OP9 mouse stromal cells rapidly differentiate into adipocytes: characterization of a useful new model of adipogenesis

Abstract

Much knowledge of adipocyte biology has been learned from cell culture models, most notably 3T3-L1 cells. The 3T3-L1 model has several limitations, including the requirement of 2 weeks to generate adipocytes and the waning of adipogenic potential in culture. We have investigated the capacity of OP9 cells, a line of bone marrowderived mouse stromal cells, to recapitulate adipogenesis. When OP9 cells are given any one of three adipogenic stimuli, they rapidly accumulate triacylglycerol, assume adipocyte morphology, and express adipocyte late marker proteins, including glucose transporter 4 and adiponectin. OP9 cells can differentiate into adipocytes within 2 days. This rapid rate of differentiation allows for the detection of transiently expressed proteins in mature OP9 adipocytes. Adipogenesis in OP9 cells involves the master transcriptional regulator of adipocyte differentiation, peroxisome proliferator-activated receptor g (PPARg). OP9 cells are late preadipocytes in that, before the addition of adipogenic stimuli, they express the adipocyte proteins CCAAT/enhancer binding proteins a and b, PPARg, sterol-regulatory element binding protein-1, S3-12, and perilipin. OP9 differentiation is not diminished by maintenance in culture at high cell density or by long periods in continuous culture, thereby facilitating the generation of stable cell lines that retain adipogenic potential. Thus, the unique features of OP9 cells will expedite the study of adipocyte biology. The increase in obesity and the identification of adipocyte-secreted proteins that regulate energy metabolism (1) have generated interest in adipocyte biology. Adipocytes are the primary storage site for energy in vertebrate animals. During fasting, adipocytes release energy-rich molecules that provide metabolic fuels to other tissues. Adipocytes also secrete hormones that orchestrate the storage, release, and oxidation of energy-rich molecules throughout the body and that control behavior, including feeding (2). Primary adipocytes maintain a large dynamic triacylglycerol (TAG) pool and express a specific set of proteins to maintain circulating metabolic fuel levels. Primary adipocytes and adipose tissue have been used to study basic adipocyte biology. However, these systems have several limitations: they do not propagate in culture, they are difficult to transfect with DNA, they have a huge TAG store that interferes with biochemistry and microscopy, they vary as a result of the genetics and conditions of the animals from which they are isolated, and the isolation procedure is tedious and introduces variation. In addition, harvesting primary adipocytes or adipose from animals generally requires the euthanasia of a vertebrate animal and the expense of specialized facilities and protocols. For these reasons and likely others, cell lines have been developed that can be induced to store TAG, to express proteins that are hallmarks of adipocytes, and presumably to recapitulate key events in adipocyte ontogeny.Three decades ago, Green and colleagues (3-...

Diacylglycerol Enrichment of Endoplasmic Reticulum or Lipid Droplets Recruits Perilipin 3/TIP47 during Lipid Storage and Mobilization

“…Cell Culture-OP9 mouse stromal cells, which have the potential to rapidly differentiate into adipocytes, were cultured as described previously (18). However, these cells were used as preadipocytes in all experiments.…”

“…In this study we use OP9 preadipocytes, which express both perilipin 3 and perilipin 4 (18). These preadipocytes have small lipid droplets as well as large flat processes with defined ER, which makes them suitable for imaging the relationship between these organelles.…”

Identification of an Insulin-regulated Lysophospholipase with Homology to Neuropathy Target Esterase

“…These bone marrow-derived mouse stromal cells can be differentiated into adipocytes accumulating TG and expressing adipocyte late marker proteins (26). Cells were cultivated in Minimum Essential Medium (MEM)␣ (Invitrogen) with 20% FCS, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin at 37°C in humidified air (89 -91% saturation) and 5% CO 2 .…”

Adipocytic Cells Augment the Support of Primitive Hematopoietic Cells In Vitro But Have No Effect in the Bone Marrow Niche Under Homeostatic Conditions

“…To achieve this, we performed CAFC assays at 5 weeks with five different cell lines. These included M2-10B4 (a mouse BM stromal cell line derived from a C57BL/6J · C3H/HeJ F1 mouse [22]), MC3T3-E1 (a preosteoblast cell line derived from a C57Bl/6 mouse [23]), ST2 (a BM stromal cell line derived from BC8 mice that can differentiate into osteoblasts and adipocytes [24][25][26]), 3T3-L1 (a preadipocyte substrain of the embryonic fibroblast cell line 3T3 [27]), and OP9 (a newborn calvaria BM stromal cell line derived from a C57BL/6 · C3H F2-op/op mouse [28]). As shown in Fig.…”