|Ph.D Student||Oshra Sedan|
|Subject||Functional and Developemental Properties of Human Embryonic|
and Human Induced Pluripotent Stem Cells-Derived
|Department||Department of Medicine||Supervisors||Professor Emeritus Binah Ofer|
|Professor Emeritus Itskovitz Joseph|
|Full Thesis text|
Based on previous findings that in human embryonic stem cells-derived cardiomyocytes (hESC-CM), the sarcoplasmic reticulum (SR) Ca2+-induced release of Ca2+ (CIRC) machinery is either absent or immature, we hypothesized that hESC-CM contain functional 1,4,5-IP3 operated intracellular Ca2+ ([Ca2+]i) stores that can be mobilized by the respective stimuli. Thus, we investigated the effects of angiotensin II (AT-II) and endothelin 1 (ET-1) which activate the 1,4,5-IP3 pathway, on [Ca2+]i transients and contractions in contracting clusters of hESC-CM. Our major findings are that in electrically paced hESC-CM, AT-II (10-9 - 10-7 M) increased the amplitude of [Ca2+]i transients and the maximal rates of [Ca2+]i rise and relaxation. Both AT-II and ET-1 (10-9 - 10-7 M) increased contraction amplitude, maximal contraction and relaxation rates and caused arrhythmias. The involvement of 1,4,5-IP3-dependent intracellular Ca2+ release in AT-II - induced effects was supported by the findings that: (1) hESC-CM express AT-II type 1 and IP3 type I and II receptors determined by fluorescence immunostaining. (2) The effects of AT-II were blocked by 2-aminoethoxyphenyl borate (2-APB, a 1,4,5-IP3 receptor blocker) and U73122 (a phosopholipase C blocker). In conclusion, our findings demonstrate that hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP3-operated releasable Ca2+ stores.
In view of the therapeutic potential of the new emerging source of cardiomyocytes, which are derived from induced pluripotent stem cells (iPSC-CM), we investigated their SR CIRC machinery. The two iPSC clones investigated were generated through infection of human foreskin fibroblasts (HFF) cells with retroviruses containing the four human genes: OCT4, Sox2, Klf4 and C-Myc. Molecular analysis of the iPSC colonies demonstrated that these cells have an ES-like molecular profile in addition to morphology. Cardiogenic molecular profile of the iPSC-CM clones investigated came out positive. In order to investigate excitation-contraction (E-C) coupling of iPSC-CM, we measured the [Ca2+]i transients and contractions in paced whole and dissociated clusters of iPSC-CM while exposing them to various protocols and chemicals. Our major findings are: (a) Isoproterenol exerts both inotropic and lusitropic effects in iPSC-CM; (b) IPSC-CM demonstrate negative force-frequency relations and exhibit a modest post-rest potentiation; (c) Ryanodine decreases contraction amplitude and maximal contraction and relaxation rates in iPSC-CM; (d) Brief exposure to caffeine elevates diastolic [Ca2+]i in iPSC-CM. (e) IPSC-CM express ryanodine receptor and calsequestrin. In conclusion, these findings demonstrate that in the iPSC-CM clones investigated, the SR is functional and contributes to contraction.