Method for production of mast cells from stem cells

• US 8,513,012 B2
• Filed: 05/04/2009
• Issued: 08/20/2013
• Est. Priority Date: 05/02/2008
• Status: Active Grant

• Current Assignee: FUJIFILM Cellular Dynamics, Inc. (Fujifilm Holdings Corporation)
• Original Assignee: Cellular Dynamics International, Inc. (Fujifilm Holdings Corporation)
• Inventors: Daigh, Christine, Rajesh, Deepika
• Primary Examiner(s): FALK, ANNE MARIE
• Application Number:
  US12/435,123
• Publication Number:
  US 20090275131A1
• Time in Patent Office: 1,569 Days
• Field of Search: None
• US Class Current: 435/377
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/145 Thrombopoietin [TPO]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2501/165 Vascular endothelial growth factor [VEGF]
• CPC Class Codes: C12N 2501/22 Colony stimulating factors (G-CSF, GM-CSF)
• CPC Class Codes: C12N 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2502/1394 Bone marrow stromal cells; whole marrow
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0642 Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells

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).

Claims

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.

2. 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 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; and

3) 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.

3. The method of claim 2, wherein the second cell population comprises tryptase-positive mast cells.

4. The method of claim 2, wherein at least some of the cells are at least partially separated or are substantially individualized prior to step (b).

5. The method of claim 4, wherein the cells are substantially individualized using an enzyme.

6. The method of claim 5, wherein the enzyme is a trypsin or TRYPLE.

7. The method of claim 5, wherein the cells are contacted with a ROCK inhibitor and a trypsin inhibitor subsequent to said individualization.

8. The method of claim 7, wherein the ROCK inhibitor is selected from the list consisting of HA-100, H-1152, and Y-27632.

9. The method of claim 2, wherein the method comprises culturing the cells in one or more of steps 1) through 3) at an atmospheric pressure of about 5-20% oxygen.

10. The method of claim 2, wherein step (a) comprises differentiating the pluripotent cells into embryoid bodies (EBs).

11. The method of claim 2, wherein step (a) comprises culturing the pluripotent cells in a first media comprising at least one of FLT-3 ligand, stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), and interleukin-6 (IL-6).

12. The method of claim 11, wherein the first media comprises stem cell factor.

13. The method of claim 11, wherein step (a) comprises culturing the pluripotent cells in a first media comprising at least two of FLT-3 ligand, stem cell factor, thrombopoietin (TPO), interleukin-3 (IL-3), and interleukin-6 (IL-6).

14. The method of claim 13, wherein step (a) comprises culturing the pluripotent cells in a media comprising FLT-3 ligand, stem cell factor, TPO, IL-3, and IL-6.

15. The method of claim 14, wherein FLT-3 ligand, stem cell factor, TPO, IL-3, and IL-6 are exogenously added and recombinant.

16. The method of claim 15, wherein the first media comprises about 10-100 ng/ml FL3, about 10-100 ng/ml stem cell factor, about 10-100 ng/ml TPO, about 10-100 ng/ml IL-3, and about 10-100 ng/ml IL-6 are exogenously added and recombinant.

17. The method of claim 2, wherein after step (a) a plurality of the pluripotent cells have been differentiated into either megakaryocytes or mast cells, wherein the mast cells are positive for CD117 and CD45, while being negative for CD34.

18. The method of claim 2, wherein the media further comprises interleukin-6 (IL-6).

19. The method of claim 18, where the media comprises about 10-100 ng/ml stem cell factor and about 10-100 ng/ml IL-6.

20. The method of claim 2, wherein the culturing of at least one of step (a) and/or step (b) are performed using serum-free media.

21. 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;

wherein the method further comprises purifying mast cells using MACS or FACS.

22. The method of claim 21, wherein step (a) comprises culturing cells under conditions which favor differentiation of the pluripotent cells into hematopoietic cells, wherein the resulting hematopoietic cells are cultured under conditions which favor differentiation into mast cells.

23. 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;

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.