Myocardial grafts and cellular compositions

• US 5,733,727 A
• Filed: 06/07/1995
• Issued: 03/31/1998
• Est. Priority Date: 11/16/1993
• Status: Expired due to Term

• Current Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Original Assignee: Indiana University Foundation (Indiana University)
• Inventors: Field, Loren J.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US08/477,783
• Time in Patent Office: 1,028 Days
• Field of Search: 435/172.3, 435/240.2, 435/6, 435/354, 435/377
• US Class Current: 435/6.16
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0271 Chimeric vertebrates, e.g. comprising exogenous cells
• 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 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• 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: C07K 14/4716 Muscle proteins, e.g. myosin, actin
• CPC Class Codes: C12N 2500/22 Zinc; Zn chelators
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2517/02 Cells from transgenic animals
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: C12N 5/0658 Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts

Abstract

Described are preferred myocardial grafts of skeletal myoblasts or cardiomyocytes, and cellular compositions and methods useful in obtaining the grafts. The myocardial grafts are stable and can be used, for example, to deliver recombinant proteins directly to the heart.

Claims

1. A method of obtaining a population of cells, comprising:

transfecting embryonic stem cells to introduce a marker gene enabling selection of a cardiomyocyte cell lineage from other cell lineages resulting from the differentiation of the stem cells;

causing the stem cells to differentiate in culture; and

selecting said cardiomyocyte cell lineage based on said marker gene.

2. The method of claim 1, comprising:

transfecting the stem cells to introduce (i) a first marker gene enabling selection of transfected stem cells from non-transfected stem cells and (ii) a second marker gene enabling selection of said one cell lineage from said other cell lineages;

selecting transfected stern cells based on the first marker gene;

causing the selected stem cells to differentiate; and

selecting said one cell lineage based on said marker gene.

3. An embryonic stem cell having introduced DNA encoding a marker gene enabling selection of a cardiomyocyte cell lineage from other cell lineages resulting from the differentiation of the stem cells.

4. The stem cell of claim 3 which includes introduced DNA encoding (i) a first marker gene enabling selection of embryonic stem cells having the first marker gene from embryonic stem cells not having the first marker gene and (ii) a second marker gene enabling selection of said cardiomyocyte cell lineage from said other cell lineages.

5. The stem cell of claim 3, wherein said marker gene confers antibiotic resistance to cells which express the marker gene.

6. The stem cell of claim 4, wherein said first and second marker genes each confer antibiotic resistance to cells which express the marker genes.

7. The stem cell of claim 6, wherein said second marker gene is under the control of a promoter which causes expression of the marker gene only in said cardiomyocyte cell lineage.

8. A method of obtaining a population of cells, comprising:

providing an embryonic stem cell having introduced DNA encoding a marker gene enabling selection of a cardiomyocyte cell lineage from other cell lineages resulting from the differentiation of the stem cells;

causing the stem cell to differentiate in culture to form a mixed differentiated cellular population containing said a cardiomyocyte cell lineage and said other cell lineages;

selecting from the cellular population said a cardiomyocyte cell lineage from said other cell lineages based on said marker gene.

9. The method of claim 8 in which:

said marker gene is under the control of a promoter which causes expression of the marker gene in said cardiomyocyte cell lineage but not in said other cell lineages, so as to enable selection of said cardiomyocyte cell lineage from said other cell lineages.

10. The method of claim 9, in which said marker gene confers antibiotic resistance, and wherein said selecting includes contacting the mixed differentiated cellular population with an antibiotic to which cells of said one cell lineage have been conferred resistance by the expression of said first marker gene, thereby killing cells of said other cell lineages but not cells of said one cell lineage which express the marker gene.

11. The method of claim 9, in which said marker gene confers resistance to neomycin or hygromycin.

12. A population of cardiomyocyte cells obtained by a method according to claim 8.

13. The population of cells of claim 12, which is obtained by a method according to claim 10.