Methods for directed differentiation of pluripotent stem cells to immune cells
Inventors
Vodyanyk, Maksym A. • Zhang, Xin • BRANDL, Andrew J. • Rajesh, Deepika • Swanson, Bradley • MUNN, Christie • BURTON, Sarah A. • Wang, Wen Bo
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10947502-B2
Publication Date
2021-03-16
Expiration Date
Abstract
Provided herein are methods for the efficient in vitro differentiation of somatic cell-derived pluripotent stem cells to hematopoietic precursor cells, and the further differentiation of the hematopoietic precursor cells into immune cells of various myeloid or lymphoid lineages, particularly T cells, NK cells, and dendritic cells. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the hematopoietic precursor cells.
Core Innovation
The document describes an in vitro method of producing immune cells by reprogramming somatic cells into induced pluripotent stem cells (iPSCs) and differentiating the iPSCs through defined media stages to hematopoietic precursor cells (HPCs). HPC differentiation is promoted by sequential culturing in defined media that include a GSK inhibitor stage followed by stages containing BMP4, FGF2, and VEGF for mesoderm induction, and then IL-3 and Flt3 ligand to proliferate and differentiate cells into HPCs. The resulting HPCs exhibit increased expression of one or more of CD7, DLL4, CD144, and CD235 compared to cells produced in the mesoderm induction stage.
The document further describes culturing the HPCs under conditions to promote immune cell differentiation, thereby producing immune cells. It includes lymphoid lineages, including T cells, NK cells, and T/NK cells, and myeloid lineages, including dendritic cells, using defined cytokine and growth factor conditions and Notch ligand-related culture conditions such as DLL4 or DLL4:Fc, with hypoxia also described for lymphoid differentiation.
The described approach emphasizes feeder-free and, in some embodiments, serum-free differentiation and the use of defined culture components to avoid animal products and feeder layers. It also describes isolating or enriching HPCs or lymphoid-primed progenitor fractions based on marker expression, including magnetic sorting strategies for enriched progenitor fractions.
The document states that the approach can generate large numbers of immune cells and preserves TCR V(D)J rearrangements, and it positions these immune cell outputs for screening, transplantation, disease modeling or therapy, including injury mechanisms.
Claims Coverage
The independent claim is clm-00001. It covers a multi-stage, defined-media method that produces immune cells from somatic-cell-derived iPSCs, with four inventive features centered on sequential preconditioning, mesoderm induction, HPC generation, and final immune-cell differentiation.
Defined media GSK-inhibitor preconditioning free of BMP4, IL-3, Flt3 ligand, and GM-CSF
Incubating iPSCs in a first defined media comprising a GSK inhibitor, wherein the media is free or essentially free of BMP4, IL-3, Flt3 ligand, and GM-CSF to prepare cells for HPC differentiation.
Sequential mesoderm induction with BMP4, FGF2, and VEGF
Culturing the cells produced in the GSK-inhibitor stage in a second defined media comprising BMP4, FGF2, and VEGF to promote mesoderm induction in a plurality of the cells.
HPC proliferation and differentiation in IL-3 and Flt3 ligand with increased CD7/DLL4/CD144/CD235 expression
Culturing the cells produced in the mesoderm induction stage in a third defined media comprising IL-3 and Flt3 ligand such that a plurality of the cells proliferate and differentiate into HPCs that exhibit increased expression of one or more of CD7, DLL4, CD144, and CD235 as compared to cells produced in the mesoderm induction stage.
Immune cell differentiation from HPCs to produce immune cells
Culturing the HPCs produced after the IL-3/Flt3 ligand stage under conditions to promote immune cell differentiation, thereby producing immune cells.
Across the independent claim, immune cells are produced from somatic-cell-derived iPSCs using sequential defined-media stages: a GSK inhibitor preconditioning stage, mesoderm induction with BMP4/FGF2/VEGF, HPC proliferation and differentiation with IL-3 and Flt3 ligand that yields HPCs with increased CD7, DLL4, CD144, and/or CD235 expression, and final immune-cell differentiation from the HPCs.
Stated Advantages
Avoid animal products and feeder layers.
Generate large numbers of immune cells.
Preserve TCR V(D)J rearrangements in the produced immune cells.
Documented Applications
Screening.
Transplantation.
Disease modeling or therapy, including injury mechanisms.
Interested in licensing this patent?