exam questions relevant to dissection (78-88% of total exam), the results were not statistically significant. However, a similar, small performance advantage on the course practical exams was highly significant.

In this report, we describe the distribution of the platelet-derived growth factor receptor alpha (PDGFRalpha) by immunolocalization in the embryonic day 10.5 mouse heart and defects in heart development associated with the absence of this receptor in the Patch mouse. The Patch mouse is a naturally occurring mutant that has been accepted as a model for determining the role of the PDGFRalpha in early cardiac development. Even though other genetic defects exist in this naturally occurring mutant, most defects associated with cardiac development are believed to be a result of the absence of this receptor. Gross morphological defects including improper septation of the outflow tract, dysmorphic shape of the heart, and lack of trabecular development are similar to those that have been previously described. Many of these defects have been attributed to the failure of a subset of non-neuronal neural crest cells to properly migrate into the region of the developing outflow tract. In these studies, we have also used confocal scanning laser and transmission electron microscopy to describe and compare the organization and differentiation of the cytoskeletal proteins actin and myosin in littermate control and Patch mouse hearts. Cytoskeletal organization of the cardiac myocytes in Patch mouse hearts has not previously been described. In most cardiac myocytes of Patch mice, actin was found only on the periphery of the cells, and the organization of actin, myosin, and precursor Z-band material into distinct myofibrils was greatly reduced.