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US 2026/0028588 A1Published application
Differentiation of stem cells in suspension culture
AssigneeUmoja Biopharma, Inc.
InventorsTeisha J. ROWLAND, Cassidy FRIEDMAN, Ashley M. YINGST, Samantha O'HARA, Dillon JARRELL, David T. VEREIDE, Ryan KONING
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Abstract
Provided are xenogenic-free methods and compositions for generating hematopoietic progenitors and natural killer (NK) cells in 3D suspension culture.
Claims95
A method for generating a population of CD34+/CD43+/CD45+ cells, comprising:
- (i) culturing a population of progenitor cells in a two-dimensional (2D) culture system for a period of time sufficient to form progenitor cell aggregates;
- (ii) passaging the progenitor cell aggregates from the 2D culture system to a three-dimensional (3D) suspension culture system;
- (iii) contacting the progenitor cell aggregates in the 3D suspension culture system with a differentiation media for a period of time sufficient to generate the population of CD34+/CD43+/CD45+ cells.
A method for differentiating a population of stem cells into a population of hematopoietic progenitors, comprising:
- (i) culturing the population of stem cells in a 2D culture system for a period of time sufficient to form stem cell aggregates;
- (ii) passaging the stem cell aggregates from the 2D culture system to a 3D suspension culture system;
- (iii) contacting the stem cell aggregates in the 3D suspension culture system with a differentiation media comprising a bone morphogenetic protein (BMP) pathway activator, a fibroblast growth factor (FGF), and a vascular endothelial growth factor (VEGF), for a period of time sufficient to differentiate the population of stem cells into the population of hematopoietic progenitors.
A method of generating a population of NK cells, comprising:
- (a) culturing a population of stem cells in a 2D culture system for a period of time sufficient to form stem cell aggregates;
- (b) passaging the stem cell aggregates from the 2D culture system to a 3D suspension culture system;
- (c) contacting the stem cell aggregates in the 3D suspension culture system with a first media comprising a BMP pathway activator, an FGF, a VEGF, and optionally an inhibitor of ROCK, for a period of time sufficient to generate embryoid bodies;
- (d) contacting the embryoid bodies with a first differentiation media comprising a BMP pathway activator, a FGF, VEGF, SCF, TPO, and an LDL, for a period of time sufficient to generate a population of hematopoietic progenitors;
- (e) contacting the population of hematopoietic progenitors with a second differentiation media, for a period of time sufficient to generate the population of NK cells.
2 more elements
A method of generating a population of hematopoietic progenitors, comprising:
- (a) genetically engineering a population of stem cells to express a synthetic cytokine receptor for a non-physiological ligand,
- wherein the cytokine receptor comprises:
- a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and
- a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain;
- (b) culturing the population of stem cells in a 2D culture system for a period of time sufficient to form stem cell aggregates;
- (c) passaging the stem cell aggregates from the 2D culture system to a 3D suspension culture system;
- and
- (d) contacting the stem cell aggregates in the 3D suspension culture system with a differentiation media for a period of time sufficient to generate hematopoietic progenitors.
5 more elements
A method of generating a population of NK cells, comprising:
- (a) genetically engineering a population of stem cells to express a synthetic cytokine receptor for a non-physiological ligand,
- wherein the cytokine receptor comprises:
- a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and
- a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain;
- (b) culturing the population of stem cells in a 2D culture system for a period of time sufficient to form stem cell aggregates;
- (c) passaging the stem cell aggregates from the 2D culture system to a 3D suspension culture system;
- (d) contacting the stem cell aggregates in the 3D suspension culture system with a first differentiation media for a period of time sufficient to generate hematopoietic progenitors;
- and
- (e) contacting the population of hematopoietic progenitors with a second differentiation media, for a period of time sufficient to generate the population of NK cells.
6 more elements
Dependencies are inferred from the claim text.