Pluripotent stem cell and T cell differentiated therefrom and application thereof

A method for directed differentiation of T cells using pluripotent stem cell, comprising the steps of:

• (1) integrating an expression vector wherein a nucleic acid sequence encoding Runx1 and a nucleic acid sequence encoding Hoxa9 are linked in tandem into pluripotent stem cells at a Rosa26 site by gene recombination and performing resistance screening with Hygromycin B;
• (2) culturing the pluripotent stem cells of step (1) with D0 medium, D2.5 medium, D3 medium, D4 medium, D5 medium, D6 medium and D7 medium sequentially, and directionally differentiating the same into hematopoietic stem cell precursors on day 11;
• (3) co-culturing the hematopoietic stem cell precursors of step (2) with OP9-DL1 cells and inducing with Doxycycline for at least 10 days to induce expression of Runx1 and Hoxa9 to obtain T-lineage progenitor cells;
• and
• (4) inducing the T-lineage progenitor cells of step (3) to differentiate into T cells, which are TCR β cells and/or TCR γ/δ cells,
• wherein:
• the D0 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4;
• the D2.5 medium is a basic differentiation medium containing 3-8 ng/mL activin A and 3-8 ng/mL basic fibroblast growth factor;
• the D3 medium is a basic differentiation medium containing 3-8 ng/mL Activin A, 3-8 ng/mL bone morphogenetic protein 4 and 3-8 ng/mL vascular endothelial growth factor;
• the D4 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4 and 3-8 ng/mL vascular endothelial growth factor;
• the D5 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4, 3-8 ng/mL vascular endothelial growth factor, 10-30 ng/mL recombinant mouse interleukin 3, 10-30 ng/mL recombinant mouse interleukin 6, 10-30 ng/mL recombinant mouse stem cell factor, 10-30 ng/mL recombinant human thrombopoietin and 10-30 ng/mL human Fms-associated tyrosine kinase 3 ligand;
• the D6 medium is a basic differentiation medium containing 3-8 ng/mL bone morphogenetic protein 4, 3-8 ng/mL vascular endothelial growth factor, 10-30 ng/mL recombinant mouse interleukin 3, 10-30 ng/mL recombinant mouse interleukin 6, 10-30 ng/mL recombinant mouse stem cell factor, 10-30 ng/mL recombinant human thrombopoietin, and 10-30 ng/mL human Fms-associated tyrosine kinase 3 ligand and 1-2 μg/mL Doxycycline;
• the D7 medium is a basic differentiation medium containing 10-30 ng/mL recombinant mouse interleukin 3, 10-30 ng/mL recombinant mouse interleukin 6, 10-30 ng/mL recombinant mouse stem cell factor, 10-30 ng/mL recombinant human thrombopoietin, and 10-30 ng/mL human Fms-associated tyrosine kinase 3 ligand and 1-2 μg/mL Doxycycline;
• and
• the basic differentiation medium is IMDM medium comprising 10-20% fetal calf serum, 180-220 μg/mL iron-saturated transferrin, 4.5×10−4 M thioglycerol, 2 mM L-alanyl-L-glutamine dipeptide, and 0.5 mM ascorbic acid.