Generating Single Positive CD8 T Cells from Induced Pluripotent Stem Cells
Transcript
Culture induced pluripotent stem cells on a feeder layer of stromal cells on a gelatin matrix.
The feeder layer mimics the natural extracellular environment, guiding stem cell differentiation into dome-shaped organoids containing hematopoietic progenitor cells.
Add enzymes to dissociate the co-cultured cells.
Transfer the cells to a fresh gelatin-coated dish for stromal cell adhesion.
Collect the non-adhered hematopoietic progenitor cells in a differentiation medium.
Culture them on a fresh feeder layer to promote progenitor cell differentiation into double-positive T cells with CD4 and CD8 receptors and double-negative T cells lacking both receptors.
Transfer the cells to a tube. Incubate them with magnetic beads carrying CD4-specific antibodies that selectively tag the double-positive T cells.
Use a magnetic separator to isolate the double-positive cells and culture them on a fresh feeder layer.
Supplement with a cytokine and antibody mix to stimulate the cells, inducing CD4 receptor downregulation and promoting their differentiation into CD8-single positive cells.
