Brown fat cell compositions and methods

• US 9,133,438 B2
• Filed: 06/28/2012
• Issued: 09/15/2015
• Est. Priority Date: 06/29/2011
• Status: Active Grant

• Current Assignee: Biorestorative Therapies, Inc.
• Original Assignee: Biorestorative Therapies, Inc.
• Inventors: Silva, Francisco, Weinreb, Mark, Patel, Amit N., Bull, David A.
• Primary Examiner(s): Long, Scott
• Application Number:
  US14/129,635
• Publication Number:
  US 20140370591A1
• Time in Patent Office: 1,174 Days
• US Class Current: 1/1
• 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: A61K 35/35 Fat tissue; Adipocytes; Stromal cells; Connective tissues adipose-derived stem cells A61K35/28; collagen A61K38/39
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: C12N 2500/84 from mammals
• CPC Class Codes: C12N 2500/98 Xeno-free medium and culture conditions
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/02 Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• 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 2501/39 Steroid hormones
• CPC Class Codes: C12N 2501/395 Thyroid hormones
• CPC Class Codes: C12N 2506/1384 from adipose-derived stem cells [ADSC], from adipose stromal stem cells
• CPC Class Codes: C12N 5/0653 Adipocytes; Adipose tissue
• CPC Class Codes: C12N 5/0667 Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5044 involving specific cell types
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Methods of developing and using cell lines, such as stem cell lines, for therapeutic or cosmetic use. In one embodiment the cell lines are used to treat a wide range of degenerative and metabolic disorders including, but not limited to, obesity, diabetes, hypertension, and cardiac deficiency. Also described are methods of using such cell lines to screen for compounds that play a role in regulating a variety of processes.

Claims

1. A method of generating a stem cell, comprising:

obtaining brown fat tissue;

isolating a stem cell from the brown fat tissue; and

culturing the stem cell in a differentiation medium not comprising a xenogenic animal product, wherein the differentiation medium comprises, insulin, dexamethasone, isobutylmethylxanthine, indomethacin, triiodothyronine (T3), rosiglitazone, fibronectin type III domain-containing protein 5 (FNDC5), and 0.5 to 20% human platelet lysate;

thereby generating a stem cell,

wherein the stem cell is positive for one or more of the following cell surface markers;

CD63, CD90, HLA, ABC, CD105, CD73, CD166, CD9, CD44, and

wherein the stem cell is negative for one or more of the following cell surface markers;

CD34, CD19, CD86, HLA DR, Lin, CD106, CD80, CD117.

2. The method of claim 1, wherein the brown fat tissue is from a sample obtained from a subject.

3. The method of claim 1, wherein the differentiation medium further comprises an anticoagulant selected from a group consisting of heparin and acid-citrate dextrose.

4. The method of claim 1, further comprising cold shocking the stem cell, wherein the cold shocking comprises:

exposing the stem cell to daily cycles of 4°

C. to 25°

C. for one hour followed by exposing the stem cell to 37°

C.

5. The method of claim 1, further comprising exposing the stem cell in the differentiation medium to hypoxic conditions.

6. The method of claim 1, wherein the differentiation medium comprises 10 to 20% human platelet lysate.

7. A method of generating a stem cell, comprising:

obtaining brown fat tissue;

isolating a stem cell from the brown fat tissue; and

culturing the stem cell in a differentiation medium not comprising a xenogenic animal product, wherein the differentiation medium consists essentially of insulin, dexamethasone, isobutylmethylxanthine, indomethacin, triiodothyronine (T3), rosiglitazone, fibronectin type III domain-containing protein 5 (FNDC5), and 0.5 to 20% human platelet lysate;

thereby generating a stem cell,

wherein the stem cell is positive for one or more of the following cell surface markers;

CD63, CD90, HLA, ABC, CD105, CD73, CD166, CD9, CD44, and

wherein the stem cell is negative for one or more of the following cell surface markers;

CD34, CD19, CD86, HLA DR, Lin, CD106, CD80, CD117.

8. The method of claim 7, wherein the brown fat tissue is from a sample obtained from a subject.

9. The method of claim 7, further comprising exposing the stem cell in the differentiation medium to hypoxic conditions.

10. The method of claim 7, wherein the differentiation medium comprises 10 to 20% human platelet lysate.

11. A method of generating a stem cell, comprising:

obtaining brown fat tissue;

isolating a stem cell from the brown fat tissue; and

culturing the stem cell in a differentiation medium not comprising a xenogenic animal product, wherein the differentiation medium comprises 0.5 to 20% human platelet lysate;

cold shocking the stem cell, wherein the cold shocking comprises;

exposing the stem cell to daily cycles of 4°

C. to 25°

C. for one hour followed by exposing the stem cell to 37°

C.;

thereby generating a stem cell;

wherein the stem cell is positive for one or more of the following cell surface markers;

CD63, CD90, HLA, ABC, CD105, CD73, CD166, CD9, CD44, and

wherein the stem cell is negative for one or more of the following cell surface markers;

CD34, CD19, CD86, HLA DR, Lin, CD106, CD80, CD117.

12. The method of claim 11, wherein the differentiation medium further comprises insulin, dexamethasone, isobutylmethylxanthine, indomethacin, triiodothyronine (T3), rosiglitazone, and fibronectin type III domain-containing protein 5 (FNDC5).

13. The method of claim 11, wherein the differentiation medium further comprises an anticoagulant selected from a group consisting of heparin and acid-citrate dextrose.

14. The method of claim 11, wherein the brown fat tissue is from a sample obtained from a subject.

15. The method of claim 11, further comprising exposing the stem cell in the differentiation medium to hypoxic conditions.

16. The method of claim 11, wherein the differentiation medium comprises 10 to 20% human platelet lysate.