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
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Publication Number
US-12129486-B2
Publication Date
2024-10-29
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 invention provides feeder-free, defined in vitro differentiation from somatic-cell-derived pluripotent stem cells, including iPSCs and T-cell-derived iPSCs (TiPSCs), to hematopoietic precursor cells (HPCs), and then to immune lineages. The disclosed process includes hypoxic differentiation and sequential defined HPC media compositions for generating hematopoietic precursor cells.
Lymphoid-primed HPCs are identified by marker expression patterns, including CD34/CD43 and DLL4/CD144/CD7 signatures. An enriched lymphoid potential population is obtained by enriching or isolating marker-positive fractions using magnetic-activated cell sorting (MACS) based on combinations of cell surface markers selected from CD31, CD34, CD144, CD43, CD45, CD7, CD235, Flk-1, and DLL4.
Lymphoid differentiation is driven by RetroNectin matrix with Notch ligand DLL4 (DLL4:Fc) together with cytokines such as IL-7, SCF, Flt-3 ligand (Flt-3L), and TPO. The approach is described as producing lymphoid cells and includes myeloid and dendritic cell differentiation from HPCs, with DC maturation marker phenotypes such as CD209, CD1a, HLA-DR, CD11c, CD83, and CD86.
The disclosed concept includes using enriched populations to increase efficiency of producing T cells, measured using a ratio of input of HPCs to output of T cells. The document also provides exemplary efficiency-supporting experimental results, including flow cytometry and enrichment/yield metrics, and frames application concepts for producing large numbers of immune cells for therapy and in vitro screening.
Claims Coverage
The partial content contains one independent claim (clm-00001) directed to enriching lymphoid progenitors to increase lymphoid potential, with downstream differentiation details provided only in dependent claims via further limitations. The inventive core of the independent claim is the MACS-based enrichment using at least two selected cell surface markers and defining improved lymphoid potential by increased T-cell production efficiency (HPC input to T-cell output ratio).
MACS-based lymphoid progenitor enrichment by at-least-two marker positivity
Enriching a starting cell population for lymphoid progenitors by performing magnetic-activated cell sorting (MACS) to isolate cells positive for at least two cell surface markers selected from CD31, CD34, CD144, CD43, CD45, CD7, CD235, Flk-1, and DLL4.
Increased lymphoid potential defined by T-cell production efficiency ratio
Providing a population of cells with increased lymphoid potential as compared to an unsorted cell population or a population of cells negative for two or more of the selected markers, wherein increased lymphoid potential is an increased efficiency at producing T cells measured by a ratio of input of HPCs to output of T cells.
Across the provided independent claim, the claimed inventive contribution is an enrichment workflow that isolates lymphoid progenitors using MACS for cells positive for at least two selected markers, and defines success by increased efficiency of producing T cells using an HPC input to T-cell output ratio.
Stated Advantages
Increased lymphoid potential compared to an unsorted cell population or marker-negative populations.
Increased efficiency at producing T cells, measured by a ratio of input of HPCs to output of T cells.
Documented Applications
Producing large numbers of immune cells for therapy.
In vitro screening using produced immune cells.
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