Methods of producing haemogenic endothelial cells from pluripotent stem cells
AssigneeAdaptimmune Limited
InventorEva GARCIA-ALEGRIA
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Abstract
This invention relates to the production of haemogenic endothelial cells (HECs). A population of induced pluripotent stem cells (iPSCs) is differentiated into mesoderm cells by culturing the IPSCs sequentially in first, second and third mesoderm induction media to induce differentiation into mesoderm cells. The mesoderm cells are then differentiated into HECs by culturing the mesoderm cells sequentially in first and second haemogenic endothelium (HE) induction media to induce differentiation into HECs. The HECs may be further differentiated into hematopoietic progenitor cells (HPCs) and progenitor T cells.
Claims71
A method of producing a population of haemogenic endothelial cells (HECs) comprising;
- (a) differentiating a population of induced pluripotent stem cells (iPSCs) into mesoderm cells by culturing the iPSCs sequentially in first, second and third mesoderm induction media to induce differentiation into mesoderm cells;
- wherein
- the first mesoderm induction medium stimulates SMAD1, SMAD5 and SMAD9 mediated signalling pathways;
- the second mesoderm induction medium (i) stimulates SMAD1, SMAD5 and SMAD9 mediated signalling pathways and (ii) has fibroblast growth factor (FGF) activity;
- and
- the third mesoderm induction medium (i) stimulates SMAD1, SMAD2, SMAD3, SMAD5 and SMAD9 mediated signalling pathways (ii) has fibroblast growth factor (FGF) activity and (iii) inhibits glycogen synthase kinase 3β;
- and;
- (b) differentiating the mesoderm cells into HECs by culturing the mesoderm cells sequentially in first and second HE induction media to induce differentiation into HECs;
- wherein;
- the first HE induction medium (i) stimulates VEGFR mediated signalling pathways and (ii) has fibroblast growth factor (FGF) activity;
- and
- the second HE induction medium (i) stimulates cKIT receptor (CD117;
- KIT receptor tyrosine kinase) mediated signalling pathways, (ii) stimulates VEGFR mediated signalling pathways;
- and (iii) has fibroblast growth factor (FGF) activity.
A method according to any one of the preceding claims wherein the first mesoderm induction medium comprises BMP
A method according to any one of the preceding claims wherein the iPSCs are cultured in the first mesoderm induction medium for 1 day.
A method according to any one of the preceding claims wherein the second mesoderm induction medium comprises BMP and FGF
A method according to any one of the preceding claims wherein the iPSCs are cultured in the second mesoderm induction medium for 1 day.
A method according to any one of the preceding claims wherein the third mesoderm induction medium comprises activin, BMP, FGF, and a glycogen synthase kinase 3β inhibitor.
A method according to any one of the preceding claims wherein the iPSCs are cultured in the third mesoderm induction medium for 2 days.
A method according to any one of the preceding claims wherein the iPSCs are cultured in the first second and third mesoderm induction medium for 4 days to produce the mesoderm cells.
A method according to any one of the preceding claims wherein the mesoderm cells express one or more mesoderm markers selected from Brachyury, Goosecoid, MixI1, KDR, FoxA2, GATA6, and PDGFαR.
A method according to any one of the preceding claims wherein mesoderm cells in the population are not purified following culture in the first, second and third mesoderm induction media.
A method according to any one of the preceding claims wherein the mesoderm cells are differentiated into HECs by culturing the population of mesoderm cells under suitable conditions to promote haemogenic endothelial (HE) differentiation.
A method according to any one of the preceding claims wherein the first HE induction medium comprises FGF and VEGF.
A method according to any one of the preceding claims wherein the second HE induction medium comprises SCF, FGF and VEGF.
A method according to any one of the preceding claims wherein the FGF is FGF2 (bFGF).
A method according to any one of the preceding claims wherein the BMP is BMP4.
A method according to any one of the preceding claims wherein activin is activin A.
A method according to any one of the preceding claims wherein the GSK3 inhibitor is CHIR-99021.
A method according to any one of the preceding claims wherein the HECs display a CD34+ phenotype.
A method according to any one of the preceding claims wherein HECs in the population are not purified following culture in the HE induction medium.
A method according to any one of the preceding claims wherein steps (i) and (ii) are performed without purification or isolation of cells in the population.
A method according to any one of the preceding claims further comprising
- (c) differentiating the HECs into HPCs by culturing the population of HECs under suitable conditions to promote haematopoietic differentiation.
A method according to any one of the preceding claims further comprising;
- (d) differentiating the population of HPCs into progenitor T cells.
A method according to any one of the preceding claims wherein the iPSCs are derived from T cells obtained from a donor individual.
Dependencies are inferred from the claim text.