Pluripotent embryonic-like stem cells, compositions, methods and uses thereof

• US 20040033214A1
• Filed: 05/22/2003
• Published: 02/19/2004
• Est. Priority Date: 09/24/1999
• Status: Abandoned Application

• Current Assignee: ABT Holding Company (Athersys, Inc.)
• Original Assignee: ABT Holding Company (Athersys, Inc.)
• Inventors: Young, Henry E., Lucas, Paul A.
• Application Number:
  US10/443,663
• Publication Number:
  US 20040033214A1
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: C12N 2503/00 Use of cells in diagnostics
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 5/0607 Non-embryonic pluripotent stem cells, e.g. MASC induced pluripotent stem cells C12N5/0696
• 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 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention relates to pluripotent stem cells, particularly to pluripotent embryonic-like stem cells. The invention further relates to methods of purifying pluripotent embryonic-like stem cells and to compositions, cultures and clones thereof. The present invention also relates to a method of transplanting the pluripotent stem cells of the present invention in a mammalian host, such as human, comprising introducing the stem cells, into the host. The invention further relates to methods of in vivo administration of a protein or gene of interest comprising transfecting a pluripotent stem cell with a construct comprising DNA which encodes a protein of interest and then introducing the stem cell into the host where the protein or gene of interest is expressed. The present also relates to methods of producing mesodermal, endodermal or ectodermal lineage-committed cells by culturing or transplantation of the pluripotent stem cells of the present invention.

Claims

1. A pluripotent embryonic-like stem cell, derived from non-embryonic or postnatal animal cells or tissue, capable of self-renewal and capable of differentiation to cells of endodermal, ectodermal and mesodermal lineages.

2. The stem-cell of claim 1 which is a human cell.

3. The stem cell of claim 1 which is isolated from the non-embryonic tissue selected from the group of muscle, dermis, fat, tendon, ligament, perichondrium, periosteum, heart, aorta, endocardium, myocardium, epicardium, large arteries and veins, granulation tissue, peripheral nerves, peripheral ganglia, spinal cord, dura, leptomeninges, trachea, esophagus, marrow, stomach, small intestine, large intestine, liver, spleen, pancreas, parietal peritoneum, visceral peritoneum, parietal pleura, visceral pleura, urinary bladder, gall bladder, kidney, associated connective tissues or bone marrow.

4. A pluripoent endodermal stem cell derived from the stem cell of claim 1.

5. A pluripotent mesenchymal stem cell derived from the stem cell of claim 1.

6. A pluripotent ectodermal stem cell derived from the stem cell of claim 1.

7. A endodermal, ectodermal or mesodermal lineage-committed cell derived from the stem cell of claim 1.

14. The stem cell of claim 1 genetically engineered to express a gene or protein of interest.

18. A method for detecting the presence or activity of an agent which is a lineage-commitment factor comprising the steps of:

A. contacting the stem cells of claim 1 with a sample suspected of containing an agent which is a lineage-commitment factor; and

B. determining the lineage of the so contacted cells by mRNA expression, antigen expression or other means;

wherein the lineage of the contacted cells indicates the presence or activity of a lineage-commitment factor in said sample.

19. A method of testing the ability of an agent, compound or factor to modulate the lineage-commitment of a lineage uncommitted cell which comprises

A. culturing the stem cells of claim 1 in a growth medium which maintains the stem cells as lineage uncommited cells;

B. adding the agent, compound or factor under test; and

C. determining the lineage of the so contacted cells by mRNA expression, antigen expression or other means.

20. An assay system for screening agents, compounds or factors for the ability to modulate the lineage-commitment of a lineage uncommitted cell, comprising:

A. culturing the stem cells of claim 1 in a growth medium which maintains the stem cells as lineage uncommited cells;

B. adding the agent, compound or factor under test; and

C. determining the lineage of the so contacted cells by mRNA expression, antigen expression or other means.

21. A method for detecting the presence or activity of an agent which is a proliferation factor comprising the steps of:

A. contacting the stem cells of claim 1 with a sample suspected of containing an agent which is a proliferation factor; and

B. determining the proliferation and lineage of the so contacted cells by mRNA expression, antigen expression or other means;

wherein the proliferation of the contacted cells without lineage commitment indicates the presence or activity of a proliferation factor in said sample.

22. A method of testing the ability of an agent, compound or factor to modulate the proliferation of a lineage uncommitted cell which comprises

A. culturing the stem cells of claim 1 in a growth medium which maintains the stem cells as lineage uncommited cells;

B. adding the agent, compound or factor under test; and

C. determining the proliferation and lineage of the so contacted cells by mRNA expression, antigen expression or other means.

23. An assay system for screening agents, compounds or factors for the ability to modulate the proliferation of a lineage uncommitted cell, comprising:

A. culturing the stem cells of claim 1 in a growth medium which maintains the stem cells as lineage uncommited cells;

B. adding the agent, compound or factor under test; and

C. determining the proliferation and lineage of the so contacted cells by mRNA expression, antigen expression or other means.

24. A method of transplanting pluripotent embryonic-like stem cells in a host comprising the step of introducing into the host the stem cells of claim 1.

25. A method of providing a host with purified pluripotent embryonic-like stem cells comprising the step of introducing into the host the pluripotent embryonic-like stem cells of claim 1.

26. A method of in vivo administration of a protein or gene of interest comprising the step of transfecting the pluripotent embryonic-like stem cell of claim 1 with a vector comprising DNA or RNA which expresses a protein or gene of interest.

29. A method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals, comprising administering to a mammal a therapeutically effective amount of a endodermal, ectodermal or mesodermal lineage-committed cell derived from the stem cell of claim 1.

30. A method of tissue repair or transplantation in mammals, comprising administering to a mammal a therapeutically effective amount of a endodermal, ectodermal or mesodermal lineage-committed cell derived from the stem cell of claim 1.

8. A culture comprising:

(a) Pluripotent embryonic-like stem cells, derived from postnatal animal cells or tissue, capable of self-renewal and capable of differentiation to cells of endodermal, ectodermal and mesodermal lineages; and

(b) a medium capable of supporting the proliferation of said stem cells.

9. The culture of claim 8, further comprising a proliferation factor or lineage commitment factor.

10. The culture of claim 8 wherein said stem cells are human cells.

11. A method of isolating an pluripotent embryonic-like stem cell, comprising the steps of:

(a) obtaining cells from a postnatal animal source;

(b) slow freezing said cells in medium containing 7.5% (v/v) dimethyl sulfoxide until a final temperature of −

80°

C. is reached; and

(c) culturing the cells.

12. A method of isolating a clonal pluripotent embryonic-like stem cell line, comprising the steps of:

(a) obtaining cells from a postnatal animal source;

(b) slow freezing said cells in medium containing 7.5% (v/v) dimethyl sulfoxide until a final temperature of −

80°

C. is reached;

(c) culturing the cells;

(d) diluting said cultured cells to clonal density;

(e) culturing said diluted cells;

(e) propogating those cultures having a single cell.

13. A clonal pluripotent embryonic-like stem cell line developed by the method of claim 12.

15. A method of producing a genetically engineered pluripotent embryonic-likestem cell comprising the steps of:

(a) transfecting pluripotent embryonic-like stem cells with a DNA construct comprising at least one of a marker gene or a gene of interest;

(b) selecting for expression of the marker gene or gene of interest in the pluripotent embryonic-like stem cells;

(c) culturing the stem cells selected in (b).

16. A genetically engineered pluripotent embryonic-like stem cell produced by the method of claim 15.

17. The stem cell of claim 16 which is a human cell.

27. A method of preventing and/or treating cellular debilitations, derangements and/or dysfunctions and/or other disease states in mammals, comprising administering to a mammal a therapeutically effective amount of pluripotent embryonic-like stem cells, or cells or tissues derived therefrom.

28. A method of tissue repair or transplantation in mammals, comprising administering to a-mammal a therapeutically effective amount of pluripotent embryonic-like stem cells, or cells or tissues derived therefrom.

32. The pharmaceutical composition of claim 28 further comprising a proliferation factor or lineage-commitment factor.

31. A pharmaceutical composition for the treatment of cellular debilitation, derangement and/or dysfunction in mammals, comprising:

A. a therapeutically effective amount of pluripotent embryonic-like stem cells, or cells or tissues derived therefrom; and

B. a pharmaceutically acceptable medium or carrier.