Expansion of renewable stem cell populations

• US 7,955,852 B2
• Filed: 02/09/2004
• Issued: 06/07/2011
• Est. Priority Date: 01/24/2002
• Status: Active Grant

• Current Assignee: Gamida Cell, Ltd.
• Original Assignee: Gamida Cell, Ltd.
• Inventors: Treves, Avi, Peled, Tony, Rosen, Oren
• Primary Examiner(s): LEAVITT, MARIA GOMEZ
• Application Number:
  US10/774,843
• Publication Number:
  US 20050008624A1
• Time in Patent Office: 2,675 Days
• Field of Search: 435/377, 435/325, 435/366, 435/384
• US Class Current: 435/377
• CPC Class Codes: A01N 1/0226 Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
• CPC Class Codes: A61K 2035/122 for inducing tolerance or supression of immune responses
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2039/515 Animal cells
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07C 51/09 from carboxylic acid esters or lactones
• CPC Class Codes: C07C 59/72 containing six-membered aromatic rings and other rings
• CPC Class Codes: C12M 23/14 Bags
• CPC Class Codes: C12N 2500/20 Transition metals
• CPC Class Codes: C12N 2500/38 Vitamins
• 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 2501/23 Interleukins [IL]
• CPC Class Codes: C12N 2501/2303 Interleukin-3 (IL-3)
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/26 Flt-3 ligand (CD135L, flk-2 ligand)
• CPC Class Codes: C12N 2501/385 of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
• CPC Class Codes: C12N 2501/599 with CD designations not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2503/02 : Drug screening
• 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 5/067 : Hepatocytes
• CPC Class Codes: G01N 2333/70567 : Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
• CPC Class Codes: G01N 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5023 : on expression patterns
• CPC Class Codes: G01N 33/5041 : involving analysis of members of signalling pathways
• CPC Class Codes: G01N 33/5073 : Stem cells
• CPC Class Codes: G01N 33/5094 : for blood cell populations red blood cells G01N33/80

Abstract

Ex vivo and in vivo methods of expansion of renewable stem cells, expanded populations of renewable stem cells and their uses.

Claims

1. A method of expanding a population of CD34+ hematopoietic stem cells ex-vivo, while at the same time, inhibiting differentiation of the stem cells ex-vivo, the method comprising:

(a) culturing said CD34+ hematopoietic stem cells ex-vivo under conditions allowing for cell proliferation, said conditions which comprise providing nutrients, serum and a combination of cytokines including each of stem cell factor, thrombopoietin, FLt3 ligand, and IL-6 and optionally IL-3 and,

(b) in the same culture medium providing nicotinamide in an amount between 1.0 mM to 10 mM,

wherein culturing said cells for a culture period of three weeks results in expanding the population of CD34+ hematopoietic stem cells while inhibiting differentiation of said CD34+ hematopoietic stem cells ex-vivo to produce an expanded CD34+ hematopoietic stem cell population with an increased proportion of CD34+/Lin− and

CD34+/CD38−

cells in the expanded culture as compared to CD34+ cells cultured in the presence of cytokines and nutrients without exogenously added nicotinamide.

2. The method of claim 1, wherein said population of stem cells are selected from the group consisting of:

embryonic stem cells and adult stem cells.

3. The method of claim 1, wherein said stem cells are derived from a source selected from the group consisting of:

bone marrow, peripheral blood and neonatal umbilical cord blood.

4. The method of claim 1, wherein said expanded hematopoietic cells are further characterized by an absence, or significantly diminished expression of cell surface antigens CD3, CD61, CD19, CD33, CD14, CD15 or CD4.

5. The method of claim 1, wherein said combination of cytokines further comprise at least one cytokine selected from the group consisting of:

interleukin-1, interleukin-2 interleukin-10, interleukin-12 and tumor necrosis factor-α

.

6. The method of claim 1, which method further comprises providing late acting cytokines.

7. The method of claim 6, wherein said late acting cytokines are selected from the group consisting of:

granulocyte colony stimulating factor, granulocyte/macrophage colony stimulating factor, erythropoietin, FGF, EGF, NGF, VEGF, LIF, Hepatocyte growth factor and macrophage colony stimulating factor.

8. The method of claim 1, wherein said culturing said cells in the presence of said exogenously added nicotinamide is for a period of up to three weeks.

9. The method of claim 1, wherein said cells are cultured in the presence of 1.0 mM of exogenously added nicotinamide.

10. The method of claim 1, wherein said cells are cultured in the presence of 5.0 mM of exogenously added nicotinamide.

11. The method of claim 1, wherein said cells are cultured in the presence of 10.0 mM of exogenously added nicotinamide.