Methods and compositions for the differentiation of human preadipocytes into adipocytes

• US 7,001,746 B1
• Filed: 06/01/2000
• Issued: 02/21/2006
• Est. Priority Date: 01/29/1999
• Status: Expired due to Fees

• Current Assignee: Artecel Sciences Inc.
• Original Assignee: Artecel Sciences Inc.
• Inventors: Wilkison, William O., Halvorsen, Yuan-Di Chang
• Primary Examiner(s): Saucier, Sandra E.
• Application Number:
  US09/585,821
• Time in Patent Office: 2,091 Days
• Field of Search: 435/377, 435/404, 435/405, 435/406, 435/407, 435/70.3
• US Class Current: 435/70.3
• CPC Class Codes: A61L 27/38 containing added animal cells organs or tissue containing native cells A61L27/36
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/33 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 2503/02 Drug screening
• CPC Class Codes: C12N 5/0653 Adipocytes; Adipose tissue

Abstract

The present invention provides methods and compositions for the consistent and quantitative differentiation of human preadipocytes isolated from adipose tissue into adipocytes bearing biochemical, genetic, and physiological characteristics similar to that observed in isolated primary adipocytes. The methods of the invention comprise incubating isolated human preadipocytes, plated at least about 25,000 cells/cm², in a medium containing, glucose, a cyclic AMP inducer such as isobutylmethylxanthine or forskolin, a glucocorticoid or glucocorticoid analogue, insulin or an insulin analogue and a PPARγ agonist or a RXR agonist. The compositions of the invention include media for the differentiation of human preadipocytes, human adipocytes differentiated by the methods of the invention and transfected adipocytes.

The present invention also provides methods for determining the ability of a compound to affect the differentiation of human preadipocytes to adipocytes, for determining the ability of a compound to act as a PPARγ antagonist. a glucocorticoid, a glucocoticoid analogue, or an insulin analogue, for transfecting cultured human adipocytes, and as a means to identify novel polypeptides secreted from human adipocytes into the conditioned medium. The methods and compositions have use in the drug discovery of compounds having relevance to the disease states of diabetes, obesity, and cardiovascular disease and in the studies of these diseases.

Claims

1. A method of identifying a protein, a polypeptide or a peptide secreted from a differentiated human adipocyte, said method comprising:

a) isolating human preadipocytes and culturing said preadipocytes in a preadipocyte medium for a period of time, wherein said preadipocytes are plated at a density of about 25,000 to 40,000 cells/cm², and further wherein said preadipocytes are primary cultures;

b) differentiating said human preadipocytes with a differentiation medium comprising a defined cell culture medium;

wherein said medium comprises 1.0-4.5 g/liter glucose, a cyclic AMP inducer, a glucocorticoid or a glucocorticoid analogue, insulin or an insulin analogue, and a Peroxisome Proliferator Activated Receptor gamma agonist or a retinoic acid X receptor agonist effective to stimulate differentiation of said human preadipocytes;

c) fractionating the conditioned medium of the differentiated adipocytes;

d) comparing the pattern of proteins, polypeptides or peptides in the conditioned medium of said differentiated adipocytes with the pattern of proteins, polypeptides or peptides secreted by an undifferentiated adipocyte;

e) identifying the protein, polypeptide or peptide secreted by the differentiated adipocyte.

2. The method of claim 1, wherein said differentiation medium further comprise 3-10% fetal bovine serum.

3. The method of claim 1, wherein said differentiation medium further comprise 1-100 μ

M pantothenate and 1-100 μ

M biotin.

4. The method of claim 1, wherein said differentiation medium further comprises a buffer having a pH of about 7.0 to 7.6.

5. The method of claim 1, wherein said differentiation medium is Dulbeccos Modified Eagle/Hams'"'"' F-10 Nutrient Broth (1:

1 vol/vol).

6. The method of claim 1, wherein said Peroxisome Proliferator Activated Receptor gamma agonist is thiazolidinedione.

7. The method of claim 6, wherein said thiazolidinedione is BRL 49653.

8. The method of claim 7, wherein the concentration of said BRL 49653 is about 0.5-1.0 μ

M.

9. The method of claim 6, wherein said thiazolidinedione is troglitazone.

10. The method of claim 9, wherein the concentration of said troglitazone is about 0.5-1.0 μ

M.

11. The method of claim 1, wherein said glucocorticoid is dexamethasone, hydrocortisone or cortisol.

12. The method of claim 1, wherein the concentration of said glucocorticoid is about 16 nM to 1.0 μ

M.

13. The method of claim 1, wherein said cyclic AMP inducer is isobutylmethylxanthine or forskolin.

14. The method of claim 13, wherein said isobutylmethylxanthine is present in said differentiation medium at a final concentration of about 0.2 to 0.5 mM.

15. The method of claim 1, wherein the concentration of insulin is about 100 nM to 1.0 μ

M.

16. The method of claim 1, wherein exogenous DNA has been introduced into said preadipocyte.