Pluripotent mesenchymal stem cells and methods of use thereof

• US 5,827,735 A
• Filed: 05/20/1996
• Issued: 10/27/1998
• Est. Priority Date: 06/22/1992
• Status: Expired due to Term

• Current Assignee: Cybios LLC
• Original Assignee: Morphogen Pharmaceuticals, Inc.
• Inventors: Young, Henry E., Lucas, Paul A.
• Primary Examiner(s): VANDERVEGT, FRANCOIS P
• Application Number:
  US08/650,420
• Time in Patent Office: 890 Days
• Field of Search: 435/349, 435/329, 435/379, 435/380, 435/395, 435/405, 435/325
• US Class Current: 435/325
• CPC Class Codes: A61F 2/2846 Support means for bone substitute or for bone graft implants, e.g. membranes or plates for covering bone defects nets or sleeves applied to surface of endoprostheses A61F2/30907; bone plates A61B17/80; bone regeneration in dental surgery A61C8/0006
• CPC Class Codes: A61F 2/30756 Cartilage endoprostheses A61F2/3603 takes precedence
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons

Abstract

A scar inhibitory factor protein isolate from mammalian basement membranes is provided that inhibits lineage commitment and differentiation of stem cells in vitro and in vivo. The protein isolate is characterized by its ability to inhibit stem cell commitment to a fibroblastic-scar phenotype without killing the cells, thus allowing their differentiation into normal tissue phenotypes. SIF thus limits the amount of scar tissue formation at the site of delivery, while maximizing the potential for the stem cells to differentiate into other tissue phenotypes (muscle, cartilage, bone, fat, etc.). Therefore, it is useful in treating numerous disorders and injuries that currently result in scar tissue or fibrous adhesion formation. The protein isolate can be administered in various modalities in vivo, i.e., as a transdermal patch, incorporated into wound dressings, incorporated into absorbable suture material, incorporated into a bioerodible polymer matrix by itself or interspersed with differentiation factors near the site of tissue injury, sprayed onto prosthetic implants, and can be administered directly to cells cultured in vitro.

Claims

1. Purified pluripotent mesenchymal stem cells, which cells are substantially free of multinucleated myogenic lineage-committed cells, and which cells are predominantly stellate-shaped cells, wherein the mesenchymal stem cells form predominantly fibroblastic cells when contacted with muscle morphogenic protein in tissue culture medium containing 10% fetal calf serum and form predominantly branched multinucleated structures that spontaneously contract when contacted with muscle morphogenic protein and scar inhibitory factor in tissue culture with medium containing 10% fetal calf serum.

2. The pluripotent mesenchymal stem cells of claim 1 isolated from muscle.

3. The pluripotent mesenchymal stem cells of claim 2 isolated from chick muscle.

4. Purified pluripotent mesenchymal stem cells prepared by the method comprising:

a) preparing a single cells suspension of muscle cells;

b) culturing the cells in culture medium supplemented with (i) 10% preselected serum or (ii) 10% horse serum or fetal bovine serum supplemented with an effective amount of scar inhibitory factor at 37°

C. in a humidified CO₂ incubator until myogenic lineage-committed cells form multinucleated spontaneously contracting myotubes embedded within multiple confluent layers of mononucleated stellate-shaped cells;

c) releasing the cells from the plate; and

d) separating the mesenchymal stem cells from the multinucleated cells.

5. The pluripotent mesenchymal stem cells of claim 4, which have been cryopreserved in cell culture medium containing (i) 10% pre-selected serum or (ii) 10% horse serum or fetal bovine serum supplemented with an effective amount of scar inhibitory factor, and 7.5% dimethyl sulfoxide.

6. The pluripotent mesenchymal stem cells of claim 4 wherein the pre-selected serum is pre-selected horse serum.

7. The pluripotent mesenchymal stem cells of claim 4 isolated from muscle.

8. The pluripotent mesenchymal stem cells of claim 7 isolated from chick muscle.

9. The pluripotent mesenchymal stem cells of claim 8 wherein the culture medium further comprises about 5% stage-specific embryo extract.

10. The pluripotent mesenchymal stem cells of claim 4 which are released by trypsin treatment.

11. A method for obtaining purified pluripotent mesenchymal stem cells comprising:

a) preparing a single cells suspension of muscle cells;

b) culturing the cells in culture medium supplemented with (i) 10% pre-selected serum or (ii) 10% horse serum or fetal bovine serum supplemented with an effective amount of scar inhibitory factor at 37°

C. in a humidified CO₂ incubator until myogenic lineage-committed cells had formed multinucleated spontaneously contracting myotubes embedded within multiple confluent layers of mononucleated stellate-shaped cells;

c) releasing the cells from the plate; and

d) separating the mesenchymal stem cells from the multinucleated cells.

12. The method according to claim 11, further comprising cryopreserving the pluripotent mesenchymal stem cells in cell culture medium containing (i) 10% pre-selected serum or (ii) 10% horse serum or fetal bovine serum supplemented with an effective amount of scar inhibitory factor, and 7.5% dimethyl sulfoxide.

13. The method according to claim 11 wherein the pre-selected serum is pre-selected horse serum.

14. The method according to claim 11 wherein the pluripotent mesenchymal stem cells are isolated from muscle.

15. The method according to claim 14 wherein the pluripotent mesenchymal stem cells are isolated from chick muscle.

16. The method according to claim 15 wherein the culture medium further comprises about 5% stage-specific embryo extract.

17. The method according to claim 11 wherein the pluripotent mesenchymal stem cells are released by trypsin treatment.