This is comprehensive protocol facilities, raw boosts generation, and use pouncing of patient specific, cultures from peripheral blood mononuclear cells. This tactics are reproducible and easy to follow, specially for beginners. And don't require, special expertize in stem cell ballads, or cardiovascular medicine.
Our protocol can be used to generate patient specific cardiomyocytes from a single blood draw that can be used for modeling cardiovascular diseases and testing the cardiac toxicity of novel drugs. This protocol requires a long time commitment to stem cell reprogramming maintenance and directed differentiation. However, you really appreciate your endeavors.
When you see beating cardiomyocytes in the dish. When the human iPSC colonies reach over 90%confluency, rinse each well with three milliliters of DPBS before treating the cells with one milliliter of 0.5 millimolar EDTA in DPBS per well at 37 degrees Celsius. After five to eight minutes, add one milliliter of iPSC passaging medium to each well and manually dislodge the cells.
Transfer 600 to 900 microliters of the cells to individual Wells have a basement membrane coated six well plate for an overnight incubation at 37 degrees Celsius and 5%carbon dioxide. Then refresh the cultures with two milliliters of complete E8 medium every day for three to four days. When the culture's reached confluency, replace the medium with two milliliters of cardiomyocyte differentiation medium, two per well.
On day two, replace the supernatants with two milliliters of cardiomyocyte differentiation medium one. On day three, replace the supernatants with two milliliters of cardiomyocyte differentiation medium three. On day five.
Replace the supernatant with two milliliters of cardiomyocyte differentiation medium one. On day seven through 10, replace the supernatant with two milliliters of cardiomyocyte differentiation medium four. On days 11 and 13.
When contracting cells are observed, replace the supernatant with two milliliters of cardiomyocyte differentiation medium five. On days 15, 17, 19 and 21. Replace the supernatant with two milliliters of cardiomyocyte differentiation medium four.
After pre-culture with complete blood medium per seven days, PBMC's become large with visible nuclei and cytoplasm indicating that they are ready for transfection with Sendai Virus reprogramming factors. Upon introduction to complete E8 medium. The completely reprogrammed cells will attach and start forming colonies.
After four to five passages, highly pure iPSC colonies will form from the reprogrammed cells with very few differentiated cells observed. At this stage, Most of the cells are OCT4 and NANOG positive indicative of their pluripoteny. Beating cardiomyocytes are usually observed after 12 days of differentiation.
After glucose starvation, and replating iPSC cardiomyocytes exhibits, spontaneous beating, and an aligned sarcomere structure with intercalated cardiac troponin T and alpha actinin expression. The colonies retain their purity as evidenced by their Troponin T expression. Although iPSC cardiomyocytes are relatively immature compared to adult cardiomyocytes.
They demonstrate ventricular and atrial like action potentials as measured by wholesale patch, clamp. Ventricular cardiomyocytes express myosin light chain two, whereas atrial iPSC cardiomyocytes are marked by NR2F2 expression. Wnt activation stimulate cell division and promotes the expression of cell cycle regulators.
Interestingly, Wnt activation also induces the robust proliferation of early iPSC cardiomyocytes for two passages compared to controls, although this proliferative ability diminishes over time. Make sure that the PBMS are enlarged with the clear nuclear and cytoplasm before the iPSC reprogramming vectors are introduced as the condition of the PBMs is critical for iPSC reprogramming. Patient specific cardiomyocytes are valuable for modeling cardiovascular disease in vitro and for providing a ton of donor cells for cardiac stem cell therapy.
