In vitro generation of thymic organoid from human pluripotent stem cells

A method of preparing a thymic organoid in vitro, the method comprising:

• (i) differentiating pluripotent stem cells into endodermal cells in vitro without enzymatically disassociating the pluripotent stem cells or the endodermal cells, wherein (i) comprises replating the pluripotent stem cells on Day 3 of culture;
• (ii) differentiating the endodermal cells into third pharyngeal pouch endodermal (PPE) cells in vitro without enzymatically dissociating the endodermal cells or the PPE cells;
• (iii) differentiating the third PPE cells into thymic epithelial progenitor-like cells (TEPLCs) in an in vitro, two-dimensional culture without enzymatically dissociating the third PPE cells or the TEPLCs, wherein the TEPLCs are FOXN1 protein negative;
• (iv) forming a three-dimensional spheroid by culturing the TEPLCs in an in vitro, three-dimensional culture and differentiating the TEPLCs into thymic epithelial progenitor cells (TEPCs) in an in vitro, three-dimensional culture without enzymatically dissociating the TEPLCs or the TEPCs, wherein the TEPCs are FOXN1 protein positive, wherein (iv) comprises differentiating the TEPLCs into TEPCs in the presence of one or more Mesenchymal Stem Cell (MSC) factors;
• (v) differentiating the TEPCs into thymic epithelial cells (TECs) in the in vitro, three-dimensional culture in the presence of bone morphogenic protein 4 (BMP4);
• and
• (vi) forming the TECs into a thymic organoid in the in vitro, three-dimensional culture,
• wherein the thymic organoid expresses keratin 5, keratin 8, FOXN1 protein, β5t, delta like Notch ligand 4 (DLL4), and interleukin 7, and
• wherein the method does not comprise co-culturing the cells with mesenchyme or stromal cells.