טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentBen-Ari Meital
SubjectDevelopmental Changes in Pacemaker Properties in Human
Induced Pluripotent Stem Cells (iPSC)-Derived
Cardiomyocytes (iPSC-CM)
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Ofer Binah
Full Thesis textFull thesis text - English Version


Abstract

Human[B1] [B2]  induced Pluripotent Stem Cells (iPSC) have an unlimited proliferation capacity at the pluripotent state and the ability to differentiate into any functional cell type in vitro, including bona fide cardiomyocytes (iPSC-derived cardiomyocytes, iPSC-CM). iPSC-CM display inherent automaticity underscoring its potential to serve as a biological pacemaker and in replacement therapies. Comprehnensive data regarding iPSC-CM functional characteristics is required for future clinical use. In the present study, iPSC were generated from hair keratinocytes plucked form healthy volunteers and than spontanously differentiated into cardiomyoctes. We investigated in iPSC-CM: (1) Developmental changes in electrophysiological characteristics (2) Pacemaker mechanism and currents (3) and Heart/Beat Rate Variability (HRV/BRV). The hypotheses and aims were:

          Hypothesis 1: At the young developmental stage there is a continuous spectrum (i.e., lack of distinct groups) of action potential phenotypes (ranging from ventricular-like to nodal-like), which as maturation progresses segregates into the 3 classic phenotypes. Aim 1: Investigate the electrophysiological properties and repertoire of action potential phenotypes of iPSC-CM at different ages. Under this aim, the major findings were: (1) FACS analysis of 30 and 60-day old cultures showed that an iPSC-CM population shifts from nodal into atrial/ventricular phenotype, while including significant transitional populations. (2) The action potential population consist of a spectrum of morphologies and not 3 distinct phenotypes. (3) Culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of atrial phenotype.

           Hypothesis 2: Pacemaker currents contributing to automaticity in iPSC-CM are age-dependent and phenotype-dependent. Aim 2: Investigate If, ICa,L and          IP3-induced intracellular Ca2 cycling in iPSC-CM at different ages and action potential phenotypes. The major findings were: (1) If, ICa,L and IP3-induced intracellular Ca2 cycling generates automaticity in iPSC-CM throughout 7-to-95 days of maturation. (2) Sub-populations of iPSC-CM exhibit different pacing mechanism that are either If, ICa,L or IP3-dependent. (3) An increase in If density occurs in proceeding from the young (predominantly nodal) to the older (predominantly ventricular) population. (4) If contributes to automaticity of nodal-, ventricular- and atrial-like cardiomyocytes. (5) IP3-induced Ca2 cycling generates automaticity in atrial-like cardiomyocytes but not in ventricular-like cells.

           Hypothesis 3a: Cellular BRV is the source of HRV dynamics. Aim 3a: Investigate HRV/BRV in three-levels of interaction among different cardiomyocyte entities: (1) the single pacemaker cell, (2) a network of electrically coupled pacemaker cells, and (3) in situ SAN. Hypothesis 3b: Disrupting intracellular Ca2 handling will alter BRV properties. Aim 3b: Investigate the contribution of intracellular Ca2 handling to BRV and fractal behavior of iPSC-CM. The main findings were: (1) BRV, self-similarity and power law behavior are shared at all levels. (2) BRV is greater in the single cell than the network and in situ heart. (3) BRV was more prominent in nodal than ventricular cardiomyocytes. (4) Disrupting intracellular Ca2 handling markedly augments BRV magnitude, suggesting that intracellular mechanisms contribute to BRV/HRV and the fractal behavior of the heart rhythm.

Collectively, our findings support the compatibility of iPSC-CM as biological pacemakers, as they not only fire spontaneously, but also present inherent dynamic pacemaker properties.






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