COMPOSITIONS AND METHODS FOR DIFFERENTIATING AND EXPANDING B LINEAGE CELLS
Disclosed are media, kits and methods for the directed differentiation of cells to the B cell lineage. The disclosed differentiation approaches may take primary cells or pluripotent stem cell-derived cells through one or more intermediate cell populations to yield the B lineage cells, using one or more stage-specific media formulations. Thus, media and supplements for carrying out directed differentiation workflows may be comprised in a kit that contains one or more basal media and one or more supplements to be added thereto.
1 . A method for differentiating a population of B cell precursors, comprising:
contacting a population of CD34 + hematopoietic stem or progenitor cells (HSPC) with a derivation medium comprising a basal medium, at least one of stem cell factor (SCF), thrombopoietin (TPO), and FMS-like tyrosine kinase 3 ligand (FLT3L), and at least one other cytokine; and
culturing the population of HSPC in the derivation medium under serum-free conditions to obtain a population of B cell precursors,
wherein the population of B cell precursors express one or both of CD10 or CD19.
2 . The method of claim 1 , wherein the population of HSPC are enriched from a tissue source or are differentiated from pluripotent stem cells (PSC).
3 . (canceled)
4 . The method of claim 1 , wherein the derivation medium comprises either SCF or TPO.
5 . The method according to claim 1 ,
further comprising contacting the population of B cell precursors with a differentiation medium comprising a basal medium, at least one of SCF, TPO, and FLT3L, and at least one other cytokine; and
culturing the population of B cell precursors in the differentiation medium under serum-free conditions.
6 . The method according to claim 5 , further comprising obtaining a population of CD19 + B lineage cells having more CD19 + cells than after culturing the population of HSPC in the derivation medium.
7 . (canceled)
8 . The method according to claim 5 , wherein at least a fraction of the CD19 + B lineage cells are IgM + cells.
9 . (canceled)
10 . The method according to claim 6 , further comprising:
contacting the population of CD19 + B lineage cells with a downstream differentiation medium comprising a basal medium, a ligand of human CD40, and the at least one other cytokine; and
culturing the population of CD19 + B lineage cells in the downstream differentiation medium under serum-free conditions.
11 . The method according to claim 10 , further comprising obtaining more IgM + cells than after culturing the population of B cell precursors in the differentiation medium.
12 . The method according to claim 11 , wherein at least a fraction of the IgM + cells are antibody secreting cells.
13 . (canceled)
14 . A method for differentiating a population of B lineage cells, comprising:
contacting a population of B cell precursors with a differentiation medium comprising a basal medium, at least one of SCF, TPO and FLT3L, and at least one other cytokine; and
culturing the population of B cell precursors in the differentiation medium under serum-free conditions to obtain a population of B lineage cells,
wherein the population of B cell precursors express one or both of CD10 or CD19.
15 . (canceled)
16 . The method of claim 14 , wherein the population of B cell precursors are derived from a population of CD34 + hematopoietic stem or progenitor cells (HSPC) that are either enriched from a tissue source or are differentiated from pluripotent stem cells (PSC).
17 . The method according to claim 16 , wherein the population of B lineage cells express CD19 and the population of B lineage cells comprises more CD19 + cells than after culturing the population of HSPC in a serum-free derivation medium comprising a basal medium, at least one cytokine, and one or more of SCF, TPO, and FLT3L.
18 . The method according to claim 17 , wherein at least a fraction of CD19 + B lineage cells are IgM + cells.
19 . The method according to claim 14 , further comprising:
contacting the population of B lineage cells with a downstream differentiation medium comprising a basal medium, a ligand of human CD40, and the at least one other cytokine; and
culturing the population of B lineage cells in the downstream differentiation medium under serum-free conditions.
20 . The method according to claim 19 , further comprising obtaining more IgM + cells than after culturing the population of B cell precursors in the differentiation medium.
21 . The method according to claim 20 , wherein at least a fraction of the IgM + cells are antibody secreting cells.
22 - 24 . (canceled)
25 . The method of claim 1 , wherein the at least one other cytokine is
i) one or more of IL-3, IL-6, or IL-7, or
ii) one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10 or IL-21.
26 . The method of claim 14 , wherein the at least one other cytokine is
i) one or more of IL-3, IL-6, or IL-7, or
ii) one or more of IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, or IL-21.
27 . The method according to claim 1 , wherein the methods are performed under feeder cell-free conditions.
28 - 42 . (canceled)
43 . The method according to claim 14 , wherein the methods are performed under feeder cell-free conditions.