Induced pluripotent stem cell selection method and method for inducing differentiation to blood cells

• US 10,240,126 B2
• Filed: 06/11/2014
• Issued: 03/26/2019
• Est. Priority Date: 06/12/2013
• Status: Active Grant

• Current Assignee: Kyoto University
• Original Assignee: Kyoto University
• Inventors: Yamanaka, Shinya, Yoshida, Yoshinori, Nishizawa, Masatoshi
• Primary Examiner(s): Epps-Smith, Janet L
• Application Number:
  US14/897,599
• Publication Number:
  US 20160137981A1
• Time in Patent Office: 1,749 Days
• Field of Search: 435377, 435325, 435375, 4352871, 4352872, 4352887, 4352891, 435366, 435455, 435456, 435 721
• CPC Class Codes: C07K 14/65 Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
• CPC Class Codes: C12N 2501/105 Insulin-like growth factors [IGF]
• CPC Class Codes: C12N 2501/115 Basic fibroblast growth factor (bFGF, FGF-2)
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/14 Erythropoietin [EPO]
• 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 2501/2303 Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/2311 Interleukin-11 (IL-11)
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0696 Artificially induced pluripotent stem cells, e.g. iPS
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

A method for producing hematopoietic stem cells and/or hematopoietic progenitor cells from pluripotent stem cells is described. The method includes a step of culturing pluripotent stem cells in the presence of IGF2. A method is described for selecting an induced pluripotent stem cell(s) having high capacity to differentiate into hematopoietic stem cells and/or hematopoietic progenitor cells, or into blood cells, based on the expression level(s) of one or more genes such as TRIM58, CTSF, FAM19A5, and TCERG1L genes, or on the DNA methylation state(s) of the TRIM58, CSMD1, and/or FAM19A5 gene(s).

Claims

1. A method for producing hematopoietic stem cells and/or hematopoietic progenitor cells from pluripotent stem (PS) cells, said method comprising:

(a) measuring the expression level(s) of one or more genes selected from the group consisting of TRIM58, CTSF, FAM19A5, and TCERG1L genes in candidate PS cells;

(b) selecting a PS cell(s) in which the expression level(s) of the gene(s) measured in Step (a) is/are equivalent to or higher than the expression level(s) in iPS cells or embryonic stem (ES) cells which are known to have high capacity to differentiate into blood cells, and/or selecting a PS cell(s) in which the expression level(s) of the gene(s) measured in Step (a) is/are higher than the expression level(s) in iPS cells or ES cells which are known to have low capacity to differentiate into blood cells; and

(c) culturing the selected PS cells in the presence of insulin-like growth factor 2 (IGF2).

2. The method according to claim 1, wherein said step of culturing the selected PS cells in the presence of IGF2 comprises the steps of:

(i) culturing the selected PS cells in a medium supplemented with BMP4 and IGF2;

(ii) culturing cells obtained in Step (i) in a medium supplemented with BMP4, bFGF, and IGF2;

(iii) culturing cells obtained in Step (ii) in a medium supplemented with VEGF, bFGF, IL-6, IL-3, IL-11, SCF, Flt3L, and IGF2; and

(iv) culturing cells obtained in Step (iii) in a medium supplemented with VEGF, IL-6, IL-3, IL-11, SCF, Flt3L, EPO, TPO, and IGF2.

3. The method according to claim 1, wherein, in said step of culturing pluripotent stem cells, said pluripotent stem cells are cultured in a form of embryoid bodies.

4. The method according to claim 1, wherein said pluripotent stem cells are iPS cells.

5. The method according to claim 4, wherein said iPS cells are human iPS cells.

6. The method according to claim 1, wherein said sample of induced pluripotent stem cells are human iPS cells.

7. The method according to claim 1, wherein said blood cells are one or more kinds of cells selected from the group consisting of erythrocytes, platelets, monocytes, T cells, B cells, NK cells, neutrophils, eosinophils, basophils, granulocytes, and macrophages.

8. The method according to claim 1, wherein said iPS cells or ES cells which are known to have high capacity to differentiate into blood cells are one or more kinds of cells selected from the group consisting of 751A3, 783F1, 751B4, 692D2, 783A2, 744A2, 744B9, 609A2, 585B1, 784D1, 609A1, 648B1, 665A7, 451F3, 610B1, 606A1, 648A1, and khES3.

9. The method according to claim 1, wherein said iPS cells or ES cells which are known to have low capacity to differentiate into blood cells are one or more kinds of cells selected from the group consisting of 427F1, khES1, TIG1204F1, H9, Kep1, 454E2, 253G1, KRV-1, 457C1, 7-KE2, 201B7, TIG1074F1, 246G1, 409B2, 201B6, 253G4, 404C2, and 588A4.

10. A method for producing hematopoietic stem cells and/or hematopoietic progenitor cells from pluripotent stem (PS) cells, said method comprising

(a) measuring the DNA methylation state(s) of TRIM58, CSMD1, and/or FAM19A5 gene(s) in candidate PS cells;

(b) selecting a PS cell(s) in which the DNA methylation level(s) measured in Step (a) is/are equivalent to or lower than the DNA methylation level(s) in iPS cells or ES cells which are known to have high capacity to differentiate into blood cells, and/or selecting a PS cell(s) in which the DNA methylation level(s) measured in Step (a) is/are lower than the DNA methylation level(s) in iPS cells or ES cells which are known to have low capacity to differentiate into blood cells; and

(c) culturing the selected PS cells in the presence of insulin-like growth factor 2 (IGF2).

11. The method according to claim 10, wherein said step of culturing the selected PS cells in the presence of IGF2 comprises the steps of:

(i) culturing the selected PS cells in a medium supplemented with BMP4 and IGF2;

(ii) culturing cells obtained in Step (i) in a medium supplemented with BMP4, bFGF, and IGF2;

(iii) culturing cells obtained in Step (ii) in a medium supplemented with VEGF, bFGF, IL-6, IL-3, IL-11, SCF, Flt3L, and IGF2; and

(iv) culturing cells obtained in Step (iii) in a medium supplemented with VEGF, IL-6, IL-3, IL-11, SCF, Flt3L, EPO, TPO, and IGF2.

12. The method according to claim 10, wherein, in said step of culturing pluripotent stem cells, said pluripotent stem cells are cultured in a form of embryoid bodies.

13. The method according to claim 10, wherein said pluripotent stem cells are iPS cells.

14. The method according to claim 13, wherein said iPS cells are human iPS cells.

15. The method according to claim 10, wherein said blood cells are one or more kinds of cells selected from the group consisting of erythrocytes, platelets, monocytes, T cells, B cells, NK cells, neutrophils, eosinophils, basophils, granulocytes, and macrophages.

16. The method according to claim 10, wherein said iPS cells or ES cells which are known to have high capacity to differentiate into blood cells are one or more kinds of cells selected from the group consisting of 751A3, 783F1, 751B4, 692D2, 783A2, 744A2, 744B9, 609A2, 585B1, 784D1, 609A1, 648B1, 665A7, 451F3, 610B1, 606A1, 648A1, and khES3.

17. The method according to claim 10, wherein said iPS cells or ES cells which are known to have low capacity to differentiate into blood cells are one or more kinds of cells selected from the group consisting of 427F1, khES1, TIG1204F1, H9, Kep1, 454E2, 253G1, KRV-1, 457C1, 7-KE2, 201B7, TIG1074F1, 246G1, 409B2, 201B6, 253G4, 404C2, and 588A4.