Directed differentiation of embryonic cells

• US 20060128013A1
• Filed: 01/03/2006
• Published: 06/15/2006
• Est. Priority Date: 08/01/2000
• Status: Active Grant

• Current Assignee: Yissum RES & DEV Company of Hebrew University of Jerusalem
• Original Assignee: Yissum RES & DEV Company of Hebrew University of Jerusalem
• Inventors: Benvenisty, Nissim
• Granted Patent: US 7,772,001 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 5/38 of the suprarenal hormones
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: C12N 2501/13 Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
• CPC Class Codes: C12N 2501/15 Transforming growth factor beta (TGF-β)
• CPC Class Codes: C12N 2501/155 Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0603 Embryonic cells production of embryos, nuclear transfer A01K67/027; Embryoid bodies
• CPC Class Codes: C12N 5/0619 Neurons
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• 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 33/5044 : involving specific cell types
• CPC Class Codes: G01N 33/5073 : Stem cells

Abstract

Methods are described for mapping a pathway of differentiation of a population of embryonic cells that includes exposing the cells to an exogenous factor and measuring gene expression products that are characteristic of a particular cell type or lineage. Directing differentiation of human embryonic cells relies on dissociated embryoid bodies that are then exposed to one or more exogenous factors to enrich a culture for a particular cell type. The differentiated cells may be used for treating a medical condition in a human. Kits for determining differentiation pathways and screening exogenous factors for their utility in differentiation are provided.

Claims

1. A method of directing differentiation of human embryonic cells to a specific differentiated cell type, comprising:

a. providing chemically dissociated human embryonic stem (hES) cells;

b. aggregating the dissociated hES cells in suspension in order to form embryoid bodies;

c. dissociating the embryoid bodies to provide dissociated single embryonic cells;

d. culturing said dissociated embryonic cells as a monolayer; and

e. exposing said embryonic cells to at least one exogenous factor for an effective period of time to direct differentiation of said dissociated embryonic cells to a specific differentiated cell type.

2. A method according to claim 1, wherein the embryoid bodies are formed in a suspension culture.

3. A method according to claim 1, wherein the exogenous factor is a growth factor.

4. A method according to claim 1, wherein said specific differentiated cell type is mesodermal.

5. A method according to claim 1, wherein said specific differentiated cell type is ectodermal.

6. A method according to claim 1, wherein said specific differentiated cell type is endodermal.

7. A method according to claim 1, wherein said exogenous factor is selected from the group consisting of basic fibroblast growth factor, transforming growth factor, activin-A, bone morphogenic protein-4, hepatocyte growth factor, epidermal growth factor, β

nerve growth factor and retinoic acid.

8. A method in accordance with claim 1, wherein said specific differentiated cell type is selected from the group consisting of brain cells, liver cells, pancreatic cells, muscle cells, chondrocytes, kidney cells, Mullerian duct cells, heart cells, blood cells, skin cells and adrenal cells.

9. A method according to claim 1, wherein said exogenous factor is selected from the group consisting of interleukins, hedgehog proteins, vascular endothelial growth factor, granulocyte macrophage colony stimulating factor, macrophage colony stimulating factor, wnt, int and other members of the FGF family.