Method for production of mast cells from stem cells
First Claim
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1. A method of preparing human mast cells by culturing human pluripotent cells in vitro comprising the steps of:
- a) culturing human pluripotent cells under conditions that promote differentiation of the cells into hematopoietic progenitor cells or megakaryocytes, to provide a first cell population comprising hematopoietic precursors or megakaryocytes; and
b) culturing the first cell population under conditions that promote the differentiation into mast cells, to provide a second cell population comprising mast cells;
wherein the step (a) culturing step does not employ a co-culture with murine fetal liver-derived stromal cells as a feeder layer;
wherein the step (b) culturing step comprises culturing the cells in a media comprising stem cell factor;
further wherein the pluripotent cells are cultured in a media comprising TPO.
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Abstract
Provided are methods for generating mast cells from pluripotent stem cells in vitro. Methods are disclosed for the differentiation of pluripotent cells, such as iPS cells and/or human embryonic stem cells, into mast cells. The resulting mast cells may be used for various purposes including screening cells for drug development and research. Growth factors which may be included in culture media according to the present invention include stem cell factor (SCF), FLT-3 ligand, thrombopoietin (TPO), interleukin-3 (IL-3), and/or interleukin-6 (IL-6).
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Citations
23 Claims
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1. A method of preparing human mast cells by culturing human pluripotent cells in vitro comprising the steps of:
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a) culturing human pluripotent cells under conditions that promote differentiation of the cells into hematopoietic progenitor cells or megakaryocytes, to provide a first cell population comprising hematopoietic precursors or megakaryocytes; and b) culturing the first cell population under conditions that promote the differentiation into mast cells, to provide a second cell population comprising mast cells; wherein the step (a) culturing step does not employ a co-culture with murine fetal liver-derived stromal cells as a feeder layer; wherein the step (b) culturing step comprises culturing the cells in a media comprising stem cell factor; further wherein the pluripotent cells are cultured in a media comprising TPO.
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2. A method of preparing human mast cells by culturing human pluripotent cells in vitro comprising the steps of:
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a) culturing human pluripotent cells under conditions that promote differentiation of the cells into hematopoietic progenitor cells or megakaryocytes, to provide a first cell population comprising hematopoietic precursors or megakaryocytes; and b) culturing the first cell population under conditions that promote the differentiation into mast cells, to provide a second cell population comprising mast cells; wherein the step (a) culturing step does not employ a co-culture with murine fetal liver-derived stromal cells as a feeder layer; wherein the step (b) culturing step comprises culturing the cells in a media comprising stem cell factor; further wherein step (a) comprises culturing the pluripotent cells under defined conditions and the sequential steps of; 1) culturing or maintaining a plurality of the pluripotent cells in a first defined media comprising at least one growth factor; 2) culturing the cells incubated in step
1) in a second defined media comprising an amount of BMP4 and VEGF sufficient to expand or promote differentiation in a plurality of the cells; and3) culturing the cells cultured in step
2) in a third defined media comprising an amount of IL3, Flt3 ligand, and GMCSF sufficient to expand or promote differentiation in a plurality of the cells;wherein a plurality of the pluripotent cells are differentiated into hematopoietic precursor cells. - View Dependent Claims (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method of preparing human mast cells by culturing human pluripotent cells in vitro comprising the steps of:
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a) culturing human pluripotent cells under conditions that promote differentiation of the cells into hematopoietic progenitor cells or megakaryocytes, to provide a first cell population comprising hematopoietic precursors or megakaryocytes; and b) culturing the first cell population under conditions that promote the differentiation into mast cells, to provide a second cell population comprising mast cells; wherein the step (a) culturing step does not employ a co-culture with murine fetal liver-derived stromal cells as a feeder layer; wherein the step (b) culturing step comprises culturing the cells in a media comprising stem cell factor; wherein the method further comprises purifying mast cells using MACS or FACS. - View Dependent Claims (22)
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23. A method of preparing human mast cells by culturing human pluripotent cells in vitro comprising the steps of:
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a) culturing human pluripotent cells under conditions that promote differentiation of the cells into hematopoietic progenitor cells or megakaryocytes, to provide a first cell population comprising hematopoietic precursors or megakaryocytes; and b) culturing the first cell population under conditions that promote the differentiation into mast cells, to provide a second cell population comprising mast cells; wherein the step (a) culturing step does not employ a co-culture with murine fetal liver-derived stromal cells as a feeder layer; wherein the step (b) culturing step comprises culturing the cells in a media comprising stem cell factor; wherein step (a) comprises culturing cells under conditions which favor differentiation of the pluripotent cells into hematopoietic cells and subsequently culturing the hematopoietic cells under conditions which favor differentiation into megakaryocytes, wherein the resulting cells are cultured under conditions which favor differentiation into mast cells.
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Specification