Reagents and methods for producing arterial hemogenic endothelium, hematopoietic progenitors, and lymphoid cells thereby
OwnerIgor Slukvin, Akhilesh Kumar +1inventors
InventorsIgor Slukvin, Akhilesh Kumar, Saritha Sandra D'Souza
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Abstract
The present disclosure provides reagents and methods for producing arterial hemogenic endothelium, hematopoietic progenitor, and lymphoid cell cultures. Pharmaceutical compositions comprising arterial hemogenic endothelium cells, hematopoietic progenitor cells, and lymphoid cells produced by the reagents and methods of the invention and therapeutic methods using these pharmaceutical compositions are also provided.
Claims72
A method of producing a spheroid containing pluripotent stem cells for hematopoietic progenitor cell differentiation, comprising the steps of:
- a) dispersing pluripotent stem cells (PSCs) into a single cell suspension of spheroid medium, wherein the medium contains 25% Dulbecco's Modified Eagle medium, 25% Ham's F-12 Medium, 10% BIT 9500 supplements, 2 mM L-alanyl-L-glutamine dipeptide, 0.1 mM Non-essential Amino Acids (NEAA), 100 μM monothioglycerol (MTG), 100 μg/ml ascorbic acid, and 40% ES-Cult M3120 comprising 2.6% methylcellulose in Iscove's Modified Dulbecco's Medium,
- b) apportioning the cell suspension into individual, separated droplets each comprising about 2,500-3,500 cells in 30-40 μl spheroid medium onto a solid surface in a sealable vessel, wherein the vessel is thereafter sealed and inverted wherein the droplets are hanging from the solid surface;
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- c) incubating the droplet-comprising vessel for 20-24 hours in an incubator at cell-sustaining temperature and humidity conditions to produce cell-comprising spheroids.
A method of producing T cells from pluripotent stem cells (PSCs) comprising reducing DLL4 expression or activity in the PSCs and reducing GAS5 expression in hemogenic endothelium cells (HEs) derived from the PSCs, and differentiating the HEs into the T cells.
A method of producing T cells from pluripotent stem cells (PSCs) comprising reducing DLL4 expression or activity in the PSCs and differentiating hematopoietic progenitor cells (HPs) derived from PSCs into the T cells, wherein the PSCs are cultured on JAG1-Fc fusion proteins coated plates until the HPs are obtained.
A method of producing B cells from pluripotent stem cells (PSCs) comprising reducing DLL4 expression or activity in the PSCs, and differentiating the PSCs into the B cells.
A method of producing B cells from pluripotent stem cells (PSCs) comprising reducing DLL4 expression or activity in the PSCs and differentiating hematopoietic progenitor cells (HPs) derived from PSCs into the B cells, wherein the PSCs are cultured on JAG1-Fc fusion proteins coated plates until the HPs are obtained.
Dependencies are inferred from the claim text.