Media and methods for differentiating natural killer cells
Media and methods for differentiating NK progenitor cells into NK cells are disclosed. The NK differentiation media comprises a pyrimidoindole compound such as UM171 or UM729.
1 . A method for differentiating NK cells, comprising:
providing a population of NK cell progenitors, wherein the population of NK cell progenitors arise in contact with a NK cell progenitor differentiation medium and a stroma cell replacement comprising components selected from fibronectin, whether full-length or a fragment thereof, VCAM-1, and an immobilized Notch ligand; and
contacting the population of NK cell progenitors in culture with an NK cell differentiation medium comprising a basal medium, IL-15 and a pyrimidoindole compound at a concentration between 10 nM and 3 μM and for a time sufficient to yield NK cells,
wherein the NK cells co-express i) CD56, NKG2D and NKp44, or ii) CD56, NKG2D and NKp30,
wherein an increased frequency and/or yield of NK cells co-expressing i) CD56, NKG2D and NKp44, or ii) CD56, NKG2D and NKp30 is obtained when contacting the population of NK cell progenitors with the NK cell differentiation medium as compared to when the NK cells are differentiated from the population of NK cell progenitors in a NK cell differentiation medium devoid of an added pyrimidoindole compound, and
wherein the NK cells produce intracellular interferon γ upon stimulation.
2 . The method according to claim 1 , wherein the population of NK cell progenitors express CD7 or CD5, or both.
3 . The method according to claim 1 , wherein the NK cells increase in frequency during the contacting step or increase in number during the contacting step, or both.
4 . The method according to claim 1 , wherein the population of NK cell progenitors increase or decrease in frequency or number during the contacting step.
5 . The method according to claim 1 , wherein the providing and contacting steps are not in the presence of stromal cells or a stroma cell replacement.
6 . The method according to claim 1 , wherein the providing and contacting steps are not in the presence of an aryl hydrocarbon receptor antagonist.
7 . The method according to claim 1 , wherein the time is at least 1 week or about 2 weeks.
8 . The method according to claim 1 , wherein the NK cells are cytotoxic.
9 . The method according to claim 1 , wherein the population of NK cell progenitors is derived or isolated from a primary sample.
10 . The method according to claim 1 , wherein the population of NK cells is differentiated from a pluripotent stem cell.
11 . The method of claim 1 , wherein less than 5% of the NK cells express CD3.
12 . A method for differentiating NK cells, comprising:
providing a population of NK cell progenitors, wherein the population of NK cell progenitors arise in contact with a NK cell progenitor differentiation medium and a stroma cell replacement comprising components selected from fibronectin, whether full-length or a fragment thereof, VCAM-1, and an immobilized Notch ligand; and
contacting the population of NK cell progenitors in culture with an NK cell differentiation medium comprising a basal medium, IL-15 and a pyrimidoindole compound at a concentration between 10 nM and 3 μM and for a time sufficient to yield NK cells,
wherein the NK cells co-express i) CD56, NKG2D and NKp44, or ii) CD56, NKG2D and NKp30,
wherein an increased frequency and/or yield of NK cells co-expressing i) CD56, NKG2D and NKp44, or ii) CD56, NKG2D and NKp30 is obtained when contacting the population of NK cell progenitors with the NK cell differentiation medium as compared to when the NK cells are differentiated from the population of NK cell progenitors in an NK cell differentiation medium devoid of an added pyrimidoindole compound,
wherein less than 5% of the NK cells express CD3, and
wherein the NK cells produce intracellular interferon γ upon stimulation.
13 . The method of claim 12 , wherein the population of NK cell progenitors express CD7 or CD5, or both.