|Cardiac commitment of primate embryonic stem cells. |
Leschik J. et al.
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Primate nonhuman and human embryonic stem (ES) cells provide a powerful model of early cardiogenesis. Furthermore, engineering of cardiac progenitors or cardiomyocytes from ES cells offers a tool for drug screening in toxicology or to search for molecules to improve and scale up the process of cardiac differentiation using high-throughput screening technology, as well as a source of cell therapy of heart failure. Spontaneous differentiation of ES cells into cardiomyocytes is, however, limited. Herein, we describe a simple protocol to commit both rhesus and human ES cells toward a cardiac lineage and to sort out early cardiac progenitors. Primate ES cells are challenged for 4 d with the cardiogenic morphogen bone morphogenetic protein 2 (BMP2) and sorted out using anti-SSEA-1 antibody-conjugated magnetic beads. Cardiac progenitor cells can be generated and isolated in 4 d using this protocol.
|Co-localization of angiotensin-converting enzyme 2-, octomer-4- and CD34-positive cells in rabbit atherosclerotic plaques. |
Anthony Zulli, Sudarshan Rai, Brian F Buxton, Louise M Burrell, David L Hare
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Angiotensin-converting enzyme 2 (ACE2) is a novel enzyme with possible implications in the treatment of blood pressure disorders. Recent evidence suggests that an upregulation of ACE2 can be stimulated by all-trans retinoic acid (at-RA); however, at-RA also affects regulation of the stem-cell marker octomer-4 (Oct-4) and thus cellular differentiation. We have previously shown that smooth muscle cells and macrophages present within rabbit atherosclerotic plaques are positive for ACE2, Oct-4 and the haematopoietic stem-cell marker CD34. Thus, to provide evidence that possible at-RA treatment could affect both plaque cellular biology (via effects on cellular differentiation) and blood pressure (via ACE2), it is vital to show that cells with atherosclerotic plaques co-express all three markers. Thus, we sought to provide evidence that a subset of cells within atherosclerotic plaques is positive for ACE2, Oct-4 and CD34. We used New Zealand White rabbits that were fed a control diet supplemented with 0.5% cholesterol plus 1% methionine for 4 weeks and then allowed to consume a normal diet for 10 weeks. Immunohistochemistry was performed by standard techniques. We report that ACE2, Oct-4 and CD34 were all present within atherosclerotic plaques. Although macrophages were positive for all three markers, spindle-shaped cells in the media did not show all three markers. The endothelium overlying normal arterial wall showed positive Oct-4 and ACE2 immunoreactivity, but CD34 immunoreactivity was patchy, indicating that such cells might not have fully differentiated. It is concluded that cells in atherosclerotic plaques express co-express ACE2, Oct-4 and CD34. Further studies aimed at establishing the effects of all-trans retinoic acid on blood pressure and atherosclerotic cell differentiation are warranted.
|Human diseased arteries contain cells expressing leukocytic and embryonic stem cell markers. |
Anthony Zulli,Brian F Buxton,Mervyn Merrilees,David L Hare
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Recent evidence suggests that smooth muscle cells within the intima of diseased human blood vessels of the elderly population contain the embryonic form of smooth muscle cells. We wanted to explore the idea that human diseased vessels may contain other primitive cell types, such as pluripotent embryonic stem cells and hematopoietic stem cells. Radial and internal mammary arteries were collected from patients undergoing coronary artery bypass surgery; and coronary arteries, from hearts at autopsy and transplant. Immunohistochemistry was used to identify the embryonic stem cell markers Octomer-4; stage-specific embryonic antigens 1, 3, and 4; TRA-1-60; and TRA-1-81, and the leukocytic markers CD34, CD14, CD133, and CD64 in all vessels. We found that diseased human radial arteries contained the highest numbers of cells in the media- and intima-expressing markers of embryonic and leukocytic origin compared with diseased human coronary arteries. In nondiseased human vessels (internal mammary arteries), such cells were rarely observed. Granulation tissue within the diseased human arteries contained similar cells, and the angiogenic vessel endothelial cell layer also expressed these markers. It is concluded that diseased human blood vessels contain cells that express markers from leukocytic and embryonic origin. These results suggest that cells within human arteries might be able to differentiate into various cell types and that blood vessels might be a reservoir for such cells.
|Establishment of rat embryonic stem cells and making of chimera rats. |
Ueda S. et al.
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The rat is a reference animal model for physiological studies and for the analysis of multigenic human diseases such as hypertension, diabetes, neurological disorders, and cancer. The rats have long been used in extensive chemical carcinogenesis studies. Thus, the rat embryonic stem (rES) cell is an important resource for the study of disease models. Attempts to derive ES cells from various mammals, including the rat, have not succeeded. Here we have established two independent rES cells from Wister rat blastocysts that have undifferentiated characters such as Nanog and Oct3/4 genes expression and they have stage-specific embryonic antigen (SSEA) -1, -3, -4, and TRA-1-81 expression. The cells were successfully cultured in an undifferentiated state and can be possible over 18 passages with maintaining more than 40% of normal karyotype. Their pluripotent potential was confirmed by the differentiation into derivatives of the endoderm, mesoderm, and ectoderm. Most importantly, the rES cells are capable of producing chimera rats. Therefore, we established pluripotent rES cell lines that are widely used to produce genetically modified experimental rats for study of human diseases.