STEMdiff T Cell Kit

STEMCELL Technologies has developed a complete kit for the differentiation of human pluripotent stem cells (hPSCs), including embryonic stem (ES) cells and induced pluripotent stem (iPS) cells, into T cells in serum-free culture conditions and without the use of stromal cells. The kit supports a four-stage protocol spanning approximately 40 days of culture, comprising STEMdiff Hematopoietic - EB reagents paired with a StemSpan T Cell Generation Kit.

In the first two stages, hPSCs are differentiated into CD34+ hematopoietic progenitor cells over 12 days. hPSCs are first seeded into AggreWell plates in EB Formation Medium (EB Medium A supplemented with 10 μM Y-27632) to form 600-cell aggregates and cultured for 3 days to induce mesoderm specification. The medium is then changed to EB Medium B for an additional 9 days, after which CD34+ cells are enriched using EasySep positive selection. Across multiple ES and iPS cell lines, the average frequency of viable CD34+ cells on day 12 ranged between 31% and 51%, with yields of 4.0 x 10^5 to 6.7 x 10^5 CD34+ cells per well of a 6-well AggreWell400 plate. In the third stage, CD34+ cells are cultured for 14 days in StemSpan Lymphoid Progenitor Expansion Medium on plates coated with StemSpan Lymphoid Differentiation Coating Material, generating CD7+CD5+ lymphoid progenitor cells at an average frequency of 38% to 54% and yields of 12 to 40 cells per input CD34+ cell.

In the fourth stage, lymphoid progenitor cells are reseeded in StemSpan T Cell Progenitor Maturation Medium for 14 additional days to generate CD4+CD8+ double-positive (DP) T cells, harvested after a total of 28 days from the lymphoid stage. On average, approximately 60 CD4+CD8+ DP T cells can be generated per CD34+ cell, with the average frequency of viable DP T cells on day 28 ranging between 23% and 58%, yields of 12 to 108 DP T cells per input CD34+ cell, and CD3+TCRαβ+ expression on DP T cells between 11% and 38%. An optional 7-day extension allows further maturation of DP T cells into CD8+ single-positive T cells, with the average frequency of CD3+TCRαβ+CD4-CD8+ cells ranging between 3% and 28%.