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Isolation and Differentiation of Primary White and Brown Preadipocytes from Newborn Mice
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Conditional immortalization of primary adipocyte precursor cells.

Christopher Church1, Mallory Brown2, Matthew S Rodeheffer3

  • 1Section of Comparative Medicine; School of Medicine; Yale University ; New Haven, CT, USA.

Adipocyte
|August 11, 2015
PubMed
Summary
This summary is machine-generated.

Creating new fat cells (adipocytes) requires precursor cells. Immortalizing these precursor cells with SV40 T antigen did not overcome their limited ability to multiply and differentiate in culture.

Keywords:
3-isobutyl-1-methylxanthine; MDIADASCSV40 T large antigen; WATadipocyte precursoradipocyte precursor; IBMXadipogenesisadipose tissue derived adult stem cells; APand IBMX adipogenic differentiation cocktail; SV40 T-Agcell culturedexamethonsoneimmortalizationinsulinpreadipocytewhite adipose tissue

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Area of Science:

  • Cell Biology
  • Adipogenesis Research
  • Stem Cell Biology

Background:

  • Adipocyte production is crucial for adipose tissue homeostasis.
  • Adipocyte precursor (AP) cells in the stromal-vascular compartment differentiate into new adipocytes.
  • Understanding AP cell behavior is key to metabolic research.

Purpose of the Study:

  • To establish an immortalized adipogenic cell line from committed APs.
  • To investigate the potential of immortalized APs for in vitro expansion and differentiation.
  • To assess the impact of SV40 T antigen on AP cell proliferation and adipogenesis.

Main Methods:

  • Isolation of committed APs from mouse white adipose tissue (WAT) using fluorescence-activated cell sorting (FACS).
  • Culture of APs with conditional SV40 T antigen expression for immortalization.
  • Induction of adipogenic differentiation and assessment via Oil Red O staining.
  • Evaluation of cell proliferation and key adipogenic gene expression (Pparγ, C/ebpα) across passages.

Main Results:

  • Committed APs immortalized with SV40 T antigen showed limited proliferative capacity.
  • Adipogenic potential rapidly declined with increasing passage number.
  • Expression of late adipogenic markers (Pparγ, C/ebpα) decreased significantly over time.
  • FACS-purified committed APs demonstrated restricted in vitro expansion and differentiation.

Conclusions:

  • SV40 T antigen-mediated immortalization does not confer indefinite expansion or differentiation potential to committed APs.
  • FACS-purified committed adipocyte precursors have inherent limitations for in vitro studies.
  • Further strategies are needed to overcome the senescence of APs for robust in vitro adipogenesis models.