Methods for producing enucleated erythroid cells derived from pluripotent stem cells

• US 9,988,602 B2
• Filed: 03/24/2016
• Issued: 06/05/2018
• Est. Priority Date: 05/06/2008
• Status: Active Grant

• Current Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Original Assignee: Astellas Institute for Regenerative Medicine (Astellas Pharma Incorporated)
• Inventors: Lanza, Robert, Lu, Shi-Jiang
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US15/080,486
• Publication Number:
  US 20170121681A1
• Time in Patent Office: 803 Days
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C12N 2500/25 Insulin-transferrin; Insulin-transferrin-selenium
• CPC Class Codes: C12N 2500/38 Vitamins
• 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 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/60 Transcription factors
• CPC Class Codes: C12N 2502/1358 Bone marrow mesenchymal stem cells (BM-MSC)
• 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 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2533/78 Cellulose
• CPC Class Codes: C12N 5/0641 Erythrocytes
• CPC Class Codes: C12N 5/0644 : Platelets; Megakaryocytes
• CPC Class Codes: C12N 5/0647 : Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0692 : Stem cells; Progenitor cells; Precursor cells

Abstract

Methods for generating enucleated erythroid cells using pluripotent stem cells are provided. The methods permit the production of large numbers of cells. The cells obtained by the methods disclosed may be used for a variety of research, clinical, and therapeutic applications. Methods for generating megakaryocyte and platelets are also provided.

Claims

1. A method of producing human pluripotent stem cell-derived enucleated erythroid cells in vitro, comprising:

(a) providing a human pluripotent stem cell cultured in serum free media;

(b) differentiating said pluripotent stem cell in a serum free media comprising bone morphogenic protein 4 (BMP-4) and vascular endothelial growth factor (VEGF), and producing embryoid bodies, wherein step (b) does not comprise culturing in a media comprising thrombopoietin (TPO), Flt3 ligand (FL), or stem cell factor (SCF);

(c) culturing the embryoid bodies obtained from step (b) in a serum free media comprising at least basic fibroblast growth factor (bFGF) and methylcellulose, and producing a hemangioblast, non-engrafting hemangio cell, or blast cell; and

(d) culturing said hemangioblast, non-engrafting hemangio cell, or blast cell in a serum free medium comprising at least erythropoietin (EPO), interleukin-3 (IL-3), SCF and methylcellulose;

(e) co-culturing cells obtained by culturing under the conditions of at least step (d) with stromal cells or mesenchymal stem cells (MSCs) to produce human pluripotent stem cell-derived enucleated erythroid cells,

wherein the human pluripotent stem cell-derived enucleated erythroid cells express β

-globin.

2. The method of claim 1, wherein said pluripotent stem cell is an embryonic stem cell or embryo-derived cell.

3. The method of claim 1, wherein said pluripotent stem cell is an induced pluripotent stem cell.

4. The method of claim 1, wherein said pluripotent stem cell is genetically manipulated prior to differentiation.

5. The method of claim 1, wherein said hemangioblast, non-engrafting hemangio cell, or blast cell is expanded prior to being differentiated into said enucleated erythroid cell.

6. The method of claim 5, wherein said hemangioblasts, non-engrafting hemangio cells, or blast cells are expanded in a hematopoietic cell medium, substantially free of animal components, with Epo, IL-3, and SCF.

7. The method of claim 1, wherein differentiating said human pluripotent stem cell into said hemangioblast further comprises

disaggregating said embryoid bodies into single cells.

8. The method of claim 1, wherein differentiating said human pluripotent stem cell into said non-engrafting hemangio cell further comprises culturing said embryoid body for 10-13 days.

9. The method of claim 8, wherein differentiating said pluripotent stem cell into said non-engrafting hemangio cell further comprises

disaggregating said embryoid bodies into single cells.

10. The method of claim 1, wherein differentiating said pluripotent stem cell into said enucleated erythroid cell further comprises culturing in culture medium comprising EPO in step (b).

11. The method of claim 1, wherein differentiating said hemangioblast, non-engrafting hemangio cell, or blast cell into said enucleated erythroid cell further comprises:

culturing said hemangioblast, non-engrafting hemangio cell, or blast cell in a culture medium comprising a supplement selected from the group consisting of inositol, folic acid, monothioglycerol, transferrin, insulin, ferrous nitrate, ferrous sulfate, BSA, L-glutamine, penicillin-streptomycin and combinations thereof.

12. The method of claim 1, wherein culturing with stromal cells or mesenchymal stem cells (MSCs) is in the presence of EPO.