Generation of novel adipocyte monolayer cultures from embryonic stem cells.
Here we show a simplified and improved method to produce large quantities of evenly distributed monolayer cultures that display major characteristics of adipocytes. These cultures are applicable for quantitative analysis for biochemical and molecular events in adipogenesis during development and may provide a useful system for high-throughput drug screening assays of antiobesity drugs. In our method, we treated embryoid bodies (EBs) with all-trans retinoic acid (ATRA) for 3 days, 1 day after they attached to the gelatin-coated culture plates without further transfer. The cells were maintained in insulin and trioiodothyronine (T(3))-containing medium until day 12, when they were dispersed by enzymatic digestion and replated onto multiple culture plates. Two days later, adipocyte induction factors were added for 6 days and examined 6 days later. The amount of lipid droplet-laden adipocytes in the culture reached approximately 80%, with a nearly five-fold increase in GPDH activity. The cells expressed high levels of adipose-specific proteins (adipocyte markers), including PPARgamma2, ALBP, LPL, HSL, perilipin, and DGAT1. The adipocytes are functionally active, as evidenced by their response to lipolytic agents, such as forskolin, Bt2-cAMP, and isoproterenol, with more than 20-fold increases in glycerol release.
Chen
Theresa L
TL
Veterans Affairs, Palo Alto Health Care System, Palo Alto, CA 94304, USA. Theresa.Chen@va.gov
Shen
Wen-Jun
WJ
Qiu
Xin-Wen
XW
Li
Tao
T
Hoffman
Andrew R
AR
Kraemer
Fredric B
FB
D017667:Adipocytes / Q000166:cytology* / Q000378:metabolism; D050156:Adipogenesis / Q000502:physiology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D003994:Bucladesine / Q000494:pharmacology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D051379:Mice; D014212:Tretinoin / Q000494:pharmacology
D000970:Antineoplastic Agents; D015415:Biomarkers; D005576:Colforsin; D014212:Tretinoin; D003994:Bucladesine