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References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Directed differentiation of ES cells into cardiac mesoderm Directed differentiation allows production of a desired cell type Pluripotent Multipotent Ectodermal cell ES cell References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Differentiated cells Heart muscle cells (or cardiomyocytes) derived by this process can be used for: brain Mesodermal cell heart Endodermal cell pancreas 1. replacement of damaged tissue (e.g. following a heart attack). 2. development of new therapies to treat chronic abnormalities of heart function (e.g. arrhythmia) by studying individual beating heart muscle cells in a dish. 1 Diseases of heart muscle tissue References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Cardiac muscle – Tissue composed of individual cells, unlike skeletal myofibers with many nuclei – Cells coupled by gap junctions to enable rapid spread of electrical signals – Cells joined end-to-end by adhesive discs Diseases of cardiac muscles – Congestive heart failure, or ineffective pumping due to cardiomyocyte dysfunction, affects 4.8 million people in U.S. –Heart attack, also known as myocardial infarction –Heart arrhythmias, or abnormal activity (palpitations) – 1% of live births display a congenital heart defect A four-chambered heart underlies double circulation (birds & mammals) • This system is the first functional unit that forms in the embryo. • Circulation provides nourishment to organs (nutrients and oxygen). • The heart is the main organ that pumps blood through the blood vessels. • The heart contains four chambers in birds and mammals (simpler in fish & 3 amphibians). Left atrium Left ventricle Heart Blood vessels Blood cells • Atrioventricular valves Right ventricle Lecture notes (hyperlink) Activity notes (hyperlink) More links… The circulatory system is: – – – Right atrium References Heart muscle, to perform its unique function, is different from skeletal and smooth muscle References Lecture notes (hyperlink) Activity notes (hyperlink) More links… 1. Cardiac muscle cells: Three types of muscle cells • Have visible myofibrils • Occur only in heart • Involuntary movement • Cells joined by intercalating disks to allow rapid spread of electrical signals 2. Skeletal muscle cells: • Elongated multinucleate cells called myofibrils. • Voluntary movements 3. Smooth muscle cells: • Individual, spindle-shaped mononuclear cells • Found in gut, blood vessels and ducts of glands • Involuntary movements 4 How are cardiomyocytes generated during development? References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Lineage restrictions Pluripotent Multipotent Differentiation Ectodermal cell ? ES cell Mesodermal cell Endodermal cell ? Multipotent cardiovascular progenitor Cardiomyocyte 5 Heart, blood and blood vessels are derived from the lateral mesoderm References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Heart, circulatory system, smooth muscle Skeletal muscles 6 Formation of heart tube from germ layers (in the chick) References Lecture notes (hyperlink) Activity notes (hyperlink) More links… 7 Cardiac looping and chamber formation in the human embryo References Lecture notes (hyperlink) Activity notes (hyperlink) More links… 8 Secreted factors specify heart-forming mesoderm in vivo Lineage restrictions References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Differentiation FGF8 Anterior lateral plate mesodermal cell Cerberus BMPs Wnts Lateral plate mesodermal cell Cardiogenic mesodermal cell Cardiomyocyte Noggin Posterior lateral plate mesodermal cell Hemangiogenic mesodermal cell Blood cells 9 Distinct cell signaling pathways induce cardiomyocyte-specific genes Cell signaling pathway References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Gene expression Heart muscle cell (Cardiomyocyte) BMPs BMP-RII Progenitor cell OFF OFF Heart muscle cell (Cardiomyocyte) ON Myosin light chain 2 gene 10 Markers of the cardiomyocyte progenitor lineage Cardiomyocyte lineage Bry Nkx2.5+ / Isl-1+ Cardiogenic mesodermal progenitor Nkx2.5+ / Isl-1+/ GATA4+/ Flk-1+ Multipotent cardiovascular progenitor (2nd heart field) Lecture notes (hyperlink) Activity notes (hyperlink) More links… Differentiation Nkx2.5+ / c-Kit+ 1st heart field progenitor Mesodermal Precursor References Left ventricle Nkx2.5+ / Isl-1+/ GATA4+ Cardiac progenitor Remainder of heart Isl-1+/ Flk-1+ Vascular progenitor Blood vessels and blood cells 11 Markers of the mature cardiomyocyte lineage References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Sources of cells for cardiomyocyte replacement therapies: the dish Directed differentiation Transplantation & engraftment References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Stable formation of mural grafts Sources of cells for cardiomyocyte replacement therapies: the heart References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Directed differentiation protocol for mouse ES cells into cardiomyocytes - LIF + serum mES cells - LIF + serum Lecture notes (hyperlink) Activity notes (hyperlink) More links… - LIF + serum Day 5-7 Nkx2.5+ progenitors Day 2 EBs References Day 7-10 beating cardiomyocytes Wnt/BMP/Activin and serum are often used to induce cardiomyocyte precursors ES cells 2 - 4 days 4 days 2 days EBs Nkx2.5+ cardiomyogenic progenitors Beating cardiomyocytes 15 Directed differentiation of mouse ES cells produces vascular and cardiac lineages Isl-1+/ Flk-1+ Vascular progenitors Fraction of Flk-1+ progenitors at day 3-5 % References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Nkx2.5+ / Isl-1+/ Gata4+ Cardiac progenitors Fraction of Nkx2.5+ progenitors at day 5 % % 3.84 % Nkx2.5::eGFP 16 Isolated heart muscle cells beat spontaneously References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Directed differentiation of ES cells creates specialized beating heart cells in vitro. • How do heart muscle cells (or cardiomyocytes) beat in a dish? 1. Express cardiac troponin T (cTnT). 2. Some cells become node cells (HCN4+) that beat spontaneously. 3. These cells express Ca++ ion channels that confer contractile properties. 17 Directed differentiation protocol for human ES cells to cardiomyocytes - bFGF + 20% FBS - bFGF + 20% FBS BMP-4 hES cells Lecture notes (hyperlink) Activity notes (hyperlink) More links… - bFGF + 20% FBS Day 7 Nkx2.5+ progenitors Day 4 hEBs References Day 11 beating cardiomyocytes Wnt/BMP/Activin and serum are often used to induce cardiomyocyte precursors 4 days hES cells 3 days 20% FBS BMP-4 12.5-25 ng/ml 4 days 20% FBS Human embryoid bodies 20% FBS Nkx2.5+ cardiomyogenic progenitors Beating cardiomyocytes 18 Rodent model of heart attack to test stem cell-based repair of heart tissue • References Lecture notes (hyperlink) Activity notes (hyperlink) More links… Sources for stem/progenitor cells that might be used to repair tissue following a heart attack: – – – – – – ES cells Cardiac stem cells Muscle stem cells Mesenchymal cells from adult bone marrow Endothelial progenitors Umbilical cord blood cells • Rats, mice and pigs are commonly used to test whether cells can repair hearts • Conditions mimicking those of a heart attack are achieved in animal models by ligation of the coronary artery Stem cells directly or indirectly repair damaged heart tissue References Lecture notes (hyperlink) Activity notes (hyperlink) More links… 20 Summary References Lecture notes (hyperlink) Activity notes (hyperlink) More links… • Directed differentiation of ES cells into heart muscle cells is the production of beating cardiomyocytes in a dish using defined factors. • The factors used are crucial for generating these muscle cells during normal embryonic development. • Formation of the mammalian heart involves complex tissue rearrangements as well as precise cell differentiation. • Similar experimental conditions lead to directed differentiation of cardiomyocytes from either mouse or human ES cells. • A wide variety of sources may provide cells that are effective for repairing heart muscle tissue after is has been damaged due to a heart attack. 21