Production of brown adipocytes
Inventors
Kraus, Marine • Dalton, Stephen • Avery, III, John W.
Assignees
Societe des Produits Nestle SA • University of Georgia Research Foundation Inc
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-12129488-B2
Publication Date
2024-10-29
Expiration Date
Abstract
Use of a transforming growth factor beta (TGF-β) signalling inhibitor for producing a population of brown adipocytes in vitro.
Core Innovation
The patent describes an in vitro differentiation method that produces a population of human brown adipocytes from human induced pluripotent stem (hIPSC) cells. The method includes culturing the hIPSC cells with a transforming growth factor beta (TGF-β) inhibitor to obtain a first population of cells, and then culturing the first population without the TGF-β inhibitor to obtain a second population of cells.
The two-step culture regimen culminates in culturing the second population of cells to form aggregated brown adipocytes. The approach uses inhibition of TGF-β signalling during differentiation, followed by withdrawal of the inhibitor during a subsequent culture period, and the resulting brown adipocytes are characterized using brown-fat markers and functional features associated with uncoupled respiration and lipolysis.
The disclosed strategy further describes that the TGF-β inhibition can be directed to pathway targets including SMAD2/SMAD3 inhibition and/or activin A inhibition. Aggregated brown adipocytes can be produced in a culture format such as rotational suspension culture, and optional activation of UCP1 using forskolin is described in the context of the prepared brown adipocytes.
Claims Coverage
The partial content explicitly provides one independent claim. It contains three principal inventive aspects: a two-phase in vitro culture regimen with TGF-β inhibitor exposure followed by TGF-β inhibitor withdrawal, formation of aggregated brown adipocytes after the second culture phase, and the use of hIPSC cells as the starting cell population. Dependent claims refine the independent claim by specifying inhibitor targets, timing constraints, marker-associated cell expression, and culture formats for adherent culture and rotational suspension aggregation.
Two-step TGF-β inhibitor differentiation regimen for hIPSC
A method in which hIPSC cells are cultured with a transforming growth factor beta (TGF-β) inhibitor to obtain a first population, and then the first population is cultured in the absence of the TGF-β inhibitor to obtain a second population.
Formation of aggregated brown adipocytes from the second population
The method further includes culturing the second population of cells to form aggregated brown adipocytes.
Specified timing across inhibitor and no-inhibitor phases
The method includes culturing the hIPSC cells for about 1-15 days with a TGF-β inhibitor and then culturing the first population for about 20-50 days in the absence of the TGF-β inhibitor.
TGF-β pathway inhibitor targets and/or activin inhibition
The method specifies that the TGF-β inhibitor is a SMAD2 and SMAD3 inhibitor and/or an activin inhibitor.
Marker-associated expression in contacted cells
The method further includes contacting hIPSC cells with a TGF-β inhibitor such that the contacted hIPSC cells express FOXC1, FOXC2, MRF4, MSGN1, MYF5, PAX3, PAX7, PRRX1, SIX1 and/or TBX6.
Adherent cultures for both culture phases
The method specifies that culturing the hIPSC cells with a TGF-β inhibitor and culturing the first population of cells without the TGF-β inhibitor are both done in adherent cultures.
Rotational suspension culture for aggregated brown adipocytes
The method further specifies that culturing the second population of cells to form aggregated brown adipocytes is done using rotational suspension culture.
Overall, the claims coverage centers on producing aggregated brown adipocytes from hIPSC cells using a two-phase regimen in which TGF-β inhibition is applied during an initial culture period and then removed during a subsequent culture period, with dependent refinements specifying SMAD2/SMAD3 and/or activin inhibition, timing constraints, marker expression, and culture formats including adherent culture and rotational suspension culture.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?