Heart disease research is often performed in small and mammalians such as Rodents. However, the physiology of the human heart differs significantly from that of the Rodent heart. Our protocol uses cardiomyocytes differentiated from human iPS cells to model Ischemic heart disease and to quantify the damage and functional impairment of Ischemic cardiomyocytes.
This protocol may provide a convenient method for personalized drug screening using iPS cells derived from individual patients, the appropriate handling of the iPS cells, such as in using fresh medium, and changing the medium slowly and punctually is critical to ensuring a successful differentiation into functional cardiomyocytes. Demonstrating the procedure with Yun Liu, Yin Liang and Mengxue Wang will be Chen Wang, a PhD student from a laboratory. To induce Human induced Pluripotent stem cell differentiation into cardiac cells, first coat the wells of a 96 well culture plate with 100 microliters of 1.675 micrograms per milliliter of laminin in PBS.
After a 30 minute incubation at 37 degrees Celsius, see three times 10 to the fourth stem cells in 200 microliters of IPS maintenance medium supplemented with 10 micromolar Y27632 into each well, and return the plate to the Cell Culture Incubator. After 24 hours, replace the super means with 200 microliters of IPS growth medium per well, and return the plate to the Cell Culture Incubator for an additional two to three days. When the cells reach 70 to 80%confluency.
Replace the medium in each well with 200 microliters of pre warmed differentiation medium A and return the plate to the Cell Culture Incubator for another 48 hours. At the end of the incubation slowly replaced the supernatant in each well with 200 microliters of pre warmed differentiation medium B and return the plate to the Cell Culture Incubator. After two days, replace the supernatants with 200 microliters of pre warmed cardiomyocyte maintenance medium per well and returned the plate to the Cell Culture Incubator for up to 30 days.
To assess the contractility of the cardiomyocytes, install the particle image velocimetry image j plugin and use a phase contrast microscope and the forks objective to record video images of the cells at approximately 20 frames per second for about 10 seconds. Then save the video file as analyzed our API and create a folder structure as indicated. To analyze discrete two dimensional vector fields of cellular displacement, launch VG and select plugins, macros and edit to open vector analysis.
ijm, then click run the analysis will be performed automatically. For Ischemic treatment, replace the supernatant in each well with 200 microliters of dmem without glucose and serum, then place the plate in a Hypoxic Chamber then infuse nitrogen gas into the chamber, maintaining the internal oxygen concentration at 2%and the carbon dioxide concentration at 5%for 24 hours. Successfully differentiated cells, demonstrates spontaneous contraction as observed under the microscope with 50%of the culture wells typically exhibiting spontaneous contraction in less than 20 days.
Cardiac marker protein can also be used to confirm a successful differentiation Cells from the ischemic group typically exhibit a lower viability in MTT assays, and a lower contractility than those from the normoxic control group. The ratio of propidium iodide positive cells is also higher in the Ischemic group, than in the control group, indicating a higher incidence of cellular damage. It's important that you accurately locate the regions of interest in the capture plate to allow comparison of the contractility of the cardiomyocytes.
This technique can be used for drug screening, using patient derived iPS cells. It also provides a unique platform for further elucidating the mechanisms underlying Ischemic heart diseases
