WO2013071459A2 - Composition comprenant un glucocorticoïde et une tiazolinédione pour induire une différenciation adipogène complète des cellules mères de mammifères - Google Patents

Composition comprenant un glucocorticoïde et une tiazolinédione pour induire une différenciation adipogène complète des cellules mères de mammifères Download PDF

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WO2013071459A2
WO2013071459A2 PCT/CL2012/000076 CL2012000076W WO2013071459A2 WO 2013071459 A2 WO2013071459 A2 WO 2013071459A2 CL 2012000076 W CL2012000076 W CL 2012000076W WO 2013071459 A2 WO2013071459 A2 WO 2013071459A2
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stem cells
use according
composition
glucocorticoid
thiazolinedione
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PCT/CL2012/000076
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WO2013071459A3 (fr
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Paulette CONGET MOLINA
David CONTADOR MARTINEZ
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Universidad Del Desarrollo
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Publication of WO2013071459A3 publication Critical patent/WO2013071459A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells

Definitions

  • Composition comprising a glucocorticoid and a thiazolinedione to induce complete adipogenic differentiation of mammalian stem cells
  • the adipogenesis process follows a sequence of hierarchical steps. In vitro, it has been described that prior to differentiation, the adipogenic precursors must be at proliferative rest (Gregoire et al., 1998). This is necessary but not indispensable, because it has been shown that cells seeded at low density can also initiate their differentiation. What is essential to stimulate adipogenesis in vitro is that the cells are exposed to differentiation inducers (Prokesch et al., 2009).
  • adipogenic stimulus is that patented by Osiris Therapeutics Inc. (US6322784B1). This was originally implemented to induce adipocyte differentiation from mesenchymal stem cells, and combines a glucocorticoid (dexamethasone), a phosphodiesterase (isobutyl methylxanthine) inhibitor, a cyclooxygenase (indomethacin) inhibitor and insulin.
  • glucocorticoid diexamethasone
  • phosphodiesterase isobutyl methylxanthine
  • indomethacin cyclooxygenase
  • This classic cocktail has some limitations of use, among them: i) it does not efficiently promote adipogenic differentiation of mesenchymal stem cells of some mammalian species, ii) the adipocytes that are generated using it are incompletely differentiated, iii) it must be prepared every time used because its stability over time is limited, iv) the stock solutions of its constituents change their physical-chemical properties over time (they precipitate, become cloudy, etc).
  • the present invention - detailed below - i) promotes the adipogenic differentiation of mesenchymal stem cells from different species of mammals, including those refractory to the classic cocktail, i) allows the generation of functional mature adipocytes.
  • the composition is stable during the period in which it should be used, iv) stock solutions do not undergo major changes over time. That is, the invention It has obvious advantages compared to the classic cocktail both in terms of its use and its commercialization.
  • the present invention is directed to provide a composition comprising a glucocorticoid and a thiazolinedione to induce adipogenic differentiation since:
  • Glucocorticoid receptors are expressed in primary preadipocytes and in cell lines established from them (Joyner et al., 2000).
  • glucocorticoids activate C / EBPdelta - from English CCAAT enhancer binding protein delta- (Wiper- Bergeron et al., 2003) and this one to C / EBPalpha and PPARgamma - from English Peroxisome Proliferator Activated Gamma Receptor-, both known regulators intracellular adipogenic differentiation (Wu et al., 1996).
  • PPARgamma is a nuclear receptor and acts as a transcription factor when it binds to its endogenous ligands (eg metabolite of prostaglandin J2) or exogenous (eg thiazolinediones such as rosiglitazone, pioglitazone, troglitazone).
  • endogenous ligands eg metabolite of prostaglandin J2
  • exogenous eg thiazolinediones such as rosiglitazone, pioglitazone, troglitazone.
  • the authors of the present invention have observed that the use of the composition comprising a glucocorticoid and a thiazolinedione robustly promotes adipogenic differentiation of stem cells that respond discreetly or that do not respond to conventional stimulation (cocktail classic).
  • adipocytes generated from stem cells exposed to the composition are mature and functional, that is, they have completed the differentiation process and, among other characteristics, have the ability to be highly sensitive to insulin and to secrete adipokines.
  • FIGURE 1 the invention induces adipogenic differentiation of human mesenchymal stem cells.
  • the cells were exposed to medium without stimulus (A), or to medium supplemented with DMSO (B), the classic cocktail (C), composition (D), dexamethasone (E), rosiglitazone (F), isobutyl methylxanthine + rosiglitazone (G) or indomethacin + rosiglitazone (H).
  • A medium without stimulus
  • B medium supplemented with DMSO
  • C composition
  • D dexamethasone
  • F rosiglitazone
  • G isobutyl methylxanthine + rosiglitazone
  • H indomethacin + rosiglitazone
  • the cells were exposed to medium supplemented with the composition comprising dexamethasone and rosiglitazone in the following concentrations (uM): 1 + 10 (D), 0.1 + 1 (Da), 0.5 + 5 (Db), 2 + 20 (De), 5 + 50 (Dd), 10 + 100 (De), 1 + 1 (Df), 1 + 5 (Dg), 1 + 20 (Dh), 1 + 50 (Di), 1 + 100 (Dj), 0.1 + 10 (Dk), 0.5 + 10 (DI), 2 + 10 (Dm), 5 + 10 (Dn), 10 + 10 (Do), 0.1 + 100 (Dp ), 0.5 + 50 (Dq), 1 + 20 (Dr), 2 + 10 (Ds), 5 + 5 (Dt) or 10 + 1 (Du).
  • FIGURE 3 the invention significantly induces adipogenic differentiation of human mesenchymal stem cells at different concentrations.
  • the cells were exposed to medium supplemented with the composition comprising dexamethasone and rosiglitazone in the following concentrations (uM): 1 + 10 (D), 0.1 + 1 (Da), 0.5 + 5 (Db), 2 + 20 (De), 5 + 50 (Dd), 10 + 100 (De), 1 + 1 (Df), 1 + 5 (Dg), 1 + 20 (Dh), 1 + 50 (Di), 1 + 100 (Dj), 0.1 + 10 (Dk), 0.5 + 10 (DI), 2 + 10 (Dm), 5 + 10 (Dn), 10 + 10 (Do), 0.1 + 100 (Dp ), 0.5 + 50 (Dq), 1 + 20 (Dr), 2 + 10 (Ds), 5 + 5 (Dt) or 10 + 1 (Du).
  • FIGURE 4 the invention induces adipogenic differentiation of mesenchymal stem cells from other mammals.
  • FIGURE 5 the invention significantly induces adipogenic differentiation of mouse mesenchymal stem cells.
  • FIGURE 6 the invention significantly induces adipogenic differentiation of rat mesenchymal stem cells.
  • FIGURE 7 the invention significantly induces adipogenic differentiation of rabbit mesenchymal stem cells.
  • FIGURE 8 the invention significantly induces adipogenic differentiation of dog mesenchymal stem cells.
  • FIGURE 9 adipocytes generated from human mesenchymal stem cells exposed to the invention are insulin sensitive.
  • FIGURE 10 adipocytes generated from human mesenchymal stem cells exposed to the classic cocktail are not insulin sensitive.
  • FIGURE 11 adipocytes generated from human mesenchymal stem cells exposed to the invention express the LEPTINA gene.
  • RNA was isolated from the cells and then quantified by real-time RT-PCR levels of LEPTIN and GAPDH mRNA (relative abundance expressed in arbitrary units). The data shown correspond mean ⁇ standard error, (n 7).
  • FIGURE 12 adipocytes generated from human mesenchymal stem cells exposed to the invention secrete leptin.
  • the present invention relates to a composition for inducing adipogenic differentiation of undifferentiated stem cells to the state of functional mature adipocyte.
  • This reagent consists of the combination of two synthetic components: a glucocorticoid and a thiazolinedione in a biocompatible vehicle, the addition of additional components not being necessary.
  • the stem cells used in the present invention can come from different species of mammals, not just humans.
  • the cell type to be used in the present invention corresponds to all types of adult or embryonic stem cells, among which mesenchymal stem cells are preferred.
  • composition could be produced on an industrial level and marketed as a reagent to be used for the following purposes:
  • adipocytes could be used as a source of molecules of biological interest (adipokines, cytokines, etc.).
  • adipocytes could be used to be transplanted to the same individual (autologous) or another (allogeneic).
  • adipocytes from stem cells, for example in metabolic diseases where instead of transplanting adipose tissue, adipocytes or indifferent stem cells, the composition would be administered.
  • adipocytes from stem cells, for example in reconstructive surgeries where instead of transplanting adipose tissue, adipocytes or indifferent stem cells, the composition would be administered.
  • EXAMPLE 1 Induction of adipogenic differentiation of human mesenchymal stem cells
  • the human mesenchymal stem cells were seeded at a density of 2.5x10 4 cells / cm 2 in minimal essential medium alpha supplemented with 10% fetal bovine serum (v / v) + 80ug / ml gentamicin (hereinafter alpha-10) and were kept under humid atmosphere air / C0 2 95/5% at 37 ° C.
  • alpha-10 the medium was replaced by alpha-10 supplemented with 1 mM dexamethasone + 100 mg isobutyl-methylxanthine + 100mM indomethacin + 0.2UI / mL insulin (classic cocktail) or with 1 uM dexamethasone plus 10uM rosiglitazone (composition).
  • adipogenic differentiation was assessed by staining with 0/7 Red O or with Nile Red.
  • the cells were washed three times with PBS and incubated in an aqueous solution of 60% (v / v) saturated isopropanol. with OH Red O for one hour at room temperature. Then, the cells were washed three times with PBS and observed under inverted light microscopy with phase contrast. Digital photographic record of the results was left.
  • Nile Red 1mg / mL in DMSO was added and incubated 5 min.
  • the cells were acquired by DAKO Cyan cytometer. Adipocytes were identified based on the following three criteria: size (FSC), granularity or internal complexity (SSC) and fluorescence intensity in the FU channel (Nile Red) that were previously "set” in the equipment. A total of 10,000 events were analyzed in each sample.
  • EXAMPLE 2 Induction of adipogenic differentiation of human mesenchymal stem cells using different concentrations of dexamethasone + rosiglitasone
  • EXAMPLE 3 Induction of adipogenic differentiation of mesenchymal stem cells from other mammals
  • the composition promoted the differentiation of mesenchymal stem cells derived from other mammals, in particular mouse, rat, rabbit and dog.
  • the classic cocktail and the composition induced in a similar magnitude the differentiation of the rat and rabbit stem cells ( Figures 4, 6 and 7).
  • the classic cocktail marginally induced adipogenic differentiation of mouse and dog stem cells, the composition promoted it efficiently ( Figures 4, 5 and 8).
  • Every process of differentiation consists of two stages, this is commitment and maturation.
  • a cell is functional when it completes its maturation process.
  • adipocytes when in addition to accumulating drops of triacylglycerides and expressing the genes of this lineage, loses its proliferative potential, is sensitive to insulin and secretes adipokines (eg leptin) (Pelleymounter et al., 1995; Friedman and Halaas, 1998).
  • adipokines eg leptin
  • INSULIN SENSITIVITY Human mesenchymal stem cells were seeded at a density of 2.5x10 4 cells / cm 2 and incubated for 14 days in the presence of the classic cocktail or composition. The sensitivity of these cells to insulin was evaluated based on the incorporation of the radioactive glucose analogue
  • [ 3 H] 2-deoxiglucose [ 3 H] 2-DG.
  • the cells Prior to the determination, the cells were deprived of fetal bovine serum for 4 hours. Then, they were incubated with insulin in increasing concentrations for 30 min, washed with PBS at 37 ° C and incubated with PBS solution containing 2-DG 4mM and [ 3 H] 2-DG 2uCi / ml for 1 min. The cells were washed with PBS at 4 ° C and frozen at -20 ° C. After using them with 0.5N formic acid for 1 h, an aliquot was taken to quantify the proteins and the rest was incubated with pure formic acid for 30 min. Finally, the level of radioactivity that the cells in the LKB Rackbeta 1217 scintillation counter was determined.
  • Adipocytes generated from human mesenchymal stem cells exposed to the composition are insulin sensitive ( Figure 9), however those generated with the classic cocktail are not ( Figure 10).
  • RNAs were stored at -80 ° C for later analysis.
  • the PCR reaction was performed in a LightCycler ® thermal cycler, using the LightCycler ® FastStar DNA Master SYBR Green I kit.
  • Each reaction mixture (10 uL) contained: 50ng of cDNA; each 0.5mM splitter and 3mM MgC. Data were quantified by the ACt method (Schmittgen and Livak, 2008).
  • LEPTINE SECRETION The culture medium was replaced by bovine fetal serum free medium. At 24 hrs, the conditioned medium was collected and centrifuged at 2,500xg for 10 min. Then, the supernatant was recovered and stored in a clean tube at -80 ° C. For quantification of leptin, the Quantikine ® human leptin immunoassay ELISA kit from R&D System was used. In the cultures of which the conditioned medium was collected, the percentage of adipocytes was also determined by staining with Red tea and flow cytometry.
  • adipocytes generated with the composition secrete leptin, however those generated with the classic cocktail do not.
  • osteoblast cells mesenchymal stem cells
  • nM glucocorticoid dexamethasone
  • uM dexamethasone
  • Rosiglitazone increases it i) appearance of fat drops -> in neutral epithelial cells
  • rosiglitazone does not affect it ii) induction of expression of ii) with respect to phenotype genes of adipocytes osteoblast exposure to iii) insulin sensitization dexamethasone and rosiglitazone: iv) induction of secretion of -> decrease expression two adipokines
  • Ninomiya Y Sugahara-Yamashita Y, Nakachi Y, Tokuzawa Y, Okazaki Y, Nishiyama M. Development of a rapid culture method to induce adipocyte differentiation of human bone marrow-derived mesenchymal stem cells. Biochem Biophys Res Commun. (2010) 394: 303-8.
  • Schmittgen TD Livak KJ. Analyzing real-time PCR data by the comparative C (T) method. Nat Protoc. (2008) 3: 1101-8.
  • WO2006130690A2 Title: METHODS AND COMPOSITIONS FOR INDUCING BROWN ADIPOGENESIS. Assignee / Applicant: JOSLIN DIABETES CENTER INC., US. Inventor: TSENG, Yu Hua. Priority Date: 2006-06-01. Publication Date: 2006-12-07
  • WO2000042211A Title: METHODS OF SCREENING PROTEASE INHIBITORS, OF INDUCING MICE SUSCEPTIBLE TO HIV PROTEASE INHIBITOR-INDUCED DYSLIPIDEMIA, AND GENES ASSOCIATED THEREWITH. Assignee / Applicant: GLAXO GROUP LIMITED, GB. Inventor: LENHARD, James, Martin. Priority Date: 2000-01-19. Publication Date: 2000-07-20
  • JP2008194044A Title: ENDOCRINE PANCREAS DIFFERENTIATION OF ADIPOSE TISSUE-DERIVED STROMAL CELLS AND USES THEREOF. Assignee / Applicant: ARTECEL SCIENCES INC. Inventor: CHEATHAM BENTLEY, HALVORSEN YUAN-Dl C, GIMBLE JEFFREY M. Priority Date: 2008-02-14. Publication Date: 2008-08-28

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Abstract

La présente invention concerne une composition qui comprend un glucocorticoïde et une tiazolinédione pour induire une différenciation adipogène, qui permet de générer des cellules matures fonctionnelles, à partir de cellules mères indifférenciées humaines ou d'autres mammifères, embryonnaires ou adultes, idéalement des cellules mères mésenchymateuses humaines. Le glucocorticoïde préféré correspond à une dexaméthasone et la tiazolinédione préférée à une rosiglitazone. Bien entendu, l'invention s'étend aux familles des deux composés.
PCT/CL2012/000076 2011-10-24 2012-12-24 Composition comprenant un glucocorticoïde et une tiazolinédione pour induire une différenciation adipogène complète des cellules mères de mammifères WO2013071459A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110008893A1 (en) * 2007-12-28 2011-01-13 Fujirebio Inc. Medium for mammalian somatic cells and additive therefor

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110008893A1 (en) * 2007-12-28 2011-01-13 Fujirebio Inc. Medium for mammalian somatic cells and additive therefor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HALVORSEN Y D ET AL.: 'Thiazolidinediones and glucocorticoids synergistically induce differentiation of human adipose tissue stromal cells: biochemical, cellular, and molecular analysis.' METABOLISM, CLINICAL AND EXPERIMENTAL. vol. 50, no. 4, April 2001, ISSN 0026-0495 pages 407 - 413 *
RYDEN M ET AL.: 'Functional characterization of human mesenchymal stem cell-derived adipocytes.' BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS. vol. 311, no. 2, 14 November 2003, ISSN 0006-291X pages 391 - 397 *
TCHOUKALOVA Y D ET AL.: 'Enhancing effect of troglitazone on porcine adipocyte differentiation in primary culture: a comparison with dexamethasone.' OBESITY RESEARCH. vol. 8, no. 9, 2000, ISSN 1071-7323 pages 664 - 672 *

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