# US 11,898,166 B2 · In vitro generation of thymic organoid from human pluripotent stem cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,898,166 B2 - Document ID: US11898166B2 - Status: Granted - Kind: B2 - Application number: 16/648,008 - Filed: 2018-09-19 - Published: 2020-08-27 - Granted: 2024-02-13 - Earliest claimed: 2017-09-20 - Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services - Applicants: The United States of America, as represented by the Secretary, Department of Health and Human Services - Inventors: Raul E. Vizcardo; Nicholas P. Restifo - Web page: https://patentgrep.com/patent/US11898166B2 ## CPC - C12N 5/065: {Thymocytes} - A61K 35/26: Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes - C12N 5/0062: {General methods for three-dimensional culture} - C12N 2500/98: Xeno-free medium and culture conditions - C12N 2501/105: Insulin-like growth factors [IGF] - C12N 2501/117: Keratinocyte growth factors (KGF-1, i.e. FGF-7; KGF-2, i.e. FGF-12) - C12N 2501/15: Transforming growth factor beta (TGF-β) - C12N 2501/41: Hedgehog proteins; Cyclopamine (inhibitor) - C12N 2501/415: Wnt; Frizzeled - C12N 2506/45: from artificially induced pluripotent stem cells - C12N 2513/00: 3D culture - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## Abstract Disclosed are methods of preparing thymic organoids according to embodiments of the invention. Also disclosed are methods of preparing thymic emigrant cells in vitro, according to embodiments of the invention. Also disclosed are methods of treating or preventing a condition in a mammal, e g., cancer. ## Claims ### Claim 1 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. ### Claim 2 (depends on 1) The method according to claim 1, wherein (v) comprises differentiating the TEPCs into TECs in the in vitro, three-dimensional culture in the presence of one or both of Protein Wnt-3a (Wnt3a) and fibroblast growth factor 7 (FGF7). ### Claim 3 (depends on 1) The method of claim 1, wherein the method comprises culturing the pluripotent stem cells in mammalian cellular basement matrix for at least one day prior to (i). ### Claim 4 (depends on 1) The method of claim 1, wherein the TEPLCs are keratin 5+/keratin 8+. ### Claim 5 (depends on 1) The method of claim 1, wherein the method does not comprise passaging the cells. ### Claim 6 (depends on 1) The method of claim 1, wherein the thymic organoid comprises keratin 5high/keratin 8low cells. ### Claim 7 (depends on 1) The method of claim 1, wherein the thymic organoid comprises keratin 5low/keratin 8high cells. ### Claim 8 (depends on 1) The method of claim 1, wherein any one or more of (i)-(vi) is/are carried out in xeno-free medium. ### Claim 9 (depends on 1) The method of claim 1, further comprising culturing the thymic organoid to overexpress one or more autologous proteins which promote T cell differentiation. ### Claim 10 (depends on 9) The method of claim 9, wherein the one or more autologous proteins which promote T cell differentiation comprise one or more of DLL4 and IL-7. ### Claim 11 (depends on 1) The method of claim 1, wherein the one or more MSC factors comprise one or more of FGF7, BMP4, Wnt3a, fibroblast growth factor 10 (FGF10), insulin-like growth factor-1 (IGF-1), fibroblast growth factor 8 (FGF8), transforming growth factor β inhibitor (TGFβinh), and cyclopamine. ### Claim 12 (depends on 11) The method of claim 11, wherein the one or more of the MSC factors comprise FGF10, IGF-1, FGF8, TGFβinh, and cyclopamine. ### Claim 13 (depends on 1) A method of preparing thymic emigrant cells in vitro, the method comprising: preparing a thymic organoid in vitro by the method of claim 1; egressing the cells from the thymic organoid, wherein the cells egressing from the thymic organoid are thymic emigrant cells; and isolating the thymic emigrant cells from the thymic organoid.