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allogenic ventricular cardiomyocyte spheroids derived from human induced pluripotent stem cells (iPSC). The iPSC cell bank was established from peripheral blood mononuclear cells (PBMCs) isolated from a healthy human donor by episomal reprogramming with plasmid vectors expressing octamer-binding transcription factor 3/4 (OCT-3/4), SOX2, Krueppel-like factor 4 (KLF4), L-MYC, LIN28, tumor suppressor p53 (Tp53), and Epstein- Barr nuclear antigen 1 (EBNA1). The cells were initially cultivated with a feeder-free stem cell culture media with interleukin-6 (IL-6), stem cell factor, thrombopoietin, Flt-3 ligand, IL-3, and granulocyte colony- stimulating factor. Following induction, the cells were seeded on plates coated with a truncated form of laminin in the same media. Clones were chosen based on the best efficiency for subsequent cardiac differentiation.
The iPSCs were differentiated into cardiomyocytes (CMs) in media containing a defined serum-free supplement lacking insulin, initially supplemented with bone morphogenetic protein 4 (BMP4) and CHIR-99021 (glycogen synthase kinase 3 (GSK-3) inhibitor), and subsequently supplemented with a tankyrase inhibitor. The cells were further culture expanded in medium containing foetal bovine serum. Undifferentiated iPSC were removed using an adhesive culture in glucose- and glutamine- depleted media supplemented with lactic acid. The harvested CMs (>98%) were subsequently cultured on plates to form cardiomyocyte spheroids.
The final cells are troponin T and myosin light chain 2 ventricular isoform (MLC2v) positive, negative for the undifferentiated iPSC marker OCT-3/4 and secrete vascular endothelial growth factor (VEGF) and other growth factors.

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