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Stem Cells and Heart Induction The goal of this program is to develop technology, resources and reagents that will enable the discovery of molecules that stimulate the formation of cardiomyocytes. Although our focus is on differentiation of stem cells, either in vitro or endogenous cells in vivo, we expect that molecules that induce heart formation in the embryo will be able to be used to direct stem cell cardiomyogenesis. Classical embryologists beginning in the 1920s mapped the part of the embryo that gives rise to the heart and subsequently defined some of the interactions with other tissues that provide the inductive stimuli for heart formation. In vertebrates, the heart forms in the anterior mesoderm as a result of signals from endodermal and/or extraembryonic endodermal sources, depending on the species. The use of amphibian and chick embryos has been very important historically for the identification of signaling proteins, and our work has positioned several factors, such as Dickkopf-1 and Nodal, in the hierarchy of inducing factors.
Movie. An beating heart created from normally non-cardiogenic tissue of an of the frog, Xenopus laevis by treatment with Dickkopf-1 (Schneider, V. and Mercola, M. Genes and Development, 2001).
Many of the known factors involved in heart formation are diagrammed in Figure 1.
Figure 1. Pathways that control cardiogenesis. Adapted from Guzzo et al. Advances in Developmental Biology (2007). We are interested in defining extracellular molecules that stimulate cardiogenesis, and we have been pursuing signals from Dickkopf and Nodal proteins. Both of these proteins are involved in initiating heart formation but act via different pathways: Dickkopf is an inhibitor of canonical Wnt signaling, but has other activities as well, whereas Nodal is a TGFb family member and acts to control the production of Cerberus, which antagonizes bone morphogenetic protein (BMP) and Nodal signaling. Morphogenesis of the heart is influenced by numerous other signals, notably those that regulate left-right asymmetry, neural crest and secondary heart field. Our approach is to elucidate the molecules and downstream signals that control heart development using embryos and embryonic stem cells (ESCs) of both mouse and human origin and to use biosensors to visualize signaling pathways directly in human embryoid bodies made from hESCs.
Figure 2. Comparison of heart induction in mouse embryos and ESCs. ESCs are derived from the inner cell mass of pre-implantation embryos (~E3.5, top). Under differentiation conditions, aggregates of ESCs, known as embryoid bodies (EBs), spontaneously form derivatives of all three germ layers including a small number of cardiomyocytes. Heart induction in EBs probably recapitulates the process in the ~E7.25 embryo, in which the anterior visceral and definitive endoderm initiates cardiogenesis within the adjacent heart- forming mesoderm (dark red). Most of the endoderm in this diagram is shown peeled away; the heart-inducing region (grey) consists of the extraembryonic anterior visceral endoderm as well as the anterior definitive endoderm. Currently, we are applying the embryological knowledge to the problem of generating cardiomyocytes from stem cells with the immediate goal of developing the tools and reagents to enhance the formation of cardiomyocytes from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hIPSCs). hIPSCs offer an unprecedented opportunity since they can be derived from adult cells yet share with hESCs the ability to produce all the cell types in the body. Since they can be derived from a patient’s cells, they offer a more rapid path to clinical application since cardiomyocytes derived from these cells would be expected to be immunologically compatible with the patient. An NHLBI-sponsored program in the lab is to discover natural protein and microRNA regulators of cardiomyocyte differentiation and maturation to a physiologically mature heart muscle cell. Ultimately, these factors will be used in defined conditions to efficiently produce cardiomyocytes from hESCs. Thus far, the research has resulted in protocols and lentiviral factors for the production of pure populations of cardiomyocytes. (see movie below)
Movie. Cardiomyocyte spheroid, labeled with eGFP driven by the aMHC promoter, and derived from hESCs by a genetic selection strategy. An extension of this project is to adapt hESC procedures to the engineering of hIPSCs for production of cardiomyocytes. Currently, we have generated hIPSC lines for visualization and genetic selection of cardiomyocytes, and these cells are being evaluated for therapeutic potential in pre-clinical animal models of heart disease. An additional goal is to engineer lines of hESCs and hIPSCs for visualization of cardiomyocytes and their progenitors, and to use these cells for in vitro assays to discover novel small molecules that direct differentiation and maturation (see Automated Screening page). Epicardial cells are an additional potential source of cardiomyocytes. The epicardium is the outer layer of the heart and has recently been shown to be capable of forming cardiomyocytes, both in vitro and in vivo. In collaboration with the laboratory of Dr. Pilar Ruiz-Lozano at the BIMR, we are isolating epicardial cells and evaluating known molecules for the ability to stimulate cardiomyogenesis from these cells. In addition, it might also be possible to screen directly for molecules that promote cardiomyocyte differentiation from epicardial cells, since such molecules might be useful as leads to develop drugs for endogenous regeneration.
Movie. Epicardial-derived cells induced to form cardiomyocytes in culture.
People Wenqing Cai, PhD student, BIMR PhD program Rosa Guzzo, PhD, postdoctoral fellow Ruchika Gupta, PhD, postdoctoral fellow Natalia Kan, PhD, postdoctoral fellow Hiroko Kita-Matsuo, PhD, postdoctoral fellow Frederick Lo, PhD student, UCSD Bioengineering Ramon Diaz Trelles, PhD, postdoctoral fellow Ke Wei, PhD, postdoctoral fellow
Collaborators: Pilar Ruiz-Lozano, PhD, Assistant Professor, BIMR H.S. Vincent Chen, B.M., Ph.D., Assistant Professor, BIMR Raj Krishnan, PhD student, UCSD Bioengineering, in collaborator Professor Michael Heller's laboratory
Recent publications Korol, O., Gupta, R.W., and Mercola, M. (2008). A Novel Activity of the Dickkopf-1 Amino-terminal Domain Promotes Axial and Heart Development Independently of Canonical Wnt Inhibition. Developmental Biology, Dec 1;324(1):131-8. [PubMed]. Campa, V.M., Gutiérrez-Lanza, R., Diaz-Trelles, R., Cerignoli, F., Tsuji, T., Jiang, W., and Mercola, M. (2008). Notch Activates Cell Cycle Re-entry and Progression in Postmitotic Cardiomyocytes. J. Cell Biology, Oct 6;183(1):129-41. [PubMed]. Maurer, J., Nelson, B., Ceceña, G., Bajpai, R., Mercola, M., Terskikh, A.V., and Oshima, R.G. (2008). Contrasting expression of keratins in mouse and human embryonic stem cells. PLoS ONE, 3(10) e3451. [PubMed]. Foley, A. C., Korol, O., Timmer, A. M. and Mercola, M. (2007). Multiple functions of Cerberus cooperate to induce heart downstream of Nodal. Dev Biol. [PubMed] Guzzo, R. M., Foley, A. C., Ibarra, Y. M. and Mercola, M. (2007). Signaling Pathways in Embryonic Heart Induction. Adv. Devl. Biol. 18, 117-151. Foley, A. C., Gupta, R. W., Guzzo, R. M., Korol, O. and Mercola, M. (2006). Embryonic Heart Induction. Ann N Y Acad. Sci 1080, 85-96. [PubMed] Foley, A. C. and Mercola, M. (2005). Heart induction by Wnt antagonists depends on the homeodomain transcription factor Hex. Genes Dev 19, 387-96. [PubMed] Foley, A. and Mercola, M. (2004). Heart induction: embryology to cardiomyocyte regeneration. Trends Cardiovasc Med 14, 121-5. [PubMed] Levin, M., Thorlin, T., Robinson, K., Nogi, T. and Mercola, M. (2002). Asymmetries in H(+)/K(+)-ATPase and Cell Membrane Potentials Comprise a Very Early Step in Left-Right Patterning. Cell 111, 77-89. [PubMed] Raffin, M., Leong, L. M., Rones, M. S., Sparrow, D., Mohun, T. and Mercola, M. (2000). Subdivision of the cardiac Nkx2.5 expression domain into myogenic and non-myogenic compartments. Developmental Biology 218, 326-340. [PubMed] Schneider, V. A. and Mercola, M. (2001). Wnt antagonism initiates cardiogenesis in Xenopus laevis. Genes and Development 15, 304–315. [PubMed] Schneider, V. A. and Mercola, M. (1999). Spatially distinct head and heart inducers within the Xenopus organizer region. Current Biology 9, 800-809. [PubMed]
Funding Mathers Family Charitable Trust http://www.mathersfoundation.org/ NIH Heart, Lung and Blood Institute
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