|M.Sc Student||Bookay Haddas|
|Subject||Sorting of Cardiomyocytes Derived from Human Embryonic Stem|
Cells Using Transgenic Markers
|Department||Department of Medicine||Supervisor||Professor Emeritus Joseph Itskovitz|
|Full Thesis text|
Significant progress has been made in characterizing cardiomyocytes derived from human embryonic stem cells (hESCs), which hold great promise as cell therapy for the treatment of heart diseases. However, several obstacles must be overcome prior to their application, one of which is the difficulty of obtaining pure populations of single functional transplantable cell types. This is an essential prerequisite so as to avoid the risk of teratoma formation and further impairment of tissue function, which may result from the implantation of undifferentiated hESCs or of undesired differentiated cell types. Methods such as fluorescence-activated cell sorting (FACS) or magnetic cell sorting (MACS) allow the purification of the desired cell population, but are dependent on the expression of a specific recognizable surface marker. To be fully effective, an endogenous marker needs to be absolutely cell-type specific. However, an effective endogenous marker for cardiomyocytes is yet to be revealed. Therefore, sorting methods have to rely on the introduction of a marker gene under the control of a lineage-specific promoter. In the current study we describe the establishment of transgenic hESC lines that allow identification and selection of differentiating human cardiomyocytes. This approach is based on using several cardiac-specific promoters; one of them is the atrial natriuretic factor (ANF) promoter that derives the expression of a selectable reporter gene (GFP). Another two promoters are ventricular forms of myosin light chain (MLC2v) and NKX2.5 that drive the expression of a selectable reporter gene (CD4).
The transgenic hESC lines that contain the ANF GFP vector maintain normal karyotype, and their ability to form teratomas in immunosuppressed mice, and on their ability to differentiate into spontaneously beating cardiomyocytes when compared to the wild-type lines. Functional studies of cardiomyocytes derived from the transgenic hESC lines showed normal behavior compared to the wild-type lines.
According to FACS analysis, 4% of the differentiating cells expressed GFP, and this portion of the population could be sorted by FACS Aria. Thus we conclude that pure cardiomyocytes could be obtained by sorting of these cells from hESCs using a transgenic marker.