Method for making hematopoietic cells

• US 20040110286A1
• Filed: 12/06/2002
• Published: 06/10/2004
• Est. Priority Date: 12/06/2002
• Status: Abandoned Application

• Current Assignee: Robarts Research Institute
• Original Assignee: The John P. Robarts Research Institue
• Inventors: Bhatia, Mickie
• Application Number:
  US10/313,195
• Publication Number:
  US 20040110286A1
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 2501/125 Stem cell factor [SCF], c-kit ligand [KL]
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• 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 2501/41 Hedgehog proteins; Cyclopamine (inhibitor)
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors

Abstract

This invention provides a system for producing cells of the hematopoietic lineage from embryonic stem cells. Differentiation is conducted in the presence of hematogenic cytokines and other factors listed in the disclosure. The cell population that is obtained is remarkably enriched in CD45 +ve cells, a marker of early hematopoietic precursor with self-renewing capacity. Including a bone morphogenic protein during the differentiation process enhances the ability of the cell population to form secondary colonies. Because of the enormous replicative capacity of embryonic stem cells, this provides an important new commercial source of hematopoietic cells.

Claims

1. A method for differentiating human pluripotent stem (hPS) cells into a cell population with hematopoietic potential, comprising:

a) harvesting undifferentiated hPS cells from a feeder-free culture;

b) differentiating the harvested hPS cells in a culture environment essentially free of any cells having a different genotype, but containing at least two hematopoietic growth factors selected from stem cell factor (SCF), FLT-3 ligand, IL-3, IL-6, and granulocyte colony stimulating factor (G-CSF);

c) simultaneously or subsequently culturing the differentiating cells with a bone morphogenic protein; and

d) harvesting from the culture environment a cell population that is at least 1% CD45 positive, or that forms colonies in an assay for hematopoietic colony forming units (CFU) at a plating efficiency of at least −

1 in 2000.

2. The method of claim 1, wherein at least 5% of the cells harvested from the culture environment are CD45 +ve.

3. The method of claim 1, wherein at least 70% of the cells harvested from the culture environment are CD13 +ve.

4. The method of claim 1, wherein at least 10% of the cells harvested from the culture environment are AC133 +ve.

5. The method of claim 1, wherein at least 5% of the cells harvested from the culture environment are both CD34 +ve and CD45 +ve.

6. The method of claim 1, wherein the cell population harvested from the culture environment forms colonies in an assay for hematopoietic colony forming units (CFU) at a plating efficiency of at least ˜

1 in 500

7. The method of claim 1, wherein the hPS cells are progeny of cells isolated from a human blastocyst.

8. The method of claim 1, wherein the hPS cells are human embryonic stem cells.

9. The method of claim 1, wherein the differentiating comprises forming embryoid bodies or cell aggregates.

10. The method of claim 1, wherein the differentiating comprises culturing in low density conditioned medium.

11. The method of claim 1, wherein the cells are cultured with said hematopoietic growth factors within 10 days of the onset of differentiation.

12. The method of claim 1, wherein the cells are cultured with bone morphogenic protein 4 subsequent to the culturing with said hematopoietic growth factors.

13. The method of claim 1, which is a method for producing hematopoietic progenitors.

14. The method of claim 1, which is a method for producing erythroid cells, granulocytic cells, monocytic cells, megakaryocytes, or lymphoid cells.

15. The method of claim 1, which is a method for producing hematopoietic cells suitable for rendering an individual into whom they are injected immunotolerant for an allograft that is MHC matched with the hematopoietic cells.