Brown adipocyte progenitors in human skeletal muscle
Inventors
Boss, Olivier D. • Crisan, Mihaela • Giacobino, Jean-Paul
Assignees
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Publication Number
US-11299710-B2
Publication Date
2022-04-12
Expiration Date
Abstract
This invention relates to brown adipose tissue (BAT) progenitor cells and methods for isolating BAT progenitor cells from skeletal muscle. BAT progenitor cell surface markers and medium and agents for inducing cell differentiation into brown adipocytes are also provided. In some embodiments, the BAT progenitor cell expresses a first cell surface marker associated with endothelial cells, the first cell surface marker being detectable in an antibody based assay using a first antibody. In addition, the BAT progenitor cell can be substantially free of a second cell surface marker associated with endothelial cells, the second cell surface marker being substantially undetectable in said antibody based assay using a second antibody. The BAT progenitor cell can also be substantially free of additional cell surface markers.
Core Innovation
The invention relates to a method for identifying an agent that induces differentiation of a brown adipocyte progenitor cell into a brown adipocyte. The method starts by contacting a population of skeletal muscle cells with first and second antibodies specific for CD34 and CD31, respectively, and selecting brown adipocyte progenitor cells based on CD34 expression and CD31 absence.
Selected brown adipocyte progenitor cells are then contacted with an agent that induces differentiation into a brown adipocyte. Differentiation is assessed in the brown adipocyte progenitor cell by determining the presence of an indicator of differentiation induced by the agent, including increases in brown-adipocyte markers such as UCP1 and optionally other markers.
The claim set further specifies additional negative selection using antibodies specific for CD146, CD45, and CD56, and differentiation indicators including UCP1 protein and/or UCP1 mRNA increases, as well as other brown-adipocyte or mitochondrial gene markers. The agent is defined broadly as an agent selected from named signaling pathway modulators, activators, inhibitors, and related targets, including PPAR-related agents and multiple other pathway-targeting agents.
Claims Coverage
The patent provides at least two independent methods directed to identifying agents that induce differentiation of brown adipocyte progenitor cells into brown adipocytes. Both independent claims share a core immunoselection scheme based on CD34 and CD31 and a determination of a differentiation indicator after contacting with an agent, with dependent claims adding refinements to the indicator, the selectable progenitor population, and the agent definition via specific pathway targets.
CD34 and CD31 immunoselection of brown adipocyte progenitor cells
Contacting a population of skeletal muscle cells with a first and a second antibody specific for CD34 and CD31, respectively; and selecting a brown adipocyte progenitor cell on the basis of expression of CD34 and CD31, wherein the brown adipocyte progenitor cell is positive for CD34 and negative for CD31.
Agent contact and differentiation-indicator determination
Contacting the selected brown adipocyte progenitor cell with an agent that induces differentiation into a brown adipocyte; and determining in the brown adipocyte progenitor cell the presence of an indicator of differentiation into a brown adipocyte induced by the agent.
Plurality selection and differentiation-indicator determination
Contacting a population of skeletal muscle cells with a first and a second antibody specific for CD34 and CD31, respectively; selecting a plurality of brown adipocyte progenitor cells on the basis of expression of CD34 and CD31, wherein the brown adipocyte progenitor cell is positive for CD34 and negative for CD31; contacting the selected brown adipocyte progenitor cell with an agent that induces differentiation into a brown adipocyte; and determining in the plurality of brown adipocyte progenitor cells the presence of an indicator of differentiation into a brown adipocyte induced by the agent.
Indicator readout based on UCP1 expression
Determining differentiation via an increase in uncoupling protein-1 (UCP1) protein and/or UCP1 mRNA in the plurality of brown adipocyte progenitor cells.
Agent selection from named pathway modulators
Selecting an agent from specified modulators, activators, inhibitors targeting named pathway-related proteins and receptors, including PPAR family-related agents and other signaling pathway modulators and inhibitors, and combinations thereof.
Across the independent claims, coverage centers on immunoselecting brown adipocyte progenitor cells from skeletal muscle using CD34 and CD31 antibodies, contacting the selected progenitors with an agent, and determining the presence of a differentiation indicator in the progenitors. Dependent claim refinements include UCP1-based readouts, additional negative selection markers CD146, CD45, and CD56, optional progenitor expansion/passaging, and a broad definition of the agent via named pathway-targeting modulators and combinations.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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