In vitro generation of thymic organoid from human pluripotent stem cells
Inventors
Vizcardo, Raul E. • Restifo, Nicholas P.
Assignees
US Department of Health and Human Services
Publication Number
US-11898166-B2
Publication Date
2024-02-13
Expiration Date
2038-09-19
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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.
Core Innovation
The invention provides methods of preparing a thymic organoid in vitro. This method involves differentiating pluripotent stem cells stepwise into definitive endodermal cells, third pharyngeal pouch endodermal (PPE) cells, thymic epithelial progenitor-like cells (TEPLCs), thymic epithelial progenitor cells (TEPCs), and finally thymic epithelial cells (TECs) in a three-dimensional culture. The thymic organoid expresses markers such as β5t, DLL4, and interleukin 7 and mimics the physiology and function of a human thymus. Notably, the method does not require co-culturing with mesenchyme or stromal cells and avoids enzymatic dissociation or passaging of the cells during differentiation.
The background of the invention identifies that adoptive cell therapy (ACT) using T cell lineage cells can be clinically beneficial, for example, in cancer treatment. However, producing cells of the T cell lineage in vitro or ex vivo is challenging. Thus, there is a need for improved materials and methods to prepare such cells for ACT. This invention addresses that need by providing a method for producing thymic organoids that can generate functional thymic emigrant cells, including T cells.
Advantageous features of the invention include producing human induced pluripotent stem cell (hiPSC) derived thymic organoids capable of facilitating T cell lineage differentiation and positive selection as found in the human thymus. The organoids form three-dimensional architectures and express key markers such as keratin 5, keratin 8, FOXN1, β5t, DLL4, and interleukin 7. The method permits generation of these organoids without mesenchymal or stromal co-culture, passaging, or dissociation steps, thereby potentially improving the efficiency and reproducibility of thymic organoid generation.
Claims Coverage
The patent includes one independent claim defining a method of preparing a thymic organoid in vitro and another independent claim defining a method of preparing thymic emigrant cells in vitro using the organoid.
Stepwise differentiation of pluripotent stem cells without enzymatic dissociation
Differentiating pluripotent stem cells into endodermal cells, then third pharyngeal pouch endodermal cells, then thymic epithelial progenitor-like cells (FOXN1 negative) in a two-dimensional culture, followed by forming three-dimensional spheroids and differentiating into thymic epithelial progenitor cells (FOXN1 positive), all without enzymatic dissociation or passaging, and replating on Day 3 of culture.
Use of mesenchymal stem cell factors during 3D differentiation
Differentiating thymic epithelial progenitor-like cells into thymic epithelial progenitor cells in three-dimensional culture in the presence of one or more mesenchymal stem cell (MSC) factors, such as FGF7, BMP4, Wnt3a, FGF10, IGF-1, FGF8, TGFβ inhibitor, and cyclopamine.
Differentiation into thymic epithelial cells in presence of BMP4
Differentiating thymic epithelial progenitor cells into thymic epithelial cells (TECs) in three-dimensional culture in the presence of bone morphogenetic protein 4 (BMP4), optionally combined with Wnt3a and fibroblast growth factor 7 (FGF7).
Formation of thymic organoid expressing key markers
Forming the thymic epithelial cells into a thymic organoid in three-dimensional culture that expresses keratin 5, keratin 8, FOXN1 protein, β5t, delta-like Notch ligand 4 (DLL4), and interleukin 7 without co-culturing with mesenchyme or stromal cells.
Method of preparing thymic emigrant cells using the thymic organoid
Migrating progenitor cells into the thymic organoid, allowing cells to egress from the organoid as thymic emigrant cells, and isolating these thymic emigrant cells from the organoid.
The claims define a novel method of producing thymic organoids from pluripotent stem cells without enzymatic dissociation or mesenchymal co-culture, employing MSC factors to promote differentiation, and methods of generating thymic emigrant cells from these organoids. The organoids express specific thymic markers and can be used to produce functional T cell lineage cells.
Stated Advantages
The inventive methods allow production of human iPSC-derived thymic organoids that mimic the physiology and function of a human thymus.
The generated organoids can produce cells of the T cell lineage suitable for adoptive cell therapy.
The method obviates the need for co-culturing with mesenchymal or stromal cells, enzymatic dissociation, or passaging, simplifying and potentially improving reproducibility.
The thymic organoid expresses key markers involved in T cell development, such as keratin 5, keratin 8, FOXN1, β5t, DLL4, and interleukin 7.
Documented Applications
Generating thymic emigrant cells for adoptive cell therapy in conditions such as cancer.
Producing autologous T cells for patients with rare blood types for blood banking and treatment of anemias and cytopenias.
Treatment or prevention of cancers, including a wide variety of solid and hematologic malignancies.
Treatment or prevention of immunodeficiencies, including primary and secondary immunodeficiencies such as SCID and AIDS.
Treatment or prevention of autoimmune conditions like rheumatoid arthritis, lupus, type 1 diabetes, multiple sclerosis, and others.
Treatment or prevention of infections including viral, bacterial, fungal, and protozoan infections.
Treatment or prevention of blood conditions such as anemia, leukopenia, neutropenia, bleeding disorders, and blood clots.
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