Brown adipocyte progenitors in human skeletal muscle
Inventors
Boss, Olivier D. • Giacobino, Jean-Paul
Assignees
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Publication Number
US-9410945-B2
Publication Date
2016-08-09
Expiration Date
Abstract
Brown adipose tissue (“BAT”) progenitor cells and methods for identifying BAT progenitor cells in a population of cells are provided. Methods are also provided for inducing differentiation of BAT progenitor cells into differentiated brown adipocytes, inducing expression or increased activity levels of BAT uncoupling protein-1 (“UCP1”), and for identifying agents capable of inducing differentiation of BAT progenitor cells into brown adipocytes and/or inducing expression or increased activity levels of UCP1. Differentiated brown adipocytes and agents and methods for inducing differentiation of BAT progenitor cells can be used for treatment of or the making of medicaments for the treatment of metabolic diseases or conditions in a patient such as obesity, overweight, impaired glucose tolerance, insulin-resistance, type 2 diabetes, dyslipidemia, hypertension, cardiovascular diseases, metabolic syndrome, and the like. Differentiated brown adipocytes and agents and methods for inducing differentiation of BAT progenitor cells can be used for prevention of hypothermia.
Core Innovation
The disclosure identifies and uses brown adipose tissue progenitor cells isolated from human skeletal muscle, including CD34 positive cells, to generate in vitro differentiated brown adipocytes. The differentiated brown adipocytes are produced in an adipogenic medium containing a differentiation agent that induces differentiation from the CD34 positive cells.
The resulting brown adipocytes are characterized by expression and/or increased expression of UCP1 and thermogenic mitochondrial markers, including mtTFA/TFAM, PGC-1α, and cytochrome oxidase IV (COX IV). The disclosure further describes uncoupled respiration and high metabolic activity, and uses UCP1 as a principal molecular marker for assessing differentiation and thermogenic phenotype.
The disclosure describes modulation of UCP1 in vitro using differentiation agents including rosiglitazone, including agents referred to as PPARγ agonist(s), and cAMP derivatives, including cell-permeating cAMP. It also describes increased skeletal-muscle UCP1 mRNA in humans after rosiglitazone treatment, and assays for screening differentiation and UCP1-expression agents using CD34 positive cells and a human UCP1 promoter/enhancer reporter construct.
Claims Coverage
The independent claim covers one composition comprising in vitro differentiated brown adipocytes produced in an adipogenic medium from CD34 positive cells isolated from human skeletal muscle, where the adipogenic medium comprises a differentiation agent that induces differentiation. Dependent claims further define cell selection constraints, specify example differentiation agents, and characterize resulting brown adipocytes by molecular and functional markers and by a comparative UCP1 mRNA level.
In vitro differentiated brown adipocytes from human skeletal muscle CD34 positive cells
A composition comprising a population of in vitro differentiated brown adipocytes and an adipogenic medium, wherein the brown adipocytes are differentiated in vitro in the adipogenic medium from a plurality of CD34 positive cells cultured until confluency, wherein the CD34 positive cells are isolated from human skeletal muscle.
Adipogenic medium with a differentiation agent that induces brown adipocyte differentiation
The adipogenic medium comprises a differentiation agent which induces differentiation of the CD34 positive cells into brown adipocytes.
Cell selection by excluding CD45 in CD34 positive cells
The CD34 positive cells are negative for the CD45 marker.
Brown adipocyte characterization by UCP1, mtTFA, PGC-1α, and uncoupled respiration
The brown adipocytes are characterized by one or more of the following: expression of UCP1 protein or mRNA, expression of mtTFA protein or mRNA, expression of PGC-1α protein or mRNA, uncoupled respiration, metabolic rate, glucose utilization rate, fatty acid oxidation rate, and a combination of any of the foregoing.
Comparative UCP1 mRNA level higher than in vivo human skeletal muscle
The brown adipocytes contain UCP1 mRNA at a level higher than that of in vivo brown adipocytes in human skeletal muscle.
Differentiation agent comprises rosiglitazone
The differentiation agent comprises rosiglitazone.
Overall claim coverage is directed to in vitro differentiation of CD34 positive cells isolated from human skeletal muscle into brown adipocytes using an adipogenic medium containing a differentiation agent, with further definitions of cell-selection constraints and characterization of the brown adipocytes by thermogenic and molecular markers, uncoupled respiration and metabolic performance, including a comparative UCP1 mRNA level and an example differentiation agent (rosiglitazone).
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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